Author Report for: Lester HA

The identification of proteins that are altered following nicotine/tobacco exposure can facilitate and positively impact the investigation of related diseases. In this report, we investigated the effects of chronic (-)-menthol exposure in 14 murine brain regions for changes in total beta2 subunit protein levels and changes in epibatidine binding levels using immunoblotting and radioligand binding assays. We identified the habenula as a region of interest due to the region's marked decreases in beta2 subunit and nAChR levels in response to chronic (-)-menthol alone. Thus, we further examined the habenula, a brain region associated with both the reward and withdrawal components of addiction, for additional protein level alterations using mass spectrometry. A total of 552 proteins with altered levels were identified after chronic (-)-menthol exposure. Enriched in the proteins with altered levels after (-)-menthol exposure were proteins associated with signaling, immune systems, RNA regulation, and protein transport. The continuation and expansion of the brain region-specific protein profiling in response to (-)-menthol will provide a better understanding of how this common flavorant in tobacco and e-liquid products may affect addiction and general health.

In addition to dopaminergic and motor deficits, patients with Parkinson's disease (PD) suffer from non-motor symptoms, including early cognitive and social impairment, that do not respond well to dopaminergic therapy. Cholinergic deficits may contribute to these problems, but cholinesterase inhibitors have limited efficacy. Mice over-expressing alpha-synuclein, a protein critically associated with PD, show deficits in cognitive and social interaction tests, as well as a decrease in cortical acetylcholine. We have evaluated the effects of chronic administration of nicotine in mice over-expressing wild type human alpha-synuclein under the Thy1-promoter (Thy1-aSyn mice). Nicotine was administered subcutaneously by osmotic minipump for 6months from 2 to 8months of age at 0.4mg/kg/h and 2.0mg/kg/h. The higher dose was toxic in the Thy1-aSyn mice, but the low dose was well tolerated and both doses ameliorated cognitive impairment in Y-maze performance after 5months of treatment. In a separate cohort of Thy1-aSyn mice, nicotine was administered at the lower dose for one month beginning at 5months of age. This treatment partially eliminated the cognitive deficit in novel object recognition and social impairment. In contrast, chronic nicotine did not improve motor deficits after 2, 4 or 6months of treatment, nor modified alpha-synuclein aggregation, tyrosine hydroxylase immunostaining, synaptic and dendritic markers, or microglial activation in Thy1-aSyn mice. These results suggest that cognitive and social impairment in synucleinopathies like PD may result from deficits in cholinergic neurotransmission and may benefit from chronic administration of nicotinic agonists.

Nicotinic acetylcholine receptors (nAChRs) are pentameric cation channels expressed in the mammalian CNS, in the peripheral nervous system, and in skeletal muscle. Neuronal-type nAChRs are also found in several non-neuronal cell types, including leukocytes. Granulocytes are a subtype of leukocytes that include basophils, eosinophils, and neutrophils. Granulocytes, also known as polymorphonuclear leukocytes, are characterized by their ability to produce, store, and release compounds from intracellular granules. Granulocytes are the most abundant type of leukocyte circulating in the peripheral blood. Granulocyte abundance, nAChR expression, and nAChR upregulation following chronic nicotine administration makes granulocytes interesting models for identifying protein markers of nicotine exposure. Nicotinic receptor subunits and several non-nAChR proteins have been identified as protein markers of granulocyte nicotine exposure. We review methods to isolate granulocytes from human tissue, summarize present data about the expression of nAChRs in the three granulocyte cell types (basophils, eosinophils, and neutrophils), describe current knowledge of the effects of nicotine exposure on human granulocyte protein expression, and highlight areas of interest for future investigation. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms.

Upregulation of beta2 subunit-containing (beta2*) nicotinic acetylcholine receptors (nAChRs) is implicated in several aspects of nicotine addiction, and menthol cigarette smokers tend to upregulate beta2* nAChRs more than nonmenthol cigarette smokers. We investigated the effect of long-term menthol alone on midbrain neurons containing nAChRs. In midbrain dopaminergic (DA) neurons from mice containing fluorescent nAChR subunits, menthol alone increased the number of alpha4 and alpha6 nAChR subunits, but this upregulation did not occur in midbrain GABAergic neurons. Thus, chronic menthol produces a cell-type-selective upregulation of alpha4* nAChRs, complementing that of chronic nicotine alone, which upregulates alpha4 subunit-containing (alpha4*) nAChRs in GABAergic but not DA neurons. In mouse brain slices and cultured midbrain neurons, menthol reduced DA neuron firing frequency and altered DA neuron excitability following nAChR activation. Furthermore, menthol exposure before nicotine abolished nicotine reward-related behavior in mice. In neuroblastoma cells transfected with fluorescent nAChR subunits, exposure to 500 nm menthol alone also increased nAChR number and favored the formation of (alpha4)3(beta2)2 nAChRs; this contrasts with the action of nicotine itself, which favors (alpha4)2(beta2)3 nAChRs. Menthol alone also increases the number of alpha6beta2 receptors that exclude the beta3 subunit. Thus, menthol stabilizes lower-sensitivity alpha4* and alpha6 subunit-containing nAChRs, possibly by acting as a chemical chaperone. The abolition of nicotine reward-related behavior may be mediated through menthol's ability to stabilize lower-sensitivity nAChRs and alter DA neuron excitability. We conclude that menthol is more than a tobacco flavorant: administered alone chronically, it alters midbrain DA neurons of the nicotine reward-related pathway.

A number of mutations in alpha4beta2-containing (alpha4beta2*) nicotinic acetylcholine (ACh) receptors (nAChRs) are linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), including one in the beta2 subunit called beta2V287L. Two alpha4beta2* subtypes with different subunit stoichiometries and ACh sensitivities co-exist in the brain, a high-sensitivity subtype with (alpha4)2(beta2)3 subunit stoichiometry and a low-sensitivity subtype with (alpha4)3(beta2)2 stoichiometry. The alpha5 nicotinic subunit also co-assembles with alpha4beta2 to form a high-sensitivity alpha5alpha4beta2 nAChR. Previous studies suggest that the beta2V287L mutation suppresses low-sensitivity alpha4beta2* nAChR expression in a knock-in mouse model and also that alpha5 co-expression improves the surface expression of ADNFLE mutant nAChRs in a cell line. To test these hypotheses further, we expressed mutant and wild-type (WT) nAChRs in oocytes and mammalian cell lines, and measured the effects of the beta2V287L mutation on surface receptor expression and the ACh response using electrophysiology, a voltage-sensitive fluorescent dye, and superecliptic pHluorin (SEP). The beta2V287L mutation reduced the EC50 values of high- and low-sensitivity alpha4beta2 nAChRs expressed in Xenopus oocytes for ACh by a similar factor and suppressed low-sensitivity alpha4beta2 expression. In contrast, it did not affect the EC50 of alpha5alpha4beta2 nAChRs for ACh. Measurements of the ACh responses of WT and mutant nAChRs expressed in mammalian cell lines using a voltage-sensitive fluorescent dye and whole-cell patch-clamping confirm the oocyte data. They also show that, despite reducing the maximum response, beta2V287L increased the alpha4beta2 response to a sub-saturating ACh concentration (1 muM). Finally, imaging SEP-tagged alpha5, alpha4, beta2, and beta2V287L subunits showed that beta2V287L reduced total alpha4beta2 nAChR surface expression, increased the number of beta2 subunits per alpha4beta2 receptor, and increased surface alpha5alpha4beta2 nAChR expression. Thus, the beta2V287L mutation alters the subunit composition and sensitivity of alpha4beta2 nAChRs, and increases alpha5alpha4beta2 surface expression.

Retrospective epidemiological studies show an inverse correlation between susceptibility to Parkinson's disease and a person's history of tobacco use. Animal model studies suggest nicotine as a neuroprotective agent and nicotinic acetylcholine (ACh) receptors (nAChRs) as targets for neuroprotection, but the underlying neuroprotective mechanism(s) are unknown. We cultured mouse ventral midbrain neurons for 3 weeks. Ten to 20% of neurons were dopaminergic (DA), revealed by tyrosine hydroxylase (TH) immunoreactivity. We evoked mild endoplasmic reticulum (ER) stress with tunicamycin (Tu), producing modest increases in the level of nuclear ATF6, phosphorylated eukaryotic initiation factor 2alpha, nuclear XBP1, and the downstream proapoptotic effector nuclear C/EBP homologous protein. We incubated cultures for 2 weeks with 200 nm nicotine, the approximate steady-state concentration between cigarette smoking or vaping, or during nicotine patch use. Nicotine incubation suppressed Tu-induced ER stress and the unfolded protein response (UPR). Study of mice with fluorescent nAChR subunits showed that the cultured TH+ neurons displayed alpha4, alpha6, and beta3 nAChR subunit expression and ACh-evoked currents. Gene expression profile in cultures from TH-eGFP mice showed that the TH+ neurons also express several other genes associated with DA release. Nicotine also upregulated ACh-induced currents in DA neurons by approximately 2.5-fold. Thus, nicotine, at a concentration too low to activate an appreciable fraction of plasma membrane nAChRs, induces two sequelae of pharmacological chaperoning in the ER: UPR suppression and nAChR upregulation. Therefore, one mechanism of neuroprotection by nicotine is pharmacological chaperoning, leading to UPR suppression. Measuring this pathway may help in assessing neuroprotection. SIGNIFICANCE STATEMENT: Parkinson's disease (PD) cannot yet be cured or prevented. However, many retrospective epidemiological studies reveal that PD is diagnosed less frequently in tobacco users. Existing programs attempting to develop nicotinic drugs that might exert this apparent neuroprotective effect are asking whether agonists, antagonists, partial agonists, or channel blockers show the most promise. The underlying logic resembles the previous development of varenicline for smoking cessation. We studied whether, and how, nicotine produces neuroprotective effects in cultured dopaminergic neurons, an experimentally tractable, mechanistically revealing neuronal system. We show that nicotine, operating via nicotinic receptors, does protect these neurons against endoplasmic reticulum stress. However, the mechanism is probably "inside-out": pharmacological chaperoning in the endoplasmic reticulum. This cellular-level insight could help to guide neuroprotective strategies.

Upregulation of neuronal nicotinic acetylcholine receptors (AChRs) is a venerable result of chronic exposure to nicotine; but it is one of several consequences of pharmacological chaperoning by nicotine and by some other nicotinic ligands, especially agonists. Nicotinic ligands permeate through cell membranes, bind to immature AChR oligomers, elicit incompletely understood conformational reorganizations, increase the interaction between adjacent AChR subunits, and enhance the maturation process toward stable AChR pentamers. These changes and stabilizations in turn lead to increases in both anterograde and retrograde traffic within the early secretory pathway. In addition to the eventual upregulation of AChRs at the plasma membrane, other effects of pharmacological chaperoning include modifications to endoplasmic reticulum stress and to the unfolded protein response. Because these processes depend on pharmacological chaperoning within intracellular organelles, we group them as "inside-out pharmacology". This term contrasts with the better-known, acute, "outside-in" effects of activating and desensitizing plasma membrane AChRs. We review current knowledge concerning the mechanisms and consequences of inside-out pharmacology. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'.

Nicotinic acetylcholine receptors (nAChRs) are vital to neuronal signaling, are implicated in important processes such as learning and memory, and are therapeutic targets for neural diseases. The alpha7 nAChR has been implicated in Alzheimer's disease and schizophrenia, and allosteric modulators have become one focus of drug development efforts. We investigate the mode of action of the alpha7-selective positive allosteric modulator, PNU-120596, and show that the higher potency of acetylcholine in the presence of PNU-120596 is not due to an altered agonist binding site. In addition, we propose several residues in the gating interface and transmembrane region that are functionally important to transduction of allosteric properties, and link PNU-120596, the acetylcholine binding region, and the receptor gate. These results suggest global protein stabilization from a communication network through several key residues that alter the gating equilibrium of the receptor while leaving the agonist binding properties unperturbed.

The alpha6-containing subtypes of the nicotinic acetylcholine receptor (nAChR) are localized to presynaptic terminals of the dopaminergic pathways of the central nervous system. Selective ligands for these nAChRs are potentially useful in both Parkinson's disease and addiction. For these and other goals, it is important to distinguish the binding behavior of agonists at the alpha6-beta2 binding site versus other subtypes. To study this problem, we apply nonsense suppression-based non-canonical amino acid mutagenesis. We report a combination of four mutations in alpha6beta2 that yield high-level heterologous expression in Xenopus oocytes. By varying mRNA injection ratios, two populations were observed with unique characteristics, likely due to differing stoichiometries. Responses to nine known nAChR agonists were analyzed at the receptor, and their corresponding EC50 values and efficacies are reported. The system is compatible with nonsense suppression, allowing structure-function studies between Trp149 - a conserved residue on loop B found to make a cation-pi interaction at several nAChR subtypes - and several agonists. These studies reveal that acetylcholine forms a strong cation-pi interaction with the conserved tryptophan, while nicotine and TC299423 do not, suggesting a unique pharmacology for the alpha6beta2 nAChR.

The human alpha7 neuronal nicotinic acetylcholine receptor gene (CHRNA7) is ubiquitously expressed in both the central nervous system and in the periphery. CHRNA7 is genetically linked to multiple disorders with cognitive deficits, including schizophrenia, bipolar disorder, ADHD, epilepsy, Alzheimer's disease, and Rett syndrome. The regulation of CHRNA7 is complex; more than a dozen mechanisms are known, one of which is a partial duplication of the parent gene. Exons 5-10 of CHRNA7 on chromosome 15 were duplicated and inserted 1.6 Mb upstream of CHRNA7, interrupting an earlier partial duplication of two other genes. The chimeric CHRFAM7A gene product, dupalpha7, assembles with alpha7 subunits, resulting in a dominant negative regulation of function. The duplication is human specific, occurring neither in primates nor in rodents. The duplicated alpha7 sequence in exons 5-10 of CHRFAM7A is almost identical to CHRNA7, and thus is not completely queried in high throughput genetic studies (GWAS). Further, pre-clinical animal models of the alpha7nAChR utilized in drug development research do not have CHRFAM7A (dupalpha7) and cannot fully model human drug responses. The wide expression of CHRNA7, its multiple functions and modes of regulation present challenges for study of this gene in disease. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'.

Chronic pain is a highly prevalent and poorly managed human health problem. We used microarray-based expression genomics in 25 inbred mouse strains to identify dorsal root ganglion (DRG)-expressed genetic contributors to mechanical allodynia, a prominent symptom of chronic pain. We identified expression levels of Chrna6, which encodes the alpha6 subunit of the nicotinic acetylcholine receptor (nAChR), as highly associated with allodynia. We confirmed the importance of alpha6* (alpha6-containing) nAChRs by analyzing both gain- and loss-of-function mutants. We find that mechanical allodynia associated with neuropathic and inflammatory injuries is significantly altered in alpha6* mutants, and that alpha6* but not alpha4* nicotinic receptors are absolutely required for peripheral and/or spinal nicotine analgesia. Furthermore, we show that Chrna6's role in analgesia is at least partially due to direct interaction and cross-inhibition of alpha6* nAChRs with P2X2/3 receptors in DRG nociceptors. Finally, we establish the relevance of our results to humans by the observation of genetic association in patients suffering from chronic postsurgical and temporomandibular pain.

The glutamatergic subthalamic nucleus (STN) exerts control over motor output through nuclei of the basal ganglia. High-frequency electrical stimuli in the STN effectively alleviate motor symptoms in movement disorders, and cholinergic stimulation boosts this effect. To gain knowledge about the mechanisms of cholinergic modulation in the STN, we studied cellular and circuit aspects of nicotinic acetylcholine receptors (nAChRs) in mouse STN. We discovered two largely divergent microcircuits in the STN; these are regulated in part by either alpha4beta2 or alpha7 nAChRs. STN neurons containing alpha4beta2 nAChRs (alpha4beta2 neurons) received more glutamatergic inputs, and preferentially innervated GABAergic neurons in the substantia nigra pars reticulata. In contrast, STN neurons containing alpha7 nAChRs (alpha7 neurons) received more GABAergic inputs, and preferentially innervated dopaminergic neurons in the substantia nigra pars compacta. Interestingly, local electrical stimuli excited a majority (79%) of alpha4beta2 neurons but exerted strong inhibition in 58% of alpha7 neurons, indicating an additional diversity of STN neurons: responses to electrical stimulation. Chronic exposure to nicotine selectively affects alpha4beta2 nAChRs in STN: this treatment increased the number of alpha4beta2 neurons, upregulated alpha4-containing nAChR number and sensitivity, and enhanced the basal firing rate of alpha4beta2 neurons both ex vivo and in vivo. Thus, chronic nicotine enhances the function of the microcircuit involving alpha4beta2 nAChRs. This indicates chronic exposure to nicotinic agonist as a potential pharmacological intervention to alter selectively the balance between these two microcircuits, and may provide a means to inhibit substantia nigra dopaminergic neurons.

Chronic exposure to nicotine up-regulates high sensitivity nicotinic acetylcholine receptors (nAChRs) in the brain. This up-regulation partially underlies addiction and may also contribute to protection against Parkinson's disease. nAChRs containing the alpha6 subunit (alpha6* nAChRs) are expressed in neurons in several brain regions, but comparatively little is known about the effect of chronic nicotine on these nAChRs. We report here that nicotine up-regulates alpha6* nAChRs in several mouse brain regions (substantia nigra pars compacta, ventral tegmental area, medial habenula, and superior colliculus) and in neuroblastoma 2a cells. We present evidence that a coat protein complex I (COPI)-mediated process mediates this up-regulation of alpha6* or alpha4* nAChRs but does not participate in basal trafficking. We show that alpha6beta2beta3 nAChR up-regulation is prevented by mutating a putative COPI-binding motif in the beta3 subunit or by inhibiting COPI. Similarly, a COPI-dependent process is required for up-regulation of alpha4beta2 nAChRs by chronic nicotine but not for basal trafficking. Mutation of the putative COPI-binding motif or inhibition of COPI also results in reduced normalized Forster resonance energy transfer between alpha6beta2beta3 nAChRs and epsilonCOP subunits. The discovery that nicotine exploits a COPI-dependent process to chaperone high sensitivity nAChRs is novel and suggests that this may be a common mechanism in the up-regulation of nAChRs in response to chronic nicotine.

Nicotinic acetylcholine receptors have been shown to participate in neuroprotection in the aging brain. Lynx protein modulators dampen the activity of the cholinergic system through direct interaction with nicotinic receptors. Although lynx1 null mutant mice exhibit augmented learning and plasticity, they also exhibit macroscopic vacuolation in the dorsal striatum as they age, detectable at the optical microscope level. Despite the relevance of the lynx1 gene to brain function, little is known about the cellular ultrastructure of these age-related changes. In this study, we assessed degeneration in the dorsal striatum in 1-, 3-, 7-, and 13-month-old mice, using optical and transmission electron microscopy. We observed a loss of nerve fibers, a breakdown in nerve fiber bundles, and a loss of neuronal nuclei in the 13-month-old lynx1 null striatum. At higher magnification, these nerve fibers displayed intracellular vacuoles and disordered myelin sheaths. Few or none of these morphological alterations were present in younger lynx1 null mutant mice or in heterozygous lynx1 null mutant mice at any age. These data indicate that neuronal health can be maintained by titrating lynx1 dosage and that the lynx1 gene may participate in a trade-off between neuroprotection and augmented learning.

Nicotinic acetylcholine receptors (nAChRs) containing the alpha5 subunit are of interest because genome-wide association studies and candidate gene studies have identified polymorphisms in the alpha5 gene that are linked to an increased risk for nicotine dependence, lung cancer, and/or alcohol addiction. To probe the functional impact of an alpha5 subunit on nAChRs, a method to prepare a homogeneous population of alpha5-containing receptors must be developed. Here we use a gain of function (9') mutation to isolate populations of alpha5-containing nAChRs for characterization by electrophysiology. We find that the alpha5 subunit modulates nAChR rectification when co-assembled with alpha4 and beta2 subunits. We also probe the alpha5-alpha4 interface for possible ligand-binding interactions. We find that mutations expected to ablate an agonist-binding site involving the alpha5 subunit have no impact on receptor function. The most straightforward interpretation of this observation is that agonists do not bind at the alpha5-alpha4 interface, in contrast to what has recently been demonstrated for the alpha4-alpha4 interface in related receptors. In addition, our mutational results suggest that the alpha5 subunit does not replace the alpha4 or beta2 subunits and is relegated to occupying only the auxiliary position of the pentameric receptor.

Glycosylphosphatidylinositol-anchored neurotoxin-like receptor binding proteins, such as lynx modulators, are topologically positioned to exert pharmacological effects by binding to the extracellular portion of nAChRs. These actions are generally thought to proceed when both lynx and the nAChRs are on the plasma membrane. Here, we demonstrate that lynx1 also exerts effects on alpha4beta2 nAChRs within the endoplasmic reticulum. Lynx1 affects assembly of nascent alpha4 and beta2 subunits and alters the stoichiometry of the receptor population that reaches the plasma membrane. Additionally, these data suggest that lynx1 shifts nAChR stoichiometry to low sensitivity (alpha4)3(beta2)2 pentamers primarily through this interaction in the endoplasmic reticulum, rather than solely via direct modulation of activity on the plasma membrane. To our knowledge, these data represent the first test of the hypothesis that a lynx family member, or indeed any glycosylphosphatidylinositol-anchored protein, could act within the cell to alter assembly of a multisubunit protein.

Chronic exposure to nicotine results in an upregulation of neuronal nicotinic acetylcholine receptors (nAChRs) at the cellular plasma membrane. nAChR upregulation occurs via nicotine-mediated pharmacological receptor chaperoning and is thought to contribute to the addictive properties of tobacco as well as relapse following smoking cessation. At the subcellular level, pharmacological chaperoning by nicotine and nicotinic ligands causes profound changes in the structure and function of the endoplasmic reticulum (ER), ER exit sites, the Golgi apparatus and secretory vesicles of cells. Chaperoning-induced changes in cell physiology exert an overall inhibitory effect on the ER stress/unfolded protein response. Cell autonomous factors such as the repertoire of nAChR subtypes expressed by neurons and the pharmacological properties of nicotinic ligands (full or partial agonist versus competitive antagonist) govern the efficiency of receptor chaperoning and upregulation. Together, these findings are beginning to pave the way for developing pharmacological chaperones to treat Parkinson's disease and nicotine addiction.

The alpha7 nicotinic acetylcholine receptor gene (CHRNA7) is linked to schizophrenia. A partial duplication of CHRNA7 (CHRFAM7A) is found in humans on 15q13-14. Exon 6 of CHRFAM7A harbors a 2-bp deletion polymorphism, CHRFAM7ADelta2bp, which is also associated with schizophrenia. To understand the effects of the duplicated subunits on alpha7 receptors, we fused alpha7, dupalpha7, and dupDeltaalpha7 subunits with various fluorescent proteins. The duplicated subunits co-localized with full-length alpha7 subunits in mouse neuroblastoma cells (Neuro2a) as well as rat hippocampal neurons. We investigated the interaction between the duplicated subunits and full-length alpha7 by measuring Forster resonance energy transfer using donor recovery after photobleaching and fluorescence lifetime imaging microscopy. The results revealed that the duplicated proteins co-assemble with alpha7. In electrophysiological studies, Leu at the 9'-position in the M2 membrane-spanning segment was replaced with Cys in dupalpha7 or dupDeltaalpha7, and constructs were co-transfected with full-length alpha7 in Neuro2a cells. Exposure to ethylammonium methanethiosulfonate inhibited acetylcholine-induced currents, showing that the assembled functional nicotinic acetylcholine receptors (nAChRs) included the duplicated subunit. Incorporation of dupalpha7 and dupDeltaalpha7 subunits modestly changes the sensitivity of receptors to choline and varenicline. Thus, the duplicated proteins are assembled and transported to the cell membrane together with full-length alpha7 subunits and alter the function of the nAChRs. The characterization of dupalpha7 and dupDeltaalpha7 as well as their influence on alpha7 nAChRs may help explain the pathophysiology of schizophrenia and may suggest therapeutic strategies.

The agonist-binding site of nicotinic acetylcholine receptors (nAChRs) spans an interface between two subunits of the pentameric receptor. The principal component of this binding site is contributed by an alpha subunit, and it binds the cationic moiety of the nicotinic pharmacophore. The other part of the pharmacophore, a hydrogen bond acceptor, has recently been shown to bind to the complementary non-alpha subunit via the backbone NH of a conserved Leu. This interaction was predicted by studies of ACh-binding proteins and confirmed by functional studies of the neuronal (CNS) nAChR, alpha4beta2. The ACh-binding protein structures further suggested that the hydrogen bond to the backbone NH is mediated by a water molecule and that a second hydrogen bonding interaction occurs between the water molecule and the backbone CO of a conserved Asn, also on the non-alpha subunit. Here, we provide new insights into the nature of the interactions between the hydrogen bond acceptor of nicotinic agonists and the complementary subunit backbone. We studied both the nAChR of the neuromuscular junction (muscle-type) and a neuronal subtype, (alpha4)2(beta4)3. In the muscle-type receptor, both ACh and nicotine showed a strong interaction with the Leu NH, but the potent nicotine analog epibatidine did not. This interaction was much attenuated in the alpha4beta4 receptor. Surprisingly, we found no evidence for a functionally significant interaction with the backbone carbonyl of the relevant Asn in either receptor with an array of agonists.

Dopaminergic neurons in the substantia nigra pars compacta (SNc) degenerate in Parkinson's disease. These neurons robustly express several nicotinic acetylcholine receptor (nAChR) subtypes. Smoking appears to be neuroprotective for Parkinson's disease but the mechanism is unknown. To determine whether chronic nicotine-induced changes in gene expression contribute to the neuroprotective effects of smoking, we develop methods to measure the effect of prolonged nicotine exposure on the SNc neuronal transcriptome in an unbiased manner. Twenty neurons were collected using laser-capture microscopy and transcriptional changes were assessed using RNA deep sequencing. These results are the first whole-transcriptome analyses of chronic nicotine treatment in SNc neurons. Overall, 129 genes were significantly regulated: 67 upregulated, 62 downregulated. Nicotine-induced relief of endoplasmic reticulum (ER) stress has been postulated as a potential mechanism for the neuroprotective effects of smoking. Chronic nicotine did not significantly affect the expression of ER stress-related genes, nor of dopamine-related or nAChR genes, but it did modulate expression of 129 genes that could be relevant to the neuroprotective effects of smoking, including genes involved in (1) the ubiquitin-proteasome pathway, (2) cell cycle regulation, (3) chromatin modification, and (4) DNA binding and RNA regulation. We also report preliminary transcriptome data for single-cell dopaminergic and GABAergic neurons isolated from midbrain cultures. These novel techniques will facilitate advances in understanding the mechanisms taking place at the cellular level and may have applications elsewhere in the fields of neuroscience and molecular biology. The results give an emerging picture of the role of nicotine on the SNc and on dopaminergic neurons.

Several mutations in alpha4 or beta2 nicotinic receptor subunits are linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). One such missense mutation in the gene encoding the beta2 neuronal nicotinic acetylcholine receptor (nAChR) subunit (CHRNB2) is a valine-to-leucine substitution in the second transmembrane domain at position 287 (beta2VL). Previous studies indicated that the beta2VL mutation in mice alters circadian rhythm consistent with sleep alterations observed in ADNFLE patients (Xu et al., 2011). The current study investigates changes in nicotinic receptor function and expression that may explain the behavioral phenotype of beta2VL mice. No differences in beta2 mRNA expression were found between wild-type (WT) and heterozygous (HT) or homozygous mutant (MT) mice. However, antibody and ligand binding indicated that the mutation resulted in a reduction in receptor protein. Functional consequences of the beta2VL mutation were assessed biochemically using crude synaptosomes. A gene-dose dependent increase in sensitivity to activation by acetylcholine and decrease in maximal nAChR-mediated [(3)H]-dopamine release and (86)Rb efflux were observed. Maximal nAChR-mediated [(3)H]-GABA release in the cortex was also decreased in the MT, but maximal [(3)H]-GABA release was retained in the hippocampus. Behaviorally both HT and MT mice demonstrated increased sensitivity to nicotine-induced hypolocomotion and hypothermia. Furthermore, WT mice display only a tonic-clonic seizure (EEG recordable) 3 min after injection of a high dose of nicotine, while MT mice also display a dystonic arousal complex (non-EEG recordable) event 30s after nicotine injection. Data indicate decreases in maximal response for certain measures are larger than expected given the decrease in receptor expression.

The alpha7 nicotinic acetylcholine receptor shows broad pharmacology, complicating the development of subtype-specific nicotinic receptor agonists. Here we use unnatural amino acid mutagenesis to characterize binding to alpha7 by the smoking cessation drug varenicline (Chantix; Pfizer, Groton, CT), an alpha4beta2-targeted agonist that shows full efficacy and modest potency at the alpha7 receptor. We find that unlike binding to its target receptor, varenicline does not form a cation-pi interaction with TrpB, further supporting a unique binding mode for the cationic amine of nicotinic agonists at the alpha7 receptor. We also evaluate binding to the complementary face of the receptor's binding site by varenicline, the endogenous agonist acetylcholine, and the potent nicotine analog epibatidine. Interestingly, we find no evidence for functionally important interactions involving backbone NH and CO groups thought to bind the canonical agonist hydrogen bond acceptor of the nicotinic pharmacophore, perhaps reflecting a lesser importance of this pharmacophore element for alpha7 binding. We also show that the Trp55 and Leu119 side chains of the binding site's complementary face are important for the binding of the larger agonists epibatidine and varenicline, but dispensable for binding of the smaller, endogenous agonist acetylcholine.

Firing rates of dopamine (DA) neurons in substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) control DA release in target structures such as striatum and prefrontal cortex. DA neuron firing in the soma and release probability at axon terminals are tightly regulated by cholinergic transmission and nicotinic acetylcholine receptors (nAChRs). To understand the role of alpha6* nAChRs in DA transmission, we studied several strains of mice expressing differing levels of mutant, hypersensitive (leucine 9' to serine [L9'S]) alpha6 subunits. alpha6 L9'S mice harboring six or more copies of the hypersensitive alpha6 gene exhibited spontaneous home-cage hyperactivity and novelty-induced locomotor activity, whereas mice with an equal number of WT and L9'S alpha6 genes had locomotor activity resembling that of control mice. alpha6-dependent, nicotine-stimulated locomotor activation was also more robust in high-copy alpha6 L9'S mice versus low-copy mice. In wheel-running experiments, results were also bi-modal; high-copy alpha6 L9'S animals exhibited blunted total wheel rotations during each day of a 9-day experiment, but low-copy alpha6 L9'S mice ran normally on the wheel. Reduced wheel running in hyperactive strains of alpha6 L9'S mice was attributable to a reduction in both overall running time and velocity. ACh and nicotine-stimulated DA release from striatal synaptosomes in alpha6 L9'S mice was well-correlated with behavioral phenotypes, supporting the hypothesis that augmented DA release mediates the altered behavior of alpha6 L9'S mice. This study highlights the precise control that the nicotinic cholinergic system exerts on DA transmission and provides further insights into the mechanisms and consequences of enhanced DA release.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated, cation-selective ion channels expressed throughout the brain. Although these channels have been investigated for several decades, it is still challenging 1) to identify the important nAChR subunits in cholinergic transmission and nicotine dependence and 2) to develop nAChR subtype-specific ligands. To overcome these challenges, we and others have studied mice expressing mutant, gain-of-function nAChR subunits. In this review, we discuss this research approach and the results it has yielded to date. Gain-of-function mutations, including those in nAChR subunits, provide an approach that is complementary to loss-of-function studies such as gene knockouts; the former allows one to answer questions of sufficiency and the latter addresses questions of necessity. Mutant mice expressing gain-of-function nAChR subunits are commonly produced using traditional gene targeting in embryonic stem cells, but novel approaches such as bacterial artificial chromosome transgenesis have yielded important insights as well. alpha7 nAChRs were the first nAChRs to be targeted with a gain-of-function mutation, followed by a pair of alpha4 nAChR gain-of-function mutant mice. These alpha4 nAChR gain-of-function mice (alpha4 L9'S mice, followed by alpha4 L9'A mice) provided an important system to probe alpha4 nAChR function in vivo, particularly in the dopamine reward system. alpha6 nAChR gain-of-function mice provided the first robust system allowing specific manipulation of this receptor subtype. Other targeted mutations in various nAChR subunits have also been produced and have yielded important insights into nicotinic cholinergic biology. As nAChR research advances and more details associated with nAChR expression and function emerge, we expect that existing and new mouse lines expressing gain-of-function nAChR subunits will continue to provide new insights.

Nicotinic acetylcholine receptors (nAChRs) containing alpha6 subunits are expressed in only a few brain areas, including midbrain dopamine (DA) neurons, noradrenergic neurons of the locus ceruleus, and retinal ganglion cells. To better understand the regional and subcellular expression pattern of alpha6-containing nAChRs, we created and studied transgenic mice expressing a variant alpha6 subunit with green fluorescent protein (GFP) fused in-frame in the M3-M4 intracellular loop. In alpha6-GFP transgenic mice, alpha6-dependent synaptosomal DA release and radioligand binding experiments confirmed correct expression and function in vivo. In addition to strong alpha6* nAChR expression in glutamatergic retinal axons, which terminate in superficial superior colliculus (sSC), we also found alpha6 subunit expression in a subset of GABAergic cell bodies in this brain area. In patch-clamp recordings from sSC neurons in brain slices from mice expressing hypersensitive alpha6* nAChRs, we confirmed functional, postsynaptic alpha6* nAChR expression. Further, sSC GABAergic neurons expressing alpha6* nAChRs exhibit a tonic conductance mediated by standing activation of hypersensitive alpha6* nAChRs by ACh. alpha6* nAChRs also appear in a subpopulation of SC neurons in output layers. Finally, selective activation of alpha6* nAChRs in vivo induced sSC neuronal activation as measured with c-Fos expression. Together, these results demonstrate that alpha6* nAChRs are uniquely situated to mediate cholinergic modulation of glutamate and GABA release in SC. The SC has emerged as a potential key brain area responsible for transmitting short-latency salience signals to thalamus and midbrain DA neurons, and these results suggest that alpha6* nAChRs may be important for nicotinic cholinergic sensitization of this pathway.

The cholinergic system underlies both adaptive (learning and memory) and nonadaptive (addiction and dependency) behavioral changes through its ability to shape and regulate plasticity. Protein modulators such as lynx family members can fine tune the activity of the cholinergic system and contribute to the graded response of the cholinergic system, stabilizing neural circuitry through direct interaction with nicotinic receptors. Release of this molecular brake can unmask cholinergic-dependent mechanisms in the brain. Lynx proteins have the potential to provide top-down control over plasticity mechanisms, including addictive propensity. If this is indeed the case, then, what regulates the regulator? Transcriptional changes of lynx genes in response to pharmacological, physiological, and pathological alterations are explored in this review.

We investigated assembly and function of nicotinic acetylcholine receptors (nAChRs) composed of alpha7 and beta2 subunits. We measured optical and electrophysiological properties of wild-type and mutant subunits expressed in cell lines and Xenopus laevis oocytes. Laser scanning confocal microscopy indicated that fluorescently tagged alpha7 and beta2 subunits colocalize. Forster resonance energy transfer between fluorescently tagged subunits strongly suggested that alpha7 and beta2 subunits coassemble. Total internal reflection fluorescence microscopy revealed that assemblies localized to filopodia-like processes of SH-EP1 cells. Gain-of-function alpha7 and beta2 subunits confirmed that these subunits coassemble within functional receptors. Moreover, alpha7beta2 nAChRs composed of wild-type subunits or fluorescently tagged subunits had pharmacological properties similar to those of alpha7 nAChRs, although amplitudes of alpha7beta2 nAChR-mediated, agonist-evoked currents were generally ~2-fold lower than those for alpha7 nAChRs. It is noteworthy that alpha7beta2 nAChRs displayed sensitivity to low concentrations of the antagonist dihydro-beta-erythroidine that was not observed for alpha7 nAChRs at comparable concentrations. In addition, cysteine mutants revealed that the alpha7-beta2 subunit interface does not bind ligand in a functionally productive manner, partly explaining lower alpha7beta2 nAChR current amplitudes and challenges in identifying the function of native alpha7beta2 nAChRs. On the basis of our findings, we have constructed a model predicting receptor function that is based on stoichiometry and position of beta2 subunits within the alpha7beta2 nAChRs.

The nicotinic acetylcholine receptors (nAChRs) are a family of closely related but pharmacologically distinct neurotransmitter-gated ion channels. They are therapeutic targets for a wide range of neurological disorders, and a key issue in drug development is selective targeting among the more than 20 subtypes of nAChRs that are known. The present work evaluates a proposed hydrogen bonding interaction involving a residue known as the "loop B glycine" that distinguishes receptors that are highly responsive to ACh and nicotine from those that are much less so. We have performed structure-function studies on the loop B site, including unnatural amino acid mutagenesis, in three different nAChR subtypes and found that the correlation between agonist potency and this residue is strong. Low potency receptor subtypes have a glycine at this key site, and mutation to a residue with a side chain converts a low potency receptor to a high potency receptor. Innately high potency receptors have a lysine at the loop B site and show a decrease in potency for the reverse mutation (i.e., introducing a glycine). This residue lies outside of the agonist binding site, and studies of other residues at the agonist binding site show that the details of how changes at the loop B glycine site impact agonist potency vary for differing receptor subtypes. This suggests a model in which the loop B residue influences the global shape of the agonist binding site rather than modulating any specific interaction.

We exploit the optical and spatial features of subwavelength nanostructures to examine individual receptors on the plasma membrane of living cells. Receptors were sequestered in portions of the membrane projected into zero-mode waveguides. Using single-step photobleaching of green fluorescent protein incorporated into individual subunits, the resulting spatial isolation was used to measure subunit stoichiometry in alpha4beta4 and alpha4beta2 nicotinic acetylcholine and P2X2 ATP receptors. We also show that nicotine and cytisine have differential effects on alpha4beta2 stoichiometry.

We provide a theory for employing Forster resonance energy transfer (FRET) measurements to determine altered heteropentameric ion channel stoichiometries in intracellular compartments of living cells. We simulate FRET within nicotinic receptors (nAChRs) whose alpha4 and beta2 subunits contain acceptor and donor fluorescent protein moieties, respectively, within the cytoplasmic loops. We predict FRET and normalized FRET (NFRET) for the two predominant stoichiometries, (alpha4)(3)(beta2)(2)vs. (alpha4)(2)(beta2)(3). Studying the ratio between FRET or NFRET for the two stoichiometries, minimizes distortions due to various photophysical uncertainties. Within a range of assumptions concerning the distance between fluorophores, deviations from plane pentameric geometry, and other asymmetries, the predicted FRET and NFRET for (alpha4)(3)(beta2)(2) exceeds that of (alpha4)(2)(beta2)(3). The simulations account for published data on transfected Neuro2a cells in which alpha4beta2 stoichiometries were manipulated by varying fluorescent subunit cDNA ratios: NFRET decreased monotonically from (alpha4)(3)(beta2)(2) stoichiometry to mostly (alpha4)(2)(beta2)(3). The simulations also account for previous macroscopic and single-channel observations that pharmacological chaperoning by nicotine and cytisine increase the (alpha4)(2)(beta2)(3) and (alpha4)(3)(beta2)(2) populations, respectively. We also analyze sources of variability. NFRET-based monitoring of changes in subunit stoichiometry can contribute usefully to studies on Cys-loop receptors.

We report the first observation that endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) can decrease when a central nervous system drug acts as an intracellular pharmacological chaperone for its classic receptor. Transient expression of alpha4beta2 nicotinic receptors (nAChRs) in Neuro-2a cells induced the nuclear translocation of activating transcription factor 6 (ATF6), which is part of the UPR. Cells were exposed for 48 h to the full agonist nicotine, the partial agonist cytisine, or the competitive antagonist dihydro-beta-erythroidine; we also tested mutant nAChRs that readily exit the ER. Each of these four manipulations increased Sec24D-enhanced green fluorescent protein fluorescence of condensed ER exit sites and attenuated translocation of ATF6-enhanced green fluorescent protein to the nucleus. However, we found no correlation among the manipulations regarding other tested parameters [i.e., changes in nAChR stoichiometry (alpha4(2)beta2(3) versus alpha4(3)beta2(2)), changes in ER and trans-Golgi structures, or the degree of nAChR up-regulation at the plasma membrane]. The four manipulations activated 0 to 0.4% of nAChRs, which shows that activation of the nAChR channel did not underlie the reduced ER stress. Nicotine also attenuated endogenously expressed ATF6 translocation and phosphorylation of eukaryotic initiation factor 2alpha in mouse cortical neurons transfected with alpha4beta2 nAChRs. We conclude that, when nicotine accelerates ER export of alpha4beta2 nAChRs, this suppresses ER stress and the UPR. Suppression of a sustained UPR may explain the apparent neuroprotective effect that causes the inverse correlation between a person's history of tobacco use and susceptibility to developing Parkinson's disease. This suggests a novel mechanism for neuroprotection by nicotine.

Drug-receptor binding interactions of four agonists, ACh, nicotine, and the smoking cessation compounds varenicline (Chantix) and cytisine (Tabex), have been evaluated at both the 2:3 and 3:2 stoichiometries of the alpha4beta2 nicotinic acetylcholine receptor (nAChR). Previous studies have established that unnatural amino acid mutagenesis can probe three key binding interactions at the nAChR: a cation-pi interaction, and two hydrogen-bonding interactions to the protein backbone of the receptor. We find that all drugs make a cation-pi interaction to TrpB of the receptor. All drugs except ACh, which lacks an N(+)H group, make a hydrogen bond to a backbone carbonyl, and ACh and nicotine behave similarly in acting as a hydrogen-bond acceptor. However, varenicline is not a hydrogen-bond acceptor to the backbone NH that interacts strongly with the other three compounds considered. In addition, we see interesting variations in hydrogen bonding interactions with cytisine that provide a rationalization for the stoichiometry selectivity seen with this compound.

The defining feature of the alpha subunits of the family of nicotinic acetylcholine receptors is a vicinal disulfide between Cys-192 and Cys-193. Although this structure has played a pivotal role in a number of pioneering studies of nicotinic receptors, its functional role in native receptors remains uncertain. Using mutant cycle analysis and unnatural residue mutagenesis, including backbone mutagenesis of the peptide bond of the vicinal disulfide, we have established the presence of a network of hydrogen bonds that extends from that peptide NH, across a beta turn to another backbone hydrogen bond, and then across the subunit interface to the side chain of a functionally important Asp residue in the non-alpha subunit. We propose that the role of the vicinal disulfide is to distort the beta turn and thereby properly position a backbone NH for intersubunit hydrogen bonding to the key Asp.

Varenicline, a widely used and successful smoking cessation agent, acts as a partial agonist at nicotinic acetylcholine receptors. Here, we explore the effects of varenicline at human and mouse 5-Hydroxytryptamine(3) (5-HT(3)) receptors. Application of varenicline to human 5-HT(3) receptors expressed in Xenopus laevis oocytes reveal it is almost a full agonist (R(max) = 80%) with an EC(50) (5.9 muM) 3-fold higher than 5-HT. At mouse 5-HT(3) receptors varenicline is a partial agonist (R(max) = 35%) with an EC(50) (18 muM) 20-fold higher than 5-HT. Displacement of the competitive 5-HT(3) receptor antagonist [(3)H]granisetron reveals similar IC(50) values for varenicline at mouse and human receptors expressed in human embryonic kidney 293 cells, although studies in these cells using a membrane potential-sensitive dye show that again varenicline is a 4- or 35-fold less potent agonist than 5-HT in human and mouse receptors, respectively. Thus the data suggest that the efficacy, but not the affinity, of varenicline is greater at human 5-HT(3) receptors compared with mouse. Docking studies provide a possible explanation for this difference, because they suggest distinct orientations of the ligand in the mouse versus human 5-HT(3) agonist binding sites. Additional binding selectivity studies in a broad panel of recombinant receptors and enzymes confirmed an interaction with 5-HT(3) receptors but revealed no additional interactions of varenicline. Therefore, activation of human 5-HT(3) receptors may be responsible for some of the side effects that preclude use of higher doses during varenicline treatment.

Cholinergic neurons and nicotinic acetylcholine receptors (nAChRs) in the brain participate in diverse functions: reward, learning and memory, mood, sensory processing, pain, and neuroprotection. Nicotinic systems also have well-known roles in drug abuse. Here, we review recent insights into nicotinic function, linking exogenous and endogenous manipulations of nAChRs to alterations in synapses, circuits, and behavior. We also discuss how these contemporary advances can motivate attempts to exploit nicotinic systems therapeutically in Parkinson's disease, cognitive decline, epilepsy, and schizophrenia.

Cation-pi interactions have been demonstrated to play a major role in agonist-binding in Cys-loop receptors. However, neither the aromatic amino acid contributing to this interaction nor its location is conserved among Cys-loop receptors. Likewise, it is not clear how many different agonists of a given receptor form a cation-pi interaction or, if they do, whether it is with the same aromatic amino acid as the major physiological agonist. We demonstrated previously that Phe159 in the glycine receptor (GlyR) alpha1 subunit forms a strong cation-pi interaction with the principal agonist, glycine. In the current study, we investigated whether the lower efficacy agonists of the human GlyR beta-alanine and taurine also form cation-pi interactions with Phe159. By incorporating a series of unnatural amino acids, we found cation-pi interactions between Phe159 and the amino groups of beta-alanine and taurine. The strengths of these interactions were significantly weaker than for glycine. Modeling studies suggest that beta-alanine and taurine are orientated subtly differently in the binding pocket, with their amino groups further from Phe159 than that of glycine. These data therefore show that similar agonists can have similar but not identical orientations and interactions in the binding pocket and provide a possible explanation for the lower potencies of beta-alanine and taurine.

Nicotinic acetylcholine receptors (nAChRs) are pentameric, neurotransmitter-gated ion channels responsible for rapid excitatory neurotransmission in the central and peripheral nervous systems, resulting in skeletal muscle tone and various cognitive effects in the brain. These complex proteins are activated by the endogenous neurotransmitter ACh as well as by nicotine and structurally related agonists. Activation and modulation of nAChRs has been implicated in the pathology of multiple neurological disorders, and as such, these proteins are established therapeutic targets. Here we use unnatural amino acid mutagenesis to examine the ligand binding mechanisms of two homologous neuronal nAChRs: the alpha4beta4 and alpha7 receptors. Despite sequence identity among the residues that form the core of the agonist-binding site, we find that the alpha4beta4 and alpha7 nAChRs employ different agonist-receptor binding interactions in this region. The alpha4beta4 receptor utilizes a strong cation-pi interaction to a conserved tryptophan (TrpB) of the receptor for both ACh and nicotine, and nicotine participates in a strong hydrogen bond with a backbone carbonyl contributed by TrpB. Interestingly, we find that the alpha7 receptor also employs a cation-pi interaction for ligand recognition, but the site has moved to a different aromatic amino acid of the agonist-binding site depending on the agonist. ACh participates in a cation-pi interaction with TyrA, whereas epibatidine participates in a cation-pi interaction with TyrC2.

We employed a pH-sensitive GFP analog, superecliptic phluorin, to observe aspects of nicotinic acetylcholine receptor (nAChR) trafficking to the plasma membrane (PM) in cultured mouse cortical neurons. The experiments exploit differences in the pH among endoplasmic reticulum (ER), trafficking vesicles, and the extracellular solution. The data confirm that few alpha4beta4 nAChRs, but many alpha4beta2 nAChRs, remain in neutral intracellular compartments, mostly the ER. We observed fusion events between nAChR-containing vesicles and PM; these could be quantified in the dendritic processes. We also studied the beta4R348C polymorphism, linked to amyotrophic lateral sclerosis (ALS). This mutation depressed fusion rates of alpha4beta4 receptor-containing vesicles with the PM by approximately 2-fold, with only a small decrease in the number of nAChRs per vesicle. The mutation also decreased the number of ER exit sites, showing that the reduced receptor insertion results from a change at an early stage in trafficking. We confirm the previous report that the mutation leads to reduced agonist-induced currents; in the cortical neurons studied, the reduction amounts to 2-3-fold. Therefore, the reduced agonist-induced currents are caused by the reduced number of alpha4beta4-containing vesicles reaching the membrane. Chronic nicotine exposure (0.2 muM) did not alter the PM insertion frequency or trafficking behavior of alpha4beta4-laden vesicles. In contrast, chronic nicotine substantially increased the number of alpha4beta2-containing vesicle fusions at the PM; this stage in alpha4beta2 nAChR up-regulation is presumably downstream from increased ER exit. Superecliptic phluorin provides a tool to monitor trafficking dynamics of nAChRs in disease and addiction.

The up-regulation of alpha4beta2* nicotinic acetylcholine receptors (nAChRs) by chronic nicotine is a cell-delimited process and may be necessary and sufficient for the initial events of nicotine dependence. Clinical literature documents an inverse relationship between a person's history of tobacco use and his or her susceptibility to Parkinson's disease; this may also result from up-regulation. This study visualizes and quantifies the subcellular mechanisms involved in nicotine-induced nAChR up-regulation by using transfected fluorescent protein (FP)-tagged alpha4 nAChR subunits and an FP-tagged Sec24D endoplasmic reticulum (ER) exit site marker. Total internal reflection fluorescence microscopy shows that nicotine (0.1 microM for 48 h) up-regulates alpha4beta2 nAChRs at the plasma membrane (PM), despite increasing the fraction of alpha4beta2 nAChRs that remain in near-PM ER. Pixel-resolved normalized Forster resonance energy transfer microscopy between alpha4-FP subunits shows that nicotine stabilizes the (alpha4)(2)(beta2)(3) stoichiometry before the nAChRs reach the trans-Golgi apparatus. Nicotine also induces the formation of additional ER exit sites (ERES). To aid in the mechanistic analysis of these phenomena, we generated a beta2(enhanced-ER-export) mutant subunit that mimics two regions of the beta4 subunit sequence: the presence of an ER export motif and the absence of an ER retention/retrieval motif. The alpha4beta2(enhanced-ER-export) nAChR resembles nicotine-exposed nAChRs with regard to stoichiometry, intracellular mobility, ERES enhancement, and PM localization. Nicotine produces only small additional PM up-regulation of alpha4beta2(enhanced-ER-export) receptors. The experimental data are simulated with a model incorporating two mechanisms: (1) nicotine acts as a stabilizing pharmacological chaperone for nascent alpha4beta2 nAChRs in the ER, eventually increasing PM receptors despite a bottleneck(s) in ER export; and (2) removal of the bottleneck (e.g., by expression of the beta2(enhanced-ER-export) subunit) is sufficient to increase PM nAChR numbers, even without nicotine. The data also suggest that pharmacological chaperoning of nAChRs by nicotine can alter the physiology of ER processes.

alpha6* nicotinic acetylcholine receptors (nAChRs) are highly expressed in mesostriatal and nigrostriatal dopaminergic systems, and participate in motor control, reward, and learning and memory. In vitro functional expression of alpha6* nAChRs is essential for full pharmacological characterization of these receptors and for drug screening, but has been challenging. We expressed eGFP-tagged-alpha6 and beta2 nAChR subunits in Neuro-2a cells, leading to functional channels. Inward currents were elicited with 300 muM ACh in 26% (5/19) of cells with evenly expressed alpha6-eGFP in cytoplasm and periphery. We dramatically increased chances of detecting functional alpha6-eGFPbeta2 nAChRs by (i) introducing two endoplasmic reticulum (ER) export-enhancing mutations into beta2 subunits, and (ii) choosing cells with abundant Sec24D-mCherry-labeled ER exit sites. Both manipulations also modestly increased alpha6-eGFPbeta2 nAChR current amplitude. alpha6-eGFPbeta2 nAChRs were also activated by nicotine and by TC-2403. The alpha6-eGFPbeta2 currents were desensitized by 1muM nicotine, blocked by alpha-conotoxin MII, partially inhibited by dihydro-beta-erythroidine, and potentiated by extracellular Ca(2+). Single-channel recordings showed that alpha6-eGFPbeta2 nAChRs had similar single-channel conductance to, but longer open time than, alpha4-eGFPbeta2 nAChRs. These methods provide avenues for developing cell lines expressing subtypes of alpha6* nAChRs for both pharmacological study and drug screening.

High-affinity nicotinic receptors containing beta2 subunits (beta2*) are widely expressed in the brain, modulating many neuronal processes and contributing to neuropathologies such as Alzheimer's disease, Parkinson's disease and epilepsy. Mutations in both the alpha4 and beta2 subunits are associated with a rare partial epilepsy, autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). In this study, we introduced one such human missense mutation into the mouse genome to generate a knock-in strain carrying a valine-to-leucine mutation beta2V287L. beta2(V287L) mice were viable and born at an expected Mendelian ratio. Surprisingly, mice did not show an overt seizure phenotype; however, homozygous mice did show significant alterations in their activity-rest patterns. This was manifest as an increase in activity during the light cycle suggestive of disturbances in the normal sleep patterns of mice; a parallel phenotype to that found in human ADNFLE patients. Consistent with the role of nicotinic receptors in reward pathways, we found that beta2(V287L) mice did not develop a normal proclivity to voluntary wheel running, a model for natural reward. Anxiety-related behaviors were also affected by the V287L mutation. Mutant mice spent more time in the open arms on the elevated plus maze suggesting that they had reduced levels of anxiety. Together, these findings emphasize several important roles of beta2* nicotinic receptors in complex biological processes including the activity-rest cycle, natural reward and anxiety.

Pharmacophore models for nicotinic agonists have been proposed for four decades. Central to these models is the presence of a cationic nitrogen and a hydrogen bond acceptor. It is now well-established that the cationic center makes an important cation-pi interaction to a conserved tryptophan, but the donor to the proposed hydrogen bond acceptor has been more challenging to identify. A structure of nicotine bound to the acetylcholine binding protein predicted that the binding partner of the pharmacophore's second component was a water molecule, which also hydrogen bonds to the backbone of the complementary subunit of the receptors. Here we use unnatural amino acid mutagenesis coupled with agonist analogs to examine whether such a hydrogen bond is functionally significant in the alpha4beta2 neuronal nAChR, the receptor most associated with nicotine addiction. We find evidence for the hydrogen bond with the agonists nicotine, acetylcholine, carbamylcholine, and epibatidine. These data represent a completed nicotinic pharmacophore and offer insight into the design of new therapeutic agents that selectively target these receptors.

Dopamine (DA) release in striatum is governed by firing rates of midbrain DA neurons, striatal cholinergic tone, and nicotinic ACh receptors (nAChRs) on DA presynaptic terminals. DA neurons selectively express alpha6* nAChRs, which show high ACh and nicotine sensitivity. To help identify nAChR subtypes that control DA transmission, we studied transgenic mice expressing hypersensitive alpha6(L9'S)* receptors. alpha6(L9'S) mice are hyperactive, travel greater distance, exhibit increased ambulatory behaviors such as walking, turning, and rearing, and show decreased pausing, hanging, drinking, and grooming. These effects were mediated by alpha6alpha4* pentamers, as alpha6(L9'S) mice lacking alpha4 subunits displayed essentially normal behavior. In alpha6(L9'S) mice, receptor numbers are normal, but loss of alpha4 subunits leads to fewer and less sensitive alpha6* receptors. Gain-of-function nicotine-stimulated DA release from striatal synaptosomes requires alpha4 subunits, implicating alpha6alpha4beta2* nAChRs in alpha6(L9'S) mouse behaviors. In brain slices, we applied electrochemical measurements to study control of DA release by alpha6(L9'S) nAChRs. Burst stimulation of DA fibers elicited increased DA release relative to single action potentials selectively in alpha6(L9'S), but not WT or alpha4KO/alpha6(L9'S), mice. Thus, increased nAChR activity, like decreased activity, leads to enhanced extracellular DA release during phasic firing. Bursts may directly enhance DA release from alpha6(L9'S) presynaptic terminals, as there was no difference in striatal DA receptor numbers or DA transporter levels or function in vitro. These results implicate alpha6alpha4beta2* nAChRs in cholinergic control of DA transmission, and strongly suggest that these receptors are candidate drug targets for disorders involving the DA system.

Mammalian brain expresses multiple nicotinic acetylcholine receptor (nAChR) subtypes that differ in subunit composition, sites of expression and pharmacological and functional properties. Among known subtypes of receptors, alpha 4 beta 2* and alpha 6 beta 2*-nAChR have the highest affinity for nicotine (where * indicates possibility of other subunits). The alpha 4 beta 2*-nAChRs are widely distributed, while alpha 6 beta 2*-nAChR are restricted to a few regions. Both subtypes modulate release of dopamine from the dopaminergic neurons of the mesoaccumbens pathway thought to be essential for reward and addiction. alpha 4 beta 2*-nAChR also modulate GABA release in these areas. Identification of selective compounds would facilitate study of nAChR subtypes. An improved understanding of the role of nAChR subtypes may help in developing more effective smoking cessation aids with fewer side effects than current therapeutics. We have screened a series of nicotinic compounds that vary in the distance between the pyridine and the cationic center, in steric bulk, and in flexibility of the molecule. These compounds were screened using membrane binding and synaptosomal function assays, or recordings from GH4C1 cells expressing h alpha 7, to determine affinity, potency and efficacy at four subtypes of nAChRs found in brain, alpha 4 beta 2*, alpha 6 beta 2*, alpha 7 and alpha 3 beta 4*. In addition, physiological assays in gain-of-function mutant mice were used to assess in vivo activity at alpha 4 beta 2* and alpha 6 beta 2*-nAChRs. This approach has identified several compounds with agonist or partial agonist activity that display improved selectivity for alpha 6 beta 2*-nAChR.

The functions of two conserved residues, Phe(135) and Pro(136), located at the apex of the Cys loop of the nicotinic acetylcholine receptor are investigated. Both residues were substituted with natural and unnatural amino acids, focusing on the role of aromaticity at Phe(135), backbone conformation at Pro(136), side chain polarity and volume, and the specific interaction between the aromatic side chain and the proline. NMR spectroscopy studies of model peptides containing proline and unnatural proline analogues following a Phe show a consistent increase in the population of the cis conformer relative to peptides lacking the Phe. In the receptor, a strong interaction between the Phe and Pro residues is evident, as is a strong preference for aromaticity and hydrophobicity at the Phe site. A similar influence of hydrophobicity is observed at the proline site. In addition, across a simple homologous series of proline analogues, the results reveal a correlation between receptor function and cis bias at the proline backbone. This could suggest a significant role for the cis proline conformer at this site in receptor function.

Recent studies suggest that high-affinity neuronal nicotinic acetylcholine receptors (nAChRs) containing alpha4 and beta2 subunits (alpha4beta2*) functionally interact with G-protein-coupled dopamine (DA) D(2) receptors in basal ganglia. We hypothesized that if a functional interaction between these receptors exists, then mice expressing an M2 point mutation (Leu9'Ala) rendering alpha4 nAChRs hypersensitive to ACh may exhibit altered sensitivity to a D(2)-receptor agonist. When challenged with the D(2)R agonist, quinpirole (0.5-10 mg/kg), Leu9'Ala mice, but not wild-type (WT) littermates, developed severe, reversible motor impairment characterized by rigidity, catalepsy, akinesia, and tremor. While striatal DA tissue content, baseline release, and quinpirole-induced DA depletion did not differ between Leu9'Ala and WT mice, quinpirole dramatically increased activity of cholinergic striatal interneurons only in mutant animals, as measured by increased c-Fos expression in choline acetyltransferase (ChAT)-positive interneurons. Highlighting the importance of the cholinergic system in this mouse model, inhibiting the effects of ACh by blocking muscarinic receptors, or by selectively activating hypersensitive nAChRs with nicotine, rescued motor symptoms. This novel mouse model mimics the imbalance between striatal DA/ACh function associated with severe motor impairment in disorders such as Parkinson's disease, and the data suggest that a D(2)R-alpha4*-nAChR functional interaction regulates cholinergic interneuron activity.

Direct comparison of pyridine versus pyrimidine substituents on a small but diverse set of ligands indicates that the pyrimidine substitution has the potential to enhance affinity and/or functional activity at alpha6 subunit-containing neuronal nicotinic receptors (NNRs) and decrease activation of ganglionic nicotinic receptors, depending on the scaffold. The ramifications of this structure-activity relationship are discussed in the context of the design of small molecules targeting smoking cessation.

The medial habenula (MHb) exhibits exceptionally high levels of nicotinic acetylcholine receptors (nAChRs), but it remains unclear whether all expressed nAChR subunit mRNAs are translated to form functional receptors. In particular alpha4 subunits have not been reported to have any functional role, despite strong alpha4 mRNA expression in the ventrolateral MHb. We studied a strain of knock-in mice expressing fluorescent alpha4* nAChRs (alpha4YFP), as well as a knock-in strain expressing hypersensitive alpha4* nAChRs (alpha4L9'A). In alpha4YFP mice, there was strong fluorescence in the ventrolateral MHb. In hypersensitive alpha4L9'A mice, injections of a low dose of nicotine (0.1 mg/kg) led to strong c-fos expression in only the ventrolateral region of the MHb, but not in the MHb of wild-type (WT) mice. In MHb slice recordings, ventrolateral neurons from alpha4L9'A mice, but not from WT mice, responded robustly to nicotine (1 microM). Neurons in the medial aspect of the MHb had >10-fold smaller responses. Thus alpha4* nAChRs contribute to the selective activation of a subset of MHb neurons. Subunit composition analysis based on gain-of-function knock-in mice provides a useful experimental paradigm.

Probing the sheet: The network of hydrogen bonds formed in the outer beta sheet of the nicotinic acetylcholine receptor (nAChR; see figure) is fairly robust and tolerates single amide-to-ester mutations throughout. However, eliminating two proximal hydrogen bonds completely destroys receptor function; this adds further support to gating models that ascribe important roles to these beta strands of the nAChR extracellular domain.Long-range communication is essential for the function of members of the Cys-loop family of neurotransmitter-gated ion channels. The involvement of the peptide backbone in binding-induced conformational changes that lead to channel gating in these membrane proteins is an interesting, but unresolved issue. To probe the role of the peptide backbone, we incorporated a series of alpha-hydroxy acid analogues into the beta-sheet-rich extracellular domain of the muscle subtype of the nicotinic acetylcholine receptor, the prototypical Cys-loop receptor. Specifically, mutations were made in beta strands 7 and 10 of the alpha subunit. A number of single backbone mutations in this region were well tolerated. However, simultaneous introduction of two proximal backbone mutations led to surface-expressed, nonfunctional receptors. Together, these data suggest that while the receptor is remarkably robust in its ability to tolerate single amide-to-ester mutations throughout these beta strands, more substantial perturbations to this region have a profound effect on the protein. These results support a model in which backbone movements in the outer beta sheet are important for receptor function.

The functional coupling of residues that are far apart in space is the quintessential property of allosteric proteins. For example, in Cys-loop receptors, the gating of an intrinsic ion channel is allosterically regulated by the binding of small molecule neurotransmitters 50-60 A from the channel gate. Some residues near the binding site must have as their primary function the communication of the binding event to the gating region. These gating pathway residues are essential to function, but their identification and characterization can be challenging. This work introduces a simple strategy, derived from mutant cycle analysis, for identifying gating pathway residues using macroscopic measurements alone. In the exemplar Cys-loop receptor, the nicotinic acetylcholine receptor, a well-characterized reporter mutation (betaL9'S) known to impact gating, was combined with mutations of target residues in the ligand-binding domain hypothesized or previously found to be functionally significant. A mutant cycle analysis of the macroscopic EC(50) measurements can then provide insights into the role of the target residue. This new method, elucidating long-range functional coupling in allosteric receptors, can be applied to several reporter mutations in a wide variety of receptors to identify previously characterized and novel mutations that impact the gating pathway. We support our interpretation of macroscopic data with single-channel studies. Elucidating long-range functional coupling in allosteric receptors should be broadly applicable to determining functional roles of residues in allosteric receptors.

The acronym SePhaChARNS, for "selective pharmacological chaperoning of acetylcholine receptor number and stoichiometry," is introduced. We hypothesize that SePhaChARNS underlies classical observations that chronic exposure to nicotine causes "upregulation" of nicotinic receptors (nAChRs). If the hypothesis is proven, (1) SePhaChARNS is the molecular mechanism of the first step in neuroadaptation to chronic nicotine; and (2) nicotine addiction is partially a disease of excessive chaperoning. The chaperone is a pharmacological one, nicotine; and the chaperoned molecules are alpha4beta2* nAChRs. SePhaChARNS may also underlie two inadvertent therapeutic effects of tobacco use: (1) the inverse correlation between tobacco use and Parkinson's disease; and (2) the suppression of seizures by nicotine in autosomal dominant nocturnal frontal lobe epilepsy. SePhaChARNS arises from the thermodynamics of pharmacological chaperoning: ligand binding, especially at subunit interfaces, stabilizes AChRs during assembly and maturation, and this stabilization is most pronounced for the highest-affinity subunit compositions, stoichiometries, and functional states of receptors. Several chemical and pharmacokinetic characteristics render exogenous nicotine a more potent pharmacological chaperone than endogenous acetylcholine. SePhaChARNS is modified by desensitized states of nAChRs, by acid trapping of nicotine in organelles, and by other aspects of proteostasis. SePhaChARNS is selective at the cellular, and possibly subcellular, levels because of variations in the detailed nAChR subunit composition, as well as in expression of auxiliary proteins such as lynx. One important implication of the SePhaChARNS hypothesis is that therapeutically relevant nicotinic receptor drugs could be discovered by studying events in intracellular compartments rather than exclusively at the surface membrane.

We report on the first, to our knowledge, successful detection of a fluorescent unnatural amino acid (fUAA), Lys(BODIPYFL), incorporated into a membrane protein (the muscle nicotinic acetylcholine receptor, nAChR) in a living cell. Xenopus oocytes were injected with a frameshift-suppressor tRNA, amino-acylated with Lys(BODIPYFL) and nAChR (alpha/beta19'GGGU/gamma/delta) mRNAs. We measured fluorescence from oocytes expressing nAChR beta19'Lys(BODIPYFL), using time-resolved total internal reflection fluorescence microscopy. Under conditions of relatively low receptor density (<0.1 receptors/microm(2)), we observed puncta with diffraction-limited profiles that were consistent with the point-spread function of our microscope. Furthermore, diffraction-limited puncta displayed step decreases in fluorescence intensity, consistent with single-molecule photobleaching. The puncta densities agreed with macroscopic ACh-induced current densities, showing that the fUAA was incorporated, and that receptors were functional. Dose-response relations for the nAChR beta19'Lys(BODIPYFL) receptors were similar to those for wild-type receptors. We also studied nAChR beta19'Lys(BODIPYFL) receptors labeled with alpha-bungarotoxin monoconjugated with Alexa488 (alphaBtxAlexa488). The nAChR has two alphaBtx binding sites, and puncta containing the Lys(BODIPYFL) labeled with alphaBtxAlexa488 yielded the expected three discrete photobleaching steps. We also performed positive control experiments with a nAChR containing enhanced green fluorescent protein in the gamma-subunit M3-M4 loop, which confirmed our nAChR beta19'Lys(BODIPYFL) measurements. Thus, we report on the cell-based single-molecule detection of nAChR beta19'Lys(BODIPYFL).

We describe a general application of the nonsense suppression methodology for unnatural amino acid incorporation to probe drug-receptor interactions in functional G protein-coupled receptors (GPCRs), evaluating the binding sites of both the M2 muscarinic acetylcholine receptor and the D2 dopamine receptor. Receptors were expressed in Xenopus oocytes, and activation of a G protein-coupled, inward-rectifying K(+) channel (GIRK) provided, after optimization of conditions, a quantitative readout of receptor function. A number of aromatic amino acids thought to be near the agonist-binding site were evaluated. Incorporation of a series of fluorinated tryptophan derivatives at W6.48 of the D2 receptor establishes a cation-pi interaction between the agonist dopamine and W6.48, suggesting a reorientation of W6.48 on agonist binding, consistent with proposed "rotamer switch" models. Interestingly, no comparable cation-pi interaction was found at the aligning residue in the M2 receptor.

These electrophysiological experiments, in slices and intact animals, study the effects of in vivo chronic exposure to nicotine on functional alpha4beta2* nAChRs in the nigrostriatal dopaminergic (DA) pathway. Recordings were made in wild-type and alpha4 nicotinic acetylcholine receptor (nAChR) subunit knock-out mice. Chronic nicotine enhanced methyllycaconitine citrate hydrate-resistant, dihydro-beta-erythroidine hydrobromide-sensitive nicotinic currents elicited by 3-1000 mum ACh in GABAergic neurons of the substantia nigra pars reticulata (SNr), but not in DA neurons of the substantia nigra pars compacta (SNc). This enhancement leads to higher firing rates of SNr GABAergic neurons and consequently to increased GABAergic inhibition of the SNc DA neurons. In the dorsal striatum, functional alpha4* nAChRs were not found on the neuronal somata; however, nicotine acts via alpha4beta2* nAChRs in the DA terminals to modulate glutamate release onto the medium spiny neurons. Chronic nicotine also increased the number and/or function of these alpha4beta2* nAChRs. These data suggest that in nigrostriatal DA pathway, chronic nicotine enhancement of alpha4beta2* nAChRs displays selectivity in cell type and in nAChR subtype as well as in cellular compartment. These selective events augment inhibition of SNc DA neurons by SNr GABAergic neurons and also temper the release of glutamate in the dorsal striatum. The effects may reduce the risk of excitotoxicity in SNc DA neurons and may also counteract the increased effectiveness of corticostriatal glutamatergic inputs during degeneration of the DA system. These processes may contribute to the inverse correlation between tobacco use and Parkinson's disease.

Nicotine addiction begins with high-affinity binding of nicotine to acetylcholine (ACh) receptors in the brain. The end result is over 4,000,000 smoking-related deaths annually worldwide and the largest source of preventable mortality in developed countries. Stress reduction, pleasure, improved cognition and other central nervous system effects are strongly associated with smoking. However, if nicotine activated ACh receptors found in muscle as potently as it does brain ACh receptors, smoking would cause intolerable and perhaps fatal muscle contractions. Despite extensive pharmacological, functional and structural studies of ACh receptors, the basis for the differential action of nicotine on brain compared with muscle ACh receptors has not been determined. Here we show that at the alpha4beta2 brain receptors thought to underlie nicotine addiction, the high affinity for nicotine is the result of a strong cation-pi interaction to a specific aromatic amino acid of the receptor, TrpB. In contrast, the low affinity for nicotine at the muscle-type ACh receptor is largely due to the fact that this key interaction is absent, even though the immediate binding site residues, including the key amino acid TrpB, are identical in the brain and muscle receptors. At the same time a hydrogen bond from nicotine to the backbone carbonyl of TrpB is enhanced in the neuronal receptor relative to the muscle type. A point mutation near TrpB that differentiates alpha4beta2 and muscle-type receptors seems to influence the shape of the binding site, allowing nicotine to interact more strongly with TrpB in the neuronal receptor. ACh receptors are established therapeutic targets for Alzheimer's disease, schizophrenia, Parkinson's disease, smoking cessation, pain, attention-deficit hyperactivity disorder, epilepsy, autism and depression. Along with solving a chemical mystery in nicotine addiction, our results provide guidance for efforts to develop drugs that target specific types of nicotinic receptors.

Alpha6-containing (alpha6*) nicotinic ACh receptors (nAChRs) are selectively expressed in dopamine (DA) neurons and participate in cholinergic transmission. We generated and studied mice with gain-of-function alpha6* nAChRs, which isolate and amplify cholinergic control of DA transmission. In contrast to gene knockouts or pharmacological blockers, which show necessity, we show that activating alpha6* nAChRs and DA neurons is sufficient to cause locomotor hyperactivity. alpha6(L9'S) mice are hyperactive in their home cage and fail to habituate to a novel environment. Selective activation of alpha6* nAChRs with low doses of nicotine, by stimulating DA but not GABA neurons, exaggerates these phenotypes and produces a hyperdopaminergic state in vivo. Experiments with additional nicotinic drugs show that altering agonist efficacy at alpha6* provides fine tuning of DA release and locomotor responses. alpha6*-specific agonists or antagonists may, by targeting endogenous cholinergic mechanisms in midbrain or striatum, provide a method for manipulating DA transmission in neural disorders.

Neuronal nicotinic acetylcholine (ACh) receptors are ligand-gated, cation-selective ion channels. Nicotinic receptors containing alpha4, alpha6, beta2, and beta3 subunits are expressed in midbrain dopaminergic neurons, and they are implicated in the response to smoked nicotine. Here, we have studied the cell biological and biophysical properties of receptors containing alpha6 and beta3 subunits by using fluorescent proteins fused within the M3-M4 intracellular loop. Receptors containing fluorescently tagged beta3 subunits were fully functional compared with receptors with untagged beta3 subunits. We find that beta3- and alpha6-containing receptors are highly expressed in neurons and that they colocalize with coexpressed, fluorescent alpha4 and beta2 subunits in neuronal soma and dendrites. Forster resonance energy transfer (FRET) reveals efficient, specific assembly of beta3 and alpha6 into nicotinic receptor pentamers of various subunit compositions. Using FRET, we demonstrate directly that only a single beta3 subunit is incorporated into nicotinic acetylcholine receptors (nAChRs) containing this subunit, whereas multiple subunit stoichiometries exist for alpha4- and alpha6-containing receptors. Finally, we demonstrate that nicotinic ACh receptors are localized in distinct microdomains at or near the plasma membrane using total internal reflection fluorescence (TIRF) microscopy. We suggest that neurons contain large, intracellular pools of assembled, functional nicotinic receptors, which may provide them with the ability to rapidly up-regulate nicotinic responses to endogenous ligands such as ACh, or to exogenous agents such as nicotine. Furthermore, this report is the first to directly measure nAChR subunit stoichiometry using FRET and plasma membrane localization of alpha6- and beta3-containing receptors using TIRF.

The muscle nicotinic acetylcholine receptor is a large, allosteric, ligand-gated ion channel with the subunit composition alpha2betagammadelta. Although much is now known about the structure of the binding site, relatively little is understood about how the binding event is communicated to the channel gate, causing the pore to open. Here we identify a key hydrogen bond near the binding site that is involved in the gating pathway. Using mutant cycle analysis with the novel unnatural residue alpha-hydroxyserine, we find that the backbone N-H of alphaSer-191 in loop C makes a hydrogen bond to an anionic side chain of the complementary subunit upon agonist binding. However, the anionic partner is not the glutamate predicted by the crystal structures of the homologous acetylcholine-binding protein. Instead, the hydrogen-bonding partner is the extensively researched aspartate gammaAsp-174/deltaAsp-180, which had originally been identified as a key binding residue for cationic agonists.

Understanding the gating mechanism of the nicotinic acetylcholine receptor (nAChR) and similar channels constitutes a significant challenge in chemical neurobiology. In the present work, we use a stereochemical probe to evaluate a proposed pin-into-hydrophobic socket mechanism for the alphaVal46 side chain of the nAChR. Utilizing nonsense suppression methodology we incorporated isoleucine (Ile), O-methyl threonine (Omt) and threonine (Thr) as well as their side chain epimers (the allo counterparts). Surprisingly, our results indicate that only the pro-S methyl group of the alphaVal46 side chain is sensitive to changes in hydrophobicity, consistent with the precise geometrical requirements of the pin-into-socket mechanism.

Acetylcholine and nicotine can modulate respiratory patterns by acting on nicotinic acetylcholine receptors (nAChRs) in the preBotzinger complex (preBotC). To further explore the molecular composition of these nAChRs, we studied a knock-in mouse strain with a leucine-to-alanine mutation in the M2 pore-lining region (L9'A) of the nAChR alpha4 subunit; this mutation renders alpha4-containing receptors hypersensitive to agonists. We recorded respiratory-related rhythmic motor activity from hypoglossal nerve (XIIn) and patch-clamped preBotC inspiratory neurons in an in vitro medullary slice preparation from neonatal mice. Nicotine affected respiratory rhythm at concentrations approximately 100-fold lower in the homozygous L9'A knock-in mice compared with wild-type mice. Bath application of 5 nm nicotine increased the excitability of preBotC inspiratory neurons, increased respiratory frequency, and induced tonic/seizure-like activities in XIIn in L9'A mice, effects similar to those induced by 1 microM nicotine in wild-type mice. In L9'A mice, microinjection of low nanomolar concentrations of nicotine into the preBotC increased respiratory frequency, whereas injection into the ipsilateral hypoglossal (XII) nucleus induced tonic/seizure-like activity. The alpha4*-selective nAChR antagonist dihydro-beta-erythroidine produced opposite effects and blocked the nicotinic responses. These data, showing that nAChRs in the preBotC and XII nucleus in L9'A mice are hypersensitive to nicotine and endogenous ACh, suggest that functional alpha4* nAChRs are present in the preBotC. They mediate cholinergic/nicotinic modulation of the excitability of preBotC inspiratory neurons and of respiratory rhythm. Furthermore, functional alpha4* nAChRs are present in XII nucleus and mediate cholinergic/nicotinic modulation of tonic activity in XIIn.

The nicotinic acetylcholine receptor and related Cys-loop receptors are ligand-gated ion channels that mediate fast synaptic transmission throughout the central and peripheral nervous system. A highly conserved aspartate residue (D89) that is near the agonist binding site but does not directly contact the ligand plays a critical part in receptor function. Here we probe the role of D89 using unnatural amino acid mutagenesis coupled with electrophysiology. Homology modeling implicates several hydrogen bonds involving D89. We find that no single hydrogen bond is essential to proper receptor function. Apparently, the side chain of D89 establishes a redundant network of hydrogen bonds; these bonds preorganize the agonist binding site by positioning a critical tryptophan residue that directly contacts the ligand. Earlier studies of the D89N mutant led to the proposal that a negative charge at this position is essential for receptor function. However, we find that receptors with neutral side chains at position 89 can function well, if the side chain is less perturbing than the amide of asparagine (nitro or keto groups allow function) or if a compensating backbone mutation is introduced to relieve unfavorable electrostatics.

The effects of nicotine on the tail-flick and hot-plate tests were determined to identify nicotinic receptor subtypes responsible for spinally and supraspinally mediated nicotine analgesia in knockin mice expressing hypersensitive alpha(4) nicotinic receptors (L9'S), in seven inbred mouse strains (C57BL/6, DBA/2, A/2, CBA/2, BALB/cByJ, C3H/HeJ, and 129/SvEv), and in two F1 hybrids (B6CBAF1 and B6D2F1). L9'S heterozygotes were approximately 6-fold more sensitive to the antinociceptive effects of nicotine than the wild-type controls in the hot-plate test but not in the tail-flick assay. Large differences in the effects of nicotine were also observed with both tests for the seven mouse strains. A/J and 129 mice were 6- to 8-fold more sensitive than CBA and BALB mice. In addition, B6CBAF1 hybrid mice were even less sensitive than CBA mice. Nicotinic binding sites were measured in three spinal cord regions and the hindbrain of the inbred strains. Significant differences in cytisine-sensitive, high affinity [(125)I]epibatidine binding site levels (alpha(4)beta(2)(*) subtypes), but not in (125)I-alpha-bungarotoxin binding (alpha(7)(*) subtypes), were observed. Significant negative correlations between cytisine-sensitive [(125)I]epibatidine binding and nicotine ED(50) for both tests were noted. Our results indicate that alpha(4)beta(2)(*) acetylcholine nicotinic receptors (nAChR) are important in mediating nicotine analgesia in supraspinal responses, while also showing that alpha(4)beta(2)(*)-nAChR and at least one other nAChR subtype appear to modulate spinal actions.

Understanding effects of chronic nicotine requires identifying the neurons and synapses whose responses to nicotine itself, and to endogenous acetylcholine, are altered by continued exposure to the drug. To address this problem, we developed mice whose alpha4 nicotinic receptor subunits are replaced by normally functioning fluorescently tagged subunits, providing quantitative studies of receptor regulation at micrometer resolution. Chronic nicotine increased alpha4 fluorescence in several regions; among these, midbrain and hippocampus were assessed functionally. Although the midbrain dopaminergic system dominates reward pathways, chronic nicotine does not change alpha4* receptor levels in dopaminergic neurons of ventral tegmental area (VTA) or substantia nigra pars compacta. Instead, upregulated, functional alpha4* receptors localize to the GABAergic neurons of the VTA and substantia nigra pars reticulata. In consequence, GABAergic neurons from chronically nicotine-treated mice have a higher basal firing rate and respond more strongly to nicotine; because of the resulting increased inhibition, dopaminergic neurons have lower basal firing and decreased response to nicotine. In hippocampus, chronic exposure to nicotine also increases alpha4* fluorescence on glutamatergic axons of the medial perforant path. In hippocampal slices from chronically treated animals, acute exposure to nicotine during tetanic stimuli enhances induction of long-term potentiation in the medial perforant path, showing that the upregulated alpha4* receptors in this pathway are also functional. The pattern of cell-specific upregulation of functional alpha4* receptors therefore provides a possible explanation for two effects of chronic nicotine: sensitization of synaptic transmission in forebrain and tolerance of dopaminergic neuron firing in midbrain.

Chronic nicotine exposure, in smokers or in experimental rodents administered nicotine, produces elevated levels of nicotinic acetylcholine receptors in several brain regions. However, there are few data on up-regulation of receptors in specific neuronal subtypes. We tested whether functional up-regulation of nicotinic responses occurs in cultured GABAergic neurons of the ventral midbrain. Fura-2 measurements of nicotinic responses were made on ventral midbrain neurons from knock-in mice heterozygous for the alpha4-M2 domain Leu9'Ala mutation, which confers nicotine hypersensitivity. Chronic nicotine exposure at a concentration (10 nM for 3 days) that activates only the hypersensitive alpha4* (Leu9'Ala) receptors, but not wild-type receptors, resulted in significant potentiation of ACh (100 microM)-elicited responses. Experiments were also performed on midbrain neuronal cultures heterozygous for the alpha4* (Leu9'Ala) mutation as well as for a GFP protein fused to a GABA transporter that reliably reveals GABAergic neurons. In cultures chronically treated with 10nM nicotine, there was significantly increased alpha4* nicotinic-induced Ca(2+) influx elicited by low concentration of ACh (3 microM). Furthermore, chronic exposure to the competitive antagonist dihydro-beta-erythroidine, but not to the noncompetitive antagonist mecamylamine, induced up-regulation of ACh elicited nicotinic responses. These results suggest that occupation of alpha4* nicotinic receptor binding site(s), at the interface between two subunits, is sufficient to promote assembly and/or up-regulation of functional receptors in GABAergic neurons. Up-regulation in neurons is both "cell-autonomous", occurring at the cell itself, and "receptor autonomous", occurring at the receptor itself, and may be a thermodynamic necessity of ligand-protein interactions.

Nicotinic receptors containing the alpha 4 subunit (alpha 4* nAChRs) have high sensitivity and are widely expressed in the central nervous system, yet their contributions to behavioral tolerance, a hallmark of nicotine dependence, are unclear. To evaluate the contribution of alpha 4* and non-alpha 4 nAChRs in the development of tolerance to hypothermia and locomotor suppression, alpha 4 knockout (KO), hypersensitive Leu9'Ala alpha 4 knock-in, and wild-type (WT) mice received daily nicotine injections, and their behaviors were compared. Repeated selective activation of alpha 4* nAChRs in Leu9'Ala mice produced profound tolerance to hypothermia over 7 days, whereas no tolerance was observed in alpha 4 KO animals. The summed time course and temperature response (after appropriate normalizations) from these two mutant mouse strains resembled the time course of WT tolerance. In addition, daily selective activation of alpha 4* nAChRs elicited locomotor activation in Leu9'Ala mice, but nicotine suppressed activity in alpha 4 KO mice and this did not change with daily drug exposure. Again, appropriately combined responses from the two mutant strains resembled the biphasic nicotine-induced activity in WT animals. Thus, by analyzing nicotinic responses in two complementary mouse lines, one lacking alpha 4* nAChRs, the other expressing hypersensitive alpha 4* nAChRs, one can accurately separate non-alpha 4 nAChR responses from alpha 4 nAChR responses, and one can also account for WT tolerance to both hypothermia and locomotor suppression. Our study suggests a new paradigm for bridging the gap between genetic manipulation of a single receptor and whole animal behavioral studies and shows that activation of alpha 4* nAChRs is both necessary and sufficient for the expression of tolerance.

We generated a mouse line harboring an autosomal-dominant nocturnal frontal lobe epilepsy (ADNFLE) mutation: the alpha4 nicotinic receptor S248F knock-in strain. In this mouse, modest nicotine doses (1-2 mg/kg) elicit a novel behavior termed the dystonic arousal complex (DAC). The DAC includes stereotypical head movements, body jerking, and forelimb dystonia; these behaviors resemble some core features of ADNFLE. A marked Straub tail is an additional component of the DAC. Similar to attacks in ADNFLE, the DAC can be partially suppressed by the sodium channel blocker carbamazepine or by pre-exposure to a very low dose of nicotine (0.1 mg/kg). The DAC is centrally mediated, genetically highly penetrant, and, surprisingly, not associated with overt ictal electrical activity as assessed by (1) epidural or frontal lobe depth-electrode electroencephalography or (2) hippocampal c-fos-regulated gene expression. Heterozygous knock-in mice are partially protected from nicotine-induced seizures. The noncompetitive antagonist mecamylamine does not suppress the DAC, although it suppresses high-dose nicotine-induced wild-type-like seizures. Experiments on agonist-induced 86Rb+ and neurotransmitter efflux from synaptosomes and on alpha4S248Fbeta2 receptors expressed in oocytes confirm that the S248F mutation confers resistance to mecamylamine blockade. Genetic background, gender, and mutant gene expression levels modulate expression of the DAC phenotype in mice. The S248F mouse thus appears to provide a model for the paroxysmal dystonic element of ADNFLE semiology. Our model complements what is seen in other ADNFLE animal models. Together, these mice cover the spectrum of behavioral and electrographic events seen in the human condition.

Nicotinic acetylcholine receptors (nAChRs) affect a wide array of biological processes, including learning and memory, attention, and addiction. lynx1, the founding member of a family of mammalian prototoxins, modulates nAChR function in vitro by altering agonist sensitivity and desensitization kinetics. Here we demonstrate, through the generation of lynx1 null mutant mice, that lynx1 modulates nAChR signaling in vivo. Its loss decreases the EC(50) for nicotine by approximately 10-fold, decreases receptor desensitization, elevates intracellular calcium levels in response to nicotine, and enhances synaptic efficacy. lynx1 null mutant mice exhibit enhanced performance in specific tests of learning and memory. Consistent with reports that mutations resulting in hyperactivation of nAChRs can lead to neurodegeneration, aging lynx1 null mutant mice exhibit a vacuolating degeneration that is exacerbated by nicotine and ameliorated by null mutations in nAChRs. We conclude that lynx1 functions as an allosteric modulator of nAChR function in vivo, balancing neuronal activity and survival in the CNS.

Nicotinic acetylcholine receptors (nAChRs) are members of the Cys-loop superfamily of pentameric ligand-gated ion channels, which include GABA (A and C), serotonin, and glycine receptors. Currently, 12 neuronal nAChR subunits have been identified (alpha2-10 and beta2-4) and are generally grouped into alpha subunits, which contain two adjacent cysteine residues essential for ACh binding, and beta subunits, which lack these residues. The majority of neuronal nAChRs fall into two categories: those that bind agonist with high affinity (nM concentrations); and those that bind with lower affinity (microM concentrations). The low-affinity receptors are presumably homomeric alpha7 receptors that are alpha-bungarotoxin sensitive, whereas alpha4beta2 nAChRs account for >90% of the high-affinity nicotinic receptors in the brain (Whiting and Lindstrom, 1986). Their physiological contributions to neurotransmission, signaling, and behavior are not completely understood. Precise mapping of subcellular and neuroanatomical localizations of neuronal nAChR subunits will help elucidate the physiological role of neuronal nAChRs and their role in nicotine addiction.

We describe an inducible genetic model for degeneration of midbrain dopaminergic neurons in adults. In previous studies, knock-in mice expressing hypersensitive M2 domain Leu9'Ser (L9'S) alpha4 nicotinic receptors (nAChR) at near-normal levels displayed dominant neonatal lethality and dopaminergic deficits in embryonic midbrain, because the hypersensitive nAChR is excitotoxic. However, heterozygous L9'S mice that retain the neomycin resistance cassette (neo) in a neighboring intron express low levels of the mutant allele (approximately 25% of normal levels), and these neo-intact mice are therefore viable and fertile. The neo cassette is flanked by loxP sites. In adult animals, we locally injected helper-dependent adenovirus (HDA) expressing cre recombinase. Local excision of the neo cassette, via cre-mediated recombination, was verified by genomic analysis. In L9'S HDA-cre injected animals, locomotion was reduced both under baseline conditions and after amphetamine application. There was no effect in L9'S HDA-control treated animals or in wild-type (WT) littermates injected with either virus. Immunocytochemical analyses revealed marked losses (> 70%) of dopaminergic neurons in L9'S HDA-cre injected mice compared to controls. At 20-33 days postinjection in control animals, the coexpressed marker gene, yellow fluorescent protein (YFP), was expressed in many neurons and few glial cells near the injection, emphasizing the neurotropic utility of the HDA. Thus, HDA-mediated gene transfer into adult midbrain induced sufficient functional expression of cre in dopaminergic neurons to allow for postnatal deletion of neo. This produced increased L9'S mutant nAChR expression, which in turn led to nicotinic cholinergic excitotoxicity in dopaminergic neurons.

The binding of three distinct agonists-acetylcholine (ACh), nicotine, and epibatidine-to the nicotinic acetylcholine receptor has been probed using unnatural amino acid mutagenesis. ACh makes a cation-pi interaction with Trp alpha149, while nicotine employs a hydrogen bond to a backbone carbonyl in the same region of the agonist binding site. The nicotine analogue epibatidine achieves its high potency by taking advantage of both the cation-pi interaction and the backbone hydrogen bond. A simple structural model that considers only possible interactions with Trp alpha149 suggests that a novel aromatic C-H...O=C hydrogen bond further augments the binding of epibatidine. These studies illustrate the subtleties and complexities of the interactions between drugs and membrane receptors and establish a paradigm for obtaining detailed structural information.

A leucine to alanine substitution (L9'A) was introduced in the M2 region of the mouse alpha4 neuronal nicotinic acetylcholine receptor (nAChR) subunit. Expressed in Xenopus oocytes, alpha4(L9'A)beta2 nAChRs were > or =30-fold more sensitive than wild type (WT) to both ACh and nicotine. We generated knock-in mice with the L9'A mutation and studied their cellular responses, seizure phenotype, and sleep-wake cycle. Seizure studies on alpha4-mutated animals are relevant to epilepsy research because all known mutations linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) occur in the M2 region of alpha4or beta2 subunits. Thalamic cultures and synaptosomes from L9'A mice were hypersensitive to nicotine-induced ion flux. L9'A mice were approximately 15-fold more sensitive to seizures elicited by nicotine injection than their WT littermates. Seizures in L9'A mice differed qualitatively from those in WT: L9'A seizures started earlier, were prevented by nicotine pretreatment, lacked EEG spike-wave discharges, and consisted of fast repetitive movements. Nicotine-induced seizures in L9'A mice were partial, whereas WT seizures were generalized. When L9'A homozygous mice received a 10 mg/kg nicotine injection, there was temporal and phenomenological separation of mutant and WT-like seizures: an initial seizure approximately 20 s after injection was clonic and showed no EEG changes. A second seizure began 3-4 min after injection, was tonic-clonic, and had EEG spike-wave activity. No spontaneous seizures were detected in L9'A mice during chronic video/EEG recordings, but their sleep-wake cycle was altered. Our findings show that hypersensitive alpha4* nicotinic receptors in mice mediate changes in the sleep-wake cycle and nicotine-induced seizures resembling ADNFLE.

GABA(C) (rho) receptors are members of the Cys-loop superfamily of neurotransmitter receptors, which includes nicotinic acetylcholine (nACh), 5-HT(3), and glycine receptors. As in other members of this family, the agonist binding site of GABA(C) receptors is rich in aromatic amino acids, but while other receptors bind agonist through a cation-pi interaction to a tryptophan, the GABA(C) binding site has tyrosine at the aligning positions. Incorporating a series of tyrosine derivatives at position 198 using unnatural amino acid mutagenesis reveals a clear correlation between the cation-pi binding ability of the side chain and EC(50) for receptor activation, thus demonstrating a cation-pi interaction between a tyrosine side chain and a neurotransmitter. Comparisons among four homologous receptors show variations in cation-pi binding energies that reflect the nature of the cationic center of the agonist.

Extracellular Ca(2+) robustly potentiates the acetylcholine response of alpha4beta2 nicotinic receptors. Rat orthologs of five mutations linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE)-alpha4(S252F), alpha4(S256L), alpha4(+L264), beta2(V262L), and beta2(V262M)-reduced 2 mM Ca(2+) potentiation of the alpha4beta2 1 mM acetylcholine response by 55 to 74%. To determine whether altered allosteric Ca(2+) activation or enhanced Ca(2+) block caused this reduction, we coexpressed the rat ADNFLE mutations with an alpha4 N-terminal mutation, alpha4(E180Q), that abolished alpha4beta2 allosteric Ca(2+) activation. In each case, Ca(2+) inhibition of the double mutants was less than that expected from a Ca(2+) blocking mechanism. In fact, the effects of Ca(2+) on the ADNFLE mutations near the intracellular end of the M2 region-alpha4(S252F) and alpha4(S256L)-were consistent with a straightforward allosteric mechanism. In contrast, the effects of Ca(2+) on the ADNFLE mutations near the extracellular end of the M2 region-alpha4(+L264)beta2, beta2(V262L), and beta2(V262M)-were consistent with a mixed mechanism involving both altered allosteric activation and enhanced block. However, the effects of 2 mM Ca(2+) on the alpha4beta2, alpha4(+L264)beta2, and alpha4beta2(V262L) single-channel conductances, the effects of membrane potential on the beta2(V262L)-mediated reduction in Ca(2+) potentiation, and the effects of eliminating the negative charges in the extracellular ring on this reduction failed to provide any direct evidence of mutant-enhanced Ca(2+) block. Moreover, analyses of the alpha4beta2, alpha4(S256L), and alpha4(+L264) Ca(2+) concentration-potentiation relations suggested that the ADNFLE mutations reduce Ca(2+) potentiation of the alpha4beta2 acetylcholine response by altering allosteric activation rather than by enhancing block.

In the Cys loop superfamily of ligand-gated ion channels, a global conformational change, initiated by agonist binding, results in channel opening and the passage of ions across the cell membrane. The detailed mechanism of channel gating is a subject that has lent itself to both structural and electrophysiological studies. Here we defined a gating interface that incorporates elements from the ligand binding domain and transmembrane domain previously reported as integral to proper channel gating. An overall analysis of charged residues within the gating interface across the entire superfamily showed a conserved charging pattern, although no specific interacting ion pairs were conserved. We utilized a combination of conventional mutagenesis and the high precision methodology of unnatural amino acid incorporation to study extensively the gating interface of the mouse muscle nicotinic acetylcholine receptor. We found that charge reversal, charge neutralization, and charge introduction at the gating interface are often well tolerated. Furthermore, based on our data and a reexamination of previously reported data on gamma-aminobutyric acid, type A, and glycine receptors, we concluded that the overall charging pattern of the gating interface, and not any specific pairwise electrostatic interactions, controls the gating process in the Cys loop superfamily.

Mutant mice with a hypersensitive serotonin (5-HT)3A receptor were generated through targeted exon replacement. A valine to serine mutation (V13'S) in the channel-lining M2 domain of the 5-HT3A receptor subunit rendered the 5-HT3 receptor 70-fold more sensitive to serotonin and produced constitutive activity when combined with the 5-HT3B subunit. Mice homozygous for the mutant allele (5-HT3Avs/vs) had decreased levels of 5-HT3A mRNA. Measurements on sympathetic ganglion cells in these mice showed that whole-cell serotonin responses were reduced, and that the remaining 5-HT3 receptors were hypersensitive. Male 5-HT3Avs/vs mice died at 2-3 months of age, and heterozygous (5-HT3Avs/+) males and homozygous mutant females died at 4-6 months of age from an obstructive uropathy. Both male and female 5-HT3A mutant mice had urinary bladder mucosal and smooth muscle hyperplasia and hypertrophy, whereas male mutant mice had additional prostatic smooth muscle and urethral hyperplasia. 5-HT3A mutant mice had marked voiding dysfunction characterized by a loss of micturition contractions with overflow incontinence. Detrusor strips from 5-HT3Avs/vs mice failed to contract to neurogenic stimulation, despite overall normal responses to a cholinergic agonist, suggestive of altered neuronal signaling in mutant mouse bladders. Consistent with this hypothesis, decreased nerve fiber immunoreactivity was observed in the urinary bladders of 5-HT3Avs/vs compared with 5-HT3A wild-type (5-HT3A+/+) mice. These data suggest that persistent activation of the hypersensitive and constitutively active 5-HT3A receptor in vivo may lead to excitotoxic neuronal cell death and functional changes in the urinary bladder, resulting in bladder hyperdistension, urinary retention, and overflow incontinence.

To study conformational transitions at the muscle nicotinic acetylcholine (ACh) receptor (nAChR), a rhodamine fluorophore was tethered to a Cys side chain introduced at the beta 19' position in the M2 region of the nAChR expressed in Xenopus oocytes. This procedure led to only minor changes in receptor function. During agonist application, fluorescence increased by (Delta F/F) approximately 10%, and the emission peak shifted to lower wavelengths, indicating a more hydrophobic environment for the fluorophore. The dose-response relations for Delta F agreed well with those for epibatidine-induced currents, but were shifted approximately 100-fold to the left of those for ACh-induced currents. Because (i) epibatidine binds more tightly to the alpha gamma-binding site than to the alpha delta site and (ii) ACh binds with reverse-site selectivity, these data suggest that Delta F monitors an event linked to binding specifically at the alpha delta-subunit interface. In experiments with flash-applied agonists, the earliest detectable Delta F occurs within milliseconds, i.e., during activation. At low [ACh] (< or = 10 microM), a phase of Delta F occurs with the same time constant as desensitization, presumably monitoring an increased population of agonist-bound receptors. However, recovery from Delta F is complete before the slowest phase of recovery from desensitization (time constant approximately 250 s), showing that one or more desensitized states have fluorescence like that of the resting channel. That conformational transitions at the alpha delta-binding site are not tightly coupled to channel activation suggests that sequential rather than fully concerted transitions occur during receptor gating. Thus, time-resolved fluorescence changes provide a powerful probe of nAChR conformational changes.

This study analyzes the electrophysiological cause and behavioral consequence of dopaminergic cell loss in a knockin mouse strain bearing hypersensitive nicotinic alpha4-receptor subunits ("L9'S mice"). Adult brains of L9'S mice show moderate loss of substantia nigra dopaminergic neurons and of striatal dopaminergic innervation. Amphetamine-stimulated locomotion is impaired, reflecting a reduction of dopamine stored in presynaptic vesicles. Recordings from dopaminergic neurons in L9'S mice show that 10 microM nicotine depolarizes cells and increases spiking rates in L9'S cells but hyperpolarizes and decreases spiking rates in wild-type (WT) cells. Thus dopaminergic neurons of L9'S mice have an excitatory response to nicotine which is qualitatively different from that of WT neurons. The cause of dopaminergic cell death is therefore probably an increased sensitivity to acetylcholine or choline of alpha4-containing nicotinic receptors. Hypersensitive excitatory stimulation during activation of alpha4-containing receptors provides the first evidence for cholinergic excitotoxicity as a cause of dopaminergic neuron death. This novel concept may be relevant to the pathophysiology of Parkinson disease.

The identity of nicotinic receptor subtypes sufficient to elicit both the acute and chronic effects of nicotine dependence is unknown. We engineered mutant mice with a4 nicotinic subunits containing a single point mutation, Leu9' --> Ala9' in the pore-forming M2 domain, rendering a4* receptors hypersensitive to nicotine. Selective activation of a4* nicotinic acetylcholine receptors with low doses of agonist recapitulates nicotine effects thought to be important in dependence, including reinforcement in response to acute nicotine administration, as well as tolerance and sensitization elicited by chronic nicotine administration. These data indicate that activation of a4* receptors is sufficient for nicotine-induced reward, tolerance, and sensitization.

We studied a strain of exon replacement mice ("L9'S knock-in") whose alpha4 nicotinic receptor subunits have a leucine to serine mutation in the M2 region, 9' position (Labarca et al., 2001); this mutation renders alpha4-containing receptors hypersensitive to agonists. Nicotine induced seizures at concentrations (1 mg/kg) approximately eight times lower in L9'S than in wild-type (WT) littermates. At these concentrations, L9'S but not WT showed increases in EEG amplitude and theta rhythm. L9'S mice also showed higher seizure sensitivity to the nicotinic agonist epibatidine, but not to the GABA(A) receptor blocker and proconvulsant bicuculline. Dorsiflexion of the tail (Straub tail) was the most sensitive nicotine effect found in L9'S mice (0.1 mg/kg). The L9'S mice were hypersensitive to galanthamine- and tacrine-induced seizures and Straub tails. There were no apparent neuroanatomical differences between L9'S and WT mice in several brain regions. [(125)I]Epibatidine binding to brain membranes showed that the mutant allele was expressed at approximately 25% of WT levels, presumably because of the presence of a neomycin selection cassette in a nearby intron. (86)Rb efflux experiments on brain synaptosomes showed an increased fraction of function at low agonist concentrations in L9'S mice. These data support the possible involvement of gain-of-function alpha4 receptors in autosomal dominant nocturnal frontal-lobe epilepsy.

We characterized the differential accessibility of the nicotinic acetylcholine receptor alpha1 subunit in the open, closed, and desensitized states by using electrophysiology-coordinated photolabeling by several lipophilic probes followed by mass spectrometric analysis. Voltage-clamped oocytes expressing receptors were preincubated with one of the lipophilic probes and were continually exposed to acetylcholine; UV irradiation was applied during 500-ms pulses to + 40 or to -140 mV (which produced closed or approximately 50% open receptors, respectively). In the open state, there was specific probe incorporation within the N-terminal domain at residues that align with the beta8-beta9 loop of the acetylcholine-binding protein. In the closed state, probe incorporation was identified at several sites of the N-terminal domain within the conserved cysteine loop (residues 128-142), the cytoplasmic loop (M3-M4), and M4. The labeling pattern in the M4 region is consistent with previous results, further defining the lipid-exposed face of this transmembrane alpha-helix. These results show regions within the N-terminal domain that are involved in gating-dependent conformational shifts, confirm that the cysteine loop resides at or near the protein-membrane interface, and show that segments of the M3-M4 loop are near to the lipid bilayer.

In a previous study utilizing benzophenone-based topological probes to study conformationally dependent changes in mouse muscle nicotinic acetylcholine receptor (nAChR) topology, electrospray ionization tandem mass spectrometric (ESI-MS/MS) analysis led to a consistent -2.0 Da mass deviation from expected values. In the present study a synthetic peptide, corresponding to nAChR alpha1 subunit residues 130-139, was photolabeled. MS/MS analysis of this peptide using an ion trap confirmed the previously observed mass deviation, associated only with fragment ions that contain the incorporated benzophenone moiety. Analysis of peak profiles for the photolabeled ions does not indicate the typical 'peak fronting' that produces a mass shift when labile ions are prematurely ejected from the ion trap. Rather, hydrogen/deuterium (H/D) exchange experiments support the hypothesis that a chemical rearrangement involving phenyl migration and ketone formation has formed an unexpected oxidized peptide, with molecular mass 2 Da less than that expected, that is isolated for collision-induced dissociation in the ion trap together with the predicted precursor due to the broad ion isolation window specified.

Two series of knockin mouse strains have been constructed with point mutations that result in hypersensitive neuronal nicotinic acetylcholine receptors containing alpha 4- or alpha 7-subunits. The full expression of the stronger alleles produces neonatal excitotoxic lethality; however, mice with attenuated expression or milder alleles are viable, and display a range of hypersensitive responses to nicotine. To date, measurements have been made on nicotine-induced seizures, Straub tail, hypothermia, antinociception, electroencephalograms and cellular electrophysiological responses. These strains are helping to define the occurrence of these important receptor subtypes, and their role in the acute and chronic actions of nicotine. The hypersensitive strains may be useful for the development of nicotinic drug therapy.

We describe an approach to achieve unnatural amino acid incorporation into channels and receptors expressed in mammalian cells. We show that microelectroporation provides a general method to deliver DNA, mRNA, and tRNA simultaneously. In both CHO cells and cultured neurons, microelectroporation efficiently delivers an in vitro transcribed, serine amber suppressor tRNA, leading to nonsense suppression in a mutant EGFP gene. In CHO cells, both natural and unnatural amino acids chemically appended to a suppressor tRNA are site specifically incorporated into the nicotinic acetylcholine receptor (nAChR). Electrophysiology confirms the expected functional consequences of the unnatural residue. The microelectroporation strategy described here is more general, less tedious, and less damaging to mammalian neuronal and nonneuronal cells than previous approaches to nonsense suppression in small cells and provides the first example of unnatural amino acid incorporation in mammalian cells using chemically aminoacylated tRNA.

Fura-2 recording of Ca2+ influx was used to show that incubation in 1 microm nicotine (2-6 d) upregulates several pharmacological components of acetylcholine (ACh) responses in ventral midbrain cultures, including a MLA-resistant, DHbetaE-sensitive component that presumably corresponds to alpha4beta2 receptors. To study changes in alpha4beta2 receptor levels and assembly during this upregulation, we incorporated yellow and cyan fluorescent proteins (YFPs and CFPs) into the alpha4 or beta2 M3-M4 intracellular loops, and these subunits were coexpressed in human embryonic kidney (HEK) 293T cells and cultured ventral midbrain neurons. The fluorescent receptors resembled wild-type receptors in maximal responses to ACh, dose-response relations, ACh-induced Ca2+ influx, and somatic and dendritic distribution. Transfected midbrain neurons that were exposed to nicotine (1 d) displayed greater levels of fluorescent alpha4 and beta2 nicotinic ACh receptor (nAChR) subunits. As expected from the hetero-multimeric nature of alpha4beta2 receptors, coexpression of the alpha4-YFP and beta2-CFP subunits resulted in robust fluorescence resonance energy transfer (FRET), with a FRET efficiency of 22%. In midbrain neurons, dendritic alpha4beta2 nAChRs displayed greater FRET than receptors inside the soma, and in HEK293T cells, a similar increase was noted for receptors that were translocated to the surface during PKC stimulation. When cultured transfected midbrain neurons were incubated in 1 microm nicotine, there was increased FRET in the cell body, denoting increased assembly of alpha4beta2 receptors. Thus, changes in alpha4beta2 receptor assembly play a role in the regulation of alpha4beta2 levels and responses in both clonal cell lines and midbrain neurons, and the regulation may result from Ca2+-stimulated pathways.

BACKGROUND: Ethanol modulates the functional activity of alpha4beta2 neuronal nicotinic cholinergic receptors (nAChR) when measured in vitro, but the potential role of alpha4beta2 nAChRs in regulating behavioral effects of ethanol is unknown. Recently, Tritto et al. (Tritto T, Stitzel JA, Marks MJ, Romm E, Collins AC (2002) Variability in response to nicotine in the LSxSS RI strains: potential role of polymorphisms in alpha4 and alpha6 nicotinic receptor genes. Pharmacogenetics 12:197-208) reported that a polymorphism (A529T) in the alpha4 nAChR subunit gene is associated with variability in nicotine's effects on startle in the LSxSS recombinant inbred (RI) strains. Ethanol also alters the acoustic startle response. Thus, we evaluated the potential role of alpha4beta2 nAChRs in modulating ethanol's effects on acoustic startle.
METHODS: The effects of ethanol on acoustic startle were determined in the LSxSS RI strains. In addition, the effects of ethanol and nicotine were also measured in alpha4 gain of function and beta2 null mutant mice. The beta2 mutants do not express the major variant of alpha4 nAChRs, alpha4beta2.
RESULTS: An association between the alpha4 A529T polymorphism and ethanol's effects on startle was found in the LSxSS RI strains; those strains that express the A529 variant of alpha4 were more sensitive to ethanol-induced depression of startle. The alpha4 gain of function mutants were more sensitive to the effects of both nicotine and ethanol and the beta2 null mutants were less sensitive to both drugs.
CONCLUSIONS: alpha4beta2-containing nAChRs may play important roles in modulating the effects of both ethanol and nicotine on the acoustic startle response. We suggest that nAChR subunit genes should be evaluated as potential contributors to both alcoholism and tobacco abuse.

Five nicotinic acetylcholine receptor (nAChR) mutations are currently linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). The similarity of their clinical symptoms suggests that a common functional anomaly of the mutations underlies ADNFLE seizures. To identify this anomaly, we constructed rat orthologues (S252F, +L264, S256L, V262L, V262M) of the human ADNFLE mutations, expressed them in Xenopus oocytes with the appropriate wild-type (WT) subunit (alpha4 or beta2), and studied the Ca2+ dependence of their ACh responses. All the mutations significantly reduced 2 mM Ca2+-induced increases in the 30 microM ACh response (P < 0.05). Consistent with a dominant mode of inheritance, this reduction persisted in oocytes injected with a 1:1 mixture of mutant and WT cRNA. BAPTA injections showed that the reduction was not due to a decrease in the secondary activation of Ca2+-activated Cl- currents. The S256L mutation also abolished 2 mM Ba2+ potentiation of the ACh response. The S256L, V262L and V262M mutations had complex effects on the ACh concentration-response relationship but all three mutations shifted the concentration-response relationship to the left at [ACh] >= 30 microM. Co-expression of the V262M mutation with a mutation (E180Q) that abolished Ca2+ potentiation resulted in 2 mM Ca2+ block, rather than potentiation, of the 30 microM ACh response, suggesting that the ADNFLE mutations reduce Ca2+ potentiation by enhancing Ca2+ block of the alpha4beta2 nAChR. Ca2+ modulation may prevent presynaptic alpha4beta2 nAChRs from overstimulating glutamate release at central excitatory synapses during bouts of synchronous, repetitive activity. Reducing the Ca2+ dependence of the ACh response could trigger seizures by increasing alpha4beta2-mediated glutamate release during such bouts.

A series of tryptophan analogues has been introduced into the binding site regions of two ion channels, the ligand-gated nicotinic acetylcholine and serotonin 5-HT(3A) receptors, using unnatural amino acid mutagenesis and heterologous expression in Xenopus oocytes. A cation-pi interaction between serotonin and Trp183 of the serotonin channel 5-HT(3A)R is identified for the first time, precisely locating the ligand-binding site of this receptor. The energetic contribution of the observed cation-pi interaction between a tryptophan and the primary ammonium ion of serotonin is estimated to be approximately 4 kcal/mol, while the comparable interaction with the quaternary ammonium of acetylcholine is approximately 2 kcal/mol. The binding mode of nicotine to the nicotinic receptor of mouse muscle is examined by the same technique and found to differ significantly from that of the natural agonist, acetylcholine.

Glutamate-gated chloride (GluCl) channels from invertebrates can be activated by ivermectin (IVM) to produce electrical silencing in mammalian neurons. To improve this GluCl/IVM strategy, we sought to mutate the Caenorhabditis elegans GluCl channels so that they become insensitive to glutamate but retain their sensitivity to IVM. Based on structure-function studies of nicotinic acetylcholine receptor superfamily members, we tested in oocytes 19 point mutants at 16 residues in the beta-subunit likely to be involved in the response to glutamate. Y182F reduces the glutamate response by greater than six-fold, with little change to IVM responses, when coexpressed with wild-type (WT) GluCl alpha. For GluCl alphabeta(Y182F), the EC(50) and Hill coefficient for glutamate are similar to those of WT, indicating that the mutant decreases the efficacy of glutamate, but not the potency. Also, fluorescent proteins (enhanced green fluorescent protein, enhanced yellow fluorescent protein, enhanced cyan fluorescent protein; XFP) were inserted into the M3-M4 loop of the GluCl alpha, beta and beta(Y182F). We found no significant functional difference between these XFP-tagged receptors and WT receptors. The modified GluCl channel, without glutamate sensitivity but with a fluorescent tag, may be more useful in GluCl silencing strategies.

A series of tethered quaternary ammonium derivatives of Tyr have been incorporated into the binding site of the nicotinic acetylcholine receptor (nAChR) using the in vivo nonsense suppression method, producing constitutively active (self-gating) receptors. We have incorporated primary, secondary, and tertiary amine tethered agonists to give receptors whose constitutive activity can be modulated by pH. Lowering the pH protonates the tethered amine, giving it a positive charge and allowing it to reversibly activate the receptor. Tertiary and secondary tethered amines, TyrO3T and TyrO3S, have been successfully incorporated at alpha149 in the nAChR. Constitutive currents at pH 5.5 are 6 times those at pH 9.0. The pKa of TyrO3T in the binding site appears to be 6 or lower, differing substantially from its pKa in solution ( approximately 9.3). This local pKa perturbation has substantial implications for pharmacological research on the nAChR: of the tertiary agonists considered, noracetylcholine experiences this pKa perturbation, while nicotine does not.

Knock-in mice were generated that harbored a leucine-to-serine mutation in the alpha4 nicotinic receptor near the gate in the channel pore. Mice with intact expression of this hypersensitive receptor display dominant neonatal lethality. These mice have a severe deficit of dopaminergic neurons in the substantia nigra, possibly because the hypersensitive receptors are continuously activated by normal extracellular choline concentrations. A strain that retains the neo selection cassette in an intron has reduced expression of the hypersensitive receptor and is viable and fertile. The viable mice display increased anxiety, poor motor learning, excessive ambulation that is eliminated by very low levels of nicotine, and a reduction of nigrostriatal dopaminergic function upon aging. These knock-in mice provide useful insights into the pathophysiology of sustained nicotinic receptor activation and may provide a model for Parkinson's disease.

BACKGROUND: The integral membrane proteins of neurons and other excitable cells are generally resistant to high resolution structural tools. Structure-function studies, especially those enhanced by the nonsense suppression methodology for unnatural amino acid incorporation, constitute one of the most powerful probes of ion channels and related structures. The nonsense suppression methodology can also be used to incorporate functional side chains designed to deliver novel structural probes to membrane proteins. In this vein, we sought to generalize a potentially powerful tool - the tethered agonist approach - for mapping the agonist binding site of ligand-gated ion channels.
RESULTS: Using the in vivo nonsense suppression method for unnatural amino acid incorporation, a series of tethered quaternary ammonium derivatives of tyrosine have been incorporated into the nicotinic acetylcholine receptor. At three sites a constitutively active receptor results, but the pattern of activation as a function of chain length is different. At position alpha149, there is a clear preference for a three-carbon tether, while at position alpha93 tethers of 2-5 carbons are comparably effective. At position gamma55/delta57 all tethers except the shortest one can activate the receptor. Based on these and other data, a model for the receptor binding site can be developed by analogy to the acetylcholine esterase crystal structure. CONCLUSION: Through the use of nonsense suppression techniques, the tethered agonist approach has been made into a general tool for probing receptor structures. When applied to the nicotinic receptor, the method places new restrictions on developing models for the agonist binding site.

Transmitter-gated cation channels are detectors of excitatory chemical signals at synapses in the nervous system. Here we show that structurally distinct alpha3beta4 nicotinic and P2X2 channels influence each other when co-activated. The activation of one channel type affects distinct kinetic and conductance states of the other, and co-activation results in non-additive responses owing to inhibition of both channel types. State-dependent inhibition of nicotinic channels is revealed most clearly with mutant P2X2 channels, and inhibition is decreased at lower densities of channel expression. In synaptically coupled myenteric neurons, nicotinic fast excitatory postsynaptic currents are occluded during activation of endogenously co-expressed P2X channels. Our data provide a molecular basis and a synaptic context for cross-inhibition between transmitter-gated channels.

Regulators of G protein signaling (RGS) stimulate the GTPase activity of G protein Galpha subunits and probably play additional roles. Some RGS proteins contain a Ggamma subunit-like (GGL) domain, which mediates a specific interaction with Gbeta5. The role of such interactions in RGS function is unclear. RGS proteins can accelerate the kinetics of coupling of G protein-coupled receptors to G-protein-gated inwardly rectifying K(+) (GIRK) channels. Therefore, we coupled m2-muscarinic acetylcholine receptors to GIRK channels in Xenopus oocytes to evaluate the effect of Gbeta5 on RGS function. Co-expression of either RGS7 or RGS9 modestly accelerated GIRK channel kinetics. When Gbeta5 was co-expressed with either RGS7 or RGS9, the acceleration of GIRK channel kinetics was strongly increased over that produced by RGS7 or RGS9 alone. RGS function was not enhanced by co-expression of Gbeta1, and co-expression of Gbeta5 alone had no effect on GIRK channel kinetics. Gbeta5 did not modulate the function either of RGS4, an RGS protein that lacks a GGL domain, or of a functional RGS7 construct in which the GGL domain was omitted. Enhancement of RGS7 function by Gbeta5 was not a consequence of an increase in the amount of plasma membrane or cytosolic RGS7 protein.

The site-specific incorporation of alpha-hydroxy acids into proteins using nonsense suppression can provide a powerful probe of protein structure and function. The resulting backbone ester may be selectively hydrolyzed in the presence of the peptide backbone, providing an "orthogonal" chemistry that can be useful both as an analytical tool and as a structural probe. Here we describe in detail a substantial substituent effect on this hydrolysis reaction. Consistent with mechanistic expectations, the steric bulk of the amino acid immediately N-terminal of the hydroxy acid has a large effect on the hydrolysis rate. On the basis of these results, we also describe a simple protocol for identifying disulfide loops in soluble and membrane proteins, exemplified by the alpha subunit of the muscle nicotinic acetylcholine receptor (nAChR). If a backbone ester is incorporated outside a disulfide loop, hydrolysis alone gives two fragments, but if the ester is incorporated within a disulfide loop, both hydrolysis and reduction are required for cleavage. This test could be useful in characterizing the disulfide topology of complex, membrane proteins.

An approach to identify backbone conformational changes underlying nicotinic acetylcholine receptor (nAChR) gating was developed. Specific backbone peptide bonds were replaced with an ester, which disrupts backbone hydrogen bonds at the site of mutation. At a conserved proline residue (alphaPro221) in the first transmembrane (M1) domain, the amide-to-ester mutation provides receptors with near-normal sensitivity, although the natural amino acids tested other than Pro produce receptors that gate with a much larger EC50 than normal. Therefore, a backbone hydrogen bond at this site may interfere with normal gating. In the alphaM2 domain, the amide-to-ester mutation yielded functional receptors at 15 positions, 3 of which provided receptors with >10-fold lower EC50 than wild type. These results support a model for gating that includes significant changes of backbone conformation within the M2 domain.

A nonsense codon suppression technique was employed to incorporate ortho-nitrobenzyl tyrosine, "caged tyrosine," in place of tyrosine at any of three positions (93, 127, or 198) in the alpha subunit of the muscle nicotinic ACh receptor (nAChR) expressed in Xenopus oocytes. The ortho-nitrobenzyl group was then removed by 1 ms flashes at 300-350 nm to yield tyrosine itself while macroscopic currents were recorded during steady ACh exposure. Responses to multiple flashes showed (1) that each flash decages up to 17% of the tyrosines and (2) that two tyrosines must be decaged per receptor for a response. The conductance relaxations showed multiple kinetic components; rate constants (<0.1 s(-1) to 10(3) s(-1)) depended on pH and the site of incorporation, and relative amplitudes depended on the number of prior flashes. This method, which is potentially quite general, (1) provides a time-resolved assay for the behavior of a protein when a mutant sidechain is abruptly changed to the wild-type residue and (2) will also allow for selective decaging of sidechains that are candidates for covalent modification (such as phosphorylation) in specific proteins in intact cells.

The nicotinic acetylcholine receptor is the prototype ligand-gated ion channel. A number of aromatic amino acids have been identified as contributing to the agonist binding site, suggesting that cation-pi interactions may be involved in binding the quaternary ammonium group of the agonist, acetylcholine. Here we show a compelling correlation between: (i) ab initio quantum mechanical predictions of cation-pi binding abilities and (ii) EC50 values for acetylcholine at the receptor for a series of tryptophan derivatives that were incorporated into the receptor by using the in vivo nonsense-suppression method for unnatural amino acid incorporation. Such a correlation is seen at one, and only one, of the aromatic residues-tryptophan-149 of the alpha subunit. This finding indicates that, on binding, the cationic, quaternary ammonium group of acetylcholine makes van der Waals contact with the indole side chain of alpha tryptophan-149, providing the most precise structural information to date on this receptor. Consistent with this model, a tethered quaternary ammonium group emanating from position alpha149 produces a constitutively active receptor.

G protein-gated inward rectifier K+ (GIRK) channels mediate hyperpolarizing postsynaptic potentials in the nervous system and in the heart during activation of Galpha(i/o)-coupled receptors. In neurons and cardiac atrial cells the time course for receptor-mediated GIRK current deactivation is 20-40 times faster than that observed in heterologous systems expressing cloned receptors and GIRK channels, suggesting that an additional component(s) is required to confer the rapid kinetic properties of the native transduction pathway. We report here that heterologous expression of "regulators of G protein signaling" (RGS proteins), along with cloned G protein-coupled receptors and GIRK channels, reconstitutes the temporal properties of the native receptor --> GIRK signal transduction pathway. GIRK current waveforms evoked by agonist activation of muscarinic m2 receptors or serotonin 1A receptors were dramatically accelerated by coexpression of either RGS1, RGS3, or RGS4, but not RGS2. For the brain-expressed RGS4 isoform, neither the current amplitude nor the steady-state agonist dose-response relationship was significantly affected by RGS expression, although the agonist-independent "basal" GIRK current was suppressed by approximately 40%. Because GIRK activation and deactivation kinetics are the limiting rates for the onset and termination of "slow" postsynaptic inhibitory currents in neurons and atrial cells, RGS proteins may play crucial roles in the timing of information transfer within the brain and to peripheral tissues.

A method for site-specific, nitrobenzyl-induced photochemical proteolysis of diverse proteins expressed in living cells has been developed based on the chemistry of the unnatural amino acid (2-nitrophenyl)glycine (Npg). Using the in vivo nonsense codon suppression method for incorporating unnatural amino acids into proteins expressed in Xenopus oocytes, Npg has been incorporated into two ion channels: the Drosophila Shaker B K+ channel and the nicotinic acetylcholine receptor. Functional studies in vivo show that irradiation of proteins containing an Npg residue does lead to peptide backbone cleavage at the site of the novel residue. Using this method, evidence is obtained for an essential functional role of the "signature" Cys128-Cys142 disulfide loop of the nAChR alpha subunit.

BACKGROUND: A key structural issue for all integral membrane proteins is the exposure of individual residues to the intracellular or extracellular media. This issue involves the basic transmembrane topology as well as more subtle variations in surface accessibility. Direct methods to evaluate the degree of exposure for residues in functional proteins expressed in living cells would be highly valuable. We sought to develop a new experimental method to determine highly surface-exposed residues, and thus transmembrane topology of membrane proteins expressed in Xenopus oocytes.
RESULTS: We have used the in vivo nonsense suppression technique to incorporate biotinylated unnatural amino acids into functional ion channels expressed in Xenopus oocytes. Binding of 125I-streptavidin to biotinylated receptors was used to determine the surface exposure of individual amino acids. In particular, we studied the main immunogenic region of the nicotinic acetylcholine receptor. The biotin-containing amino acid biocytin was efficiently incorporated into five sites in the main immunogenic region and extracellular streptavidin bound to one residue in particular, alpha 70. The position of alpha 70 as highly exposed on the receptor surface was thus established.
CONCLUSIONS: The in vivo nonsense suppression technique has been extended to provide the first in a potential series of methods to identify exposed residues and to assess their relative exposure in functional proteins expressed in Xenopus oocytes.

To provide material suitable for structural studies of the nicotinic acetylcholine receptor, we have expressed and purified the NH2-terminal extracellular domain of the mouse muscle alpha subunit. Several constructs were initially investigated using Xenopus oocytes as a convenient small scale expression system. A fusion protein (alpha210GPI) consisting of the 210 NH2-terminal amino acids of the alpha subunit and a glycosylphosphatidylinositol anchorage sequence conferred surface alpha-bungarotoxin binding in oocytes. Coexpression of alpha210GPI with an analogous construct made from the delta subunit showed no evidence of heterodimer formation. The alpha210GPI protein was chosen for large scale expression in transfected Chinese hamster ovary cells. The alpha210GPI protein was cleaved from these cells and purified on an immunoaffinity column. Gel and column chromatography show that the purified protein is processed as expected and exists as a monomer. The purified protein also retains the two distinct, conformation-specific binding sites expected for the correctly folded alpha subunit. Circular dichroism studies of alpha210GPI suggest that this region of the receptor includes considerable beta-sheet secondary structure, with a small proportion of alpha-helix.

We have studied the voltage-jump relaxation currents for a series of neuronal nicotinic acetylcholine receptors resulting from the coexpression of wild-type and chimeric beta 4/beta 2 subunits with alpha 3 subunits in Xenopus oocytes. With acetylcholine as the agonist, the wild-type alpha 3 beta 4 receptors displayed five- to eightfold slower voltage-jump relaxations than did the wild-type alpha 3 beta 2 receptors. In both cases, the relaxations could best be described by two exponential components of approximately equal amplitudes over a wide range of [ACh]'s. Relaxation rate constants increased with [ACh] and saturated at 20- to 30-fold lower concentrations for the alpha 3 beta 2 receptor than for the alpha 3 beta 4 receptor, as observed previously for the peak steady state conductance. Furthermore, the chimeric beta 4/beta 2 subunits showed a transition in the concentration dependence of the rate constants in the region between residues 94 and 109, analogous to our previous observation with steady state conductances. However, our experiments with a series of beta-subunit chimeras did not localize residues that govern the absolute value of the kinetic parameters. Hill coefficients for the relaxations also differed from those previously measured for steady state responses. The data reinforce previous conclusions that the region between residues 94 and 109 on the beta subunit plays a role in binding agonist but also show that other regions of the receptor control gating kinetics subsequent to the binding step.

Structure-function relations in the nicotinic acetylcholine receptor are probed using a recently developed method based on chemical synthesis of nonsense suppressor tRNAs with unnatural amino acid residues, site-directed incorporation at nonsense codons in Xenopus laevis oocytes, and electrophysiological measurements. A broad range of unnatural amino acids, as many as 14 at a given site, are incorporated at three sites, alpha 93, alpha 190, and alpha 198, all of which are tyrosine in the wild-type receptor and are thought to contribute to the agonist binding site. Confirming and expanding upon earlier studies using conventional mutagenesis, the three tyrosines are shown to be in substantially different structural microenvironments. In particular, a crucial role is established for the hydroxyl group of alpha Tyr93, whereas a variety of substituents are functional at the analogous position of alpha Tyr198. Interestingly, consideration of three different agonists (acetylcholine, nicotine, and tetramethylammonium) does not discriminate between these two best-characterized binding site residues. In addition, double-mutation studies establish the independent effects of mutations at the pore region (second transmembrane region) and at the agonist binding site, and this observation leads to a novel strategy for adjusting EC50 values. These results establish the broad generality and great potential of the unnatural amino acid methodology for illuminating subtle structural distinctions in neuroreceptors and related integral membrane proteins.

A nonsense suppression method was employed to incorporate a total of four natural and six unnatural residues at the 9' position of the M2 region in the beta, gamma, and delta subunits of muscle nicotinic receptors. In 33 pairwise comparisons of functional properties as influenced by structural features including side chain length, branching, and substitution of oxygen for methylene carbons, it is concluded that increased polarity in the side chains at the 9' position consistently increases the sensitivity to acetylcholine. In addition, the stereochemistry of the side chain can have marked influences on the EC50, primarily because of changes in the single-channel open time. For the case of isoleucine versus allo-isoleucine in the delta subunit, these changes are themselves modified by mutations at the 9' position in other subunits. The data suggest an especially strong interaction between the beta and delta subunits in the pore region, leading in turn to a suggested arrangement of subunits within the pentamer.

A new tRNA, THG73, has been designed and evaluated as a vehicle for incorporating unnatural amino acids site-specifically into proteins expressed in vivo using the stop codon suppression technique. The construct is a modification of tRNAGln(CUA) from Tetrahymena thermophila, which naturally recognizes the stop codon UAG. Using electrophysiological studies of mutations at several sites of the nicotinic acetylcholine receptor, it is established that THG73 represents a major improvement over previous nonsense suppressors both in terms of efficiency and fidelity of unnatural amino acid incorporation. Compared with a previous tRNA used for in vivo suppression, THG73 is as much as 100-fold less likely to be acylated by endogenous synthetases of the Xenopus oocyte. This effectively eliminates a major concern of the in vivo suppression methodology, the undesirable incorporation of natural amino acids at the suppression site. In addition, THG73 is 4-10-fold more efficient at incorporating unnatural amino acids in the oocyte system. Taken together, these two advances should greatly expand the range of applicability of the in vivo nonsense suppression methodology.

Cholinergic muscarinic, serotonergic, opioid and several other G-protein-coupled neurotransmitter receptors activate inwardly rectifying K+ channels of the GIRK family, slowing the heartbeat and decreasing the excitability of neuronal cells. Inhibitory modulation of GIRKs by G-protein-coupled receptors may have important implications in cardiac and brain physiology. Previously G alpha and G beta gamma subunits of heterotrimeric G proteins have both been implicated in channel opening, but recent studies attribute this role primarily to the G beta gamma dimer that activates GIRKs in a membrane-delimited fashion, probably by direct binding to the channel protein. We report here that free GTP gamma S-activated G alpha i 1, but not G alpha i 2 or G alpha i 3, potently inhibits G beta 1 gamma 2-induced GIRK activity in excised membrane patches of Xenopus oocytes expressing GIRK1. High-affinity but partial inhibition is produced by G alpha s-GTP gamma S. G alpha i 1-GTP gamma S also inhibits G beta 1 gamma 2-activated GIRK in atrial myocytes. Antagonistic interactions between G alpha and G beta gamma may be among the mechanisms determining specificity of G protein coupling to GIRKs.

The homozygous weaver mouse displays neuronal degeneration in several brain regions. Previous experiments in heterologous expression systems showed that the G protein-gated inward rectifier K+ channel (GIRK2) bearing the weaver pore-region GYG-to-SYG mutation (i) is not activated by G beta gamma subunits, but instead shows constitutive activation, and (ii) is no longer a K(+)-selective channel but conducts Na+ as well. The present experiments on weaverGIRK2 (wvGIRK2) expressed in Xenopus oocytes show that the level of constitutive activation depends on intracellular Na+ concentration. In particular, manipulations that decrease intracellular Na+ produce a component of Na(+)-permeable current activated via a G protein pathway. Therefore, constitutive activation may not arise because the weaver mutation directly alters the gating transitions of the channel protein. Instead, there may be a regenerative cycle of Na+ influx through the wvGIRK2 channel, leading to additional Na+ activation. We also show that the wvGIRK2 channel is permeable to Ca2+, providing an additional mechanism for the degeneration that characterizes the weaver phenotype. We further demonstrate that the GIRK4 channel bearing the analogous weaver mutation has properties similar to those of the wvGIRK2 channel, providing a glimpse of the selective pressures that have maintained the GYG sequence in nearly all known K+ channels.

We constructed chimeras of the rat beta 2 and beta 4 neuronal nicotinic subunits to locate the regions that contribute to differences between the acetylcholine (ACh) dose-response relationships of the alpha 3 beta 2 and alpha 3 beta 4 receptors. Expressed in Xenopus oocytes, the alpha 3 beta 2 receptor displays an EC50 for ACh approximately 20-fold less than the EC50 of the alpha 3 beta 4 receptor. The apparent Hill slope (n(app)) of alpha 3 beta 2 is near one whereas the alpha 3 beta 4 receptor displays an n(app) near two. Substitutions within the first 120 residues convert the EC50 for ACh from one wild-type value to the other. Exchanging just beta 2:104-120 for the corresponding region of beta 4 shifts the EC50 of ACh dose-response relationship in the expected direction but does not completely convert the EC50 of the dose-response relationship from one wild-type value to the other. However, substitutions in the beta 2:104-120 region do account for the relative sensitivity of the alpha 3 beta 2 receptor to cytisine, tetramethylammonium, and ACh. The expression of beta 4-like (strong) cooperativity requires an extensive region of beta 4 (beta 4:1-301). Relatively short beta 2 substitutions (beta 2:104-120) can reduce cooperativity to beta 2-like values. The results suggest that amino acids within the first 120 residues of beta 2 and the corresponding region of beta 4 contribute to an agonist binding site that bridges the alpha and beta subunits in neuronal nicotinic receptors.

Guanine nucleotide-binding proteins (G proteins) activate K+ conductances in cardiac atrial cells to slow heart rate and in neurons to decrease excitability. cDNAs encoding three isoforms of a G-protein-coupled, inwardly rectifying K+ channel (GIRK) have recently been cloned from cardiac (GIRK1/Kir 3.1) and brain cDNA libraries (GIRK2/Kir 3.2 and GIRK3/Kir 3.3). Here we report that GIRK2 but not GIRK3 can be activated by G protein subunits G beta 1 and G gamma 2 in Xenopus oocytes. Furthermore, when either GIRK3 or GIRK2 was coexpressed with GIRK1 and activated either by muscarinic receptors or by G beta gamma subunits, G-protein-mediated inward currents were increased by 5- to 40-fold. The single-channel conductance for GIRK1 plus GIRK2 coexpression was intermediate between those for GIRK1 alone and for GIRK2 alone, and voltage-jump kinetics for the coexpressed channels displayed new kinetic properties. On the other hand, coexpression of GIRK3 with GIRK2 suppressed the GIRK2 alone response. These studies suggest that formation of heteromultimers involving the several GIRKs is an important mechanism for generating diversity in expression level and function of neurotransmitter-coupled, inward rectifier K+ channels.

In nicotinic acetylcholine receptors (nAChR), as well as glycine, GABAA (gamma-aminobutyric acid), serotonin (5-HT3), and GluCl glutamate receptors, a leucine residue at the approximate midpoint of the M2 transmembrane domain (the 9' position) is conserved across most known subunits. Structural data for the nAChR suggest that the Leu 9' residues occupy a 'kink' in each of the five M2 helices and point into the closed channel; in the opening step, the M2 helices rotate so that Leu 9' side chains no longer occlude the conduction pathway. Mutation of Leu 9' to one of several other residues slows desensitization and increases sensitivity to agonist. We have exploited the alpha 2 beta gamma delta stoichiometry of muscle nAChR to express receptors with ms* = 0 to 5 Leu 9'Ser mutated subunits. Strikingly, each Leu 9'Ser mutation shifts the dose-response relation for ACh to the left by approximately 10-fold; a nAChR with ms* = 4 is 10(4)-fold more sensitive than the wild type. The results suggest that each of the five Leu 9' residues participates independently and symmetrically in a key step in the structural transition between the closed and open states.

An essential function of myelinating oligodendroglia in the mammalian central nervous system is the regulation of extracellular potassium levels by means of a prominent inwardly rectifying K+ current. Cardiac and neuronal K+ inward rectifiers are either activated by hyperpolarizing voltages or controlled by neurotransmitters through the action of receptor-activated G proteins. Neuromodulation of inward rectifiers has not previously been considered as a way to regulate oligodendrocyte function. Here we report the expression of serotonin, somatostatin and muscarinic acetylcholine G protein-coupled receptors in rat brain oligodendrocytes. Activation of these receptors leads to pertussis toxin-sensitive inhibition of inwardly rectifying K+ channels within < 1 s. By contrast, in the heart and in neurons, similar pathways activate an inwardly rectifying conductance. Thus, transmitter-mediated blockade of inward rectifiers appears to be an oligodendrocyte-specific variation of a common motif for convergent signalling pathways. In vivo, expression of this mechanism, which may be dependent on neuron-glia signalling, may have a regulatory role in K+ homeostasis during neuron activity in the central nervous system.

Injection of rat atrial RNA into Xenopus oocytes resulted in the expression of guanine nucleotide binding (G) protein-activated K+ channel. Current through the channel could be activated by acetylcholine or, if RNA encoding a neuronal 5HT1A receptor was coinjected with atrial RNA, by serotonin (5HT). A 5HT-evoked current (I5HT) was observed in oocytes injected with ventricle RNA fractions (of 2.5-5.5 kb) and 5HT1A receptor RNA. I5HT displayed strong inward rectification with very little conductance above the K+ equilibrium potential, was highly selective for K+ over Na+, and was blocked by 5-300 microM Ba2+. I5HT was suppressed by intracellular injection of the nonhydrolyzable analog of GDP, guanosine 5'-[beta-thio]diphosphate, but not by treatment with pertussis toxin (PTX), suggesting coupling of the receptor to the G-protein-activated K+ channel via a PTX-insensitive G protein, possibly endogenously present in the oocyte. Coexpression of the alpha subunit of a PTX-sensitive G protein, G(i2), rendered I5HT sensitive to PTX inhibition. Native oocytes displayed a constitutively active inwardly rectifying K+ current with a lower sensitivity to Ba2+ block; expression of a similar current was also directed by atrial or ventricle RNA of 1.5-3 kb. Xenopus oocytes may be employed for cloning of the G-protein-activated K+ channel cDNA and for studying the coupling between this channel and G proteins.

1. Co-injection of RNA synthesized from cloned neuronal acetylcholine receptor (nAChR) subunits (alpha 4 and beta 2) in Xenopus oocytes produced functional receptors. In macroscopic voltage-clamp experiments, the agonist-induced current exhibited a strong inward rectification. 2. Voltage jumps from +50 mV to more negative potentials produced relaxations of the agonist-induced current with a single exponential time course. The relaxation rate constant was only weakly voltage dependent. 3. At the single-channel level, three conductances were recorded of 12, 22 and 34 pS. Their burst durations were similar and varied only weakly with voltage (e-fold for 120 to 370 mV), consistent with the poorly voltage-dependent relaxation rate constants. However, the burst durations were less than 10 ms, or less than 1/5 the value expected from voltage-jump relaxations. 4. Hexamethonium (Hex, 0.5 to 8 microM) inhibited the agonist-induced current and produced voltage-jump relaxations characterized by a rapid conductance increase and a slower conductance decrease. Analysis of these relaxations suggested that the Hex-receptor interaction is open-channel blockade characterized by a forward binding rate of 1 x 10(7) M-1 s-1 and a dissociation rate constant of about 25 s-1. 5. For the relaxations produced by QX222, the slowest phase was a conductance increase, suggesting that the dissociation rate constant for QX222 is 10-30-fold greater than for Hex. 6. Hex but not QX222 produced an additional use-dependent blockade that was manifest during repetitive hyperpolarizing pulses. 7. With mouse muscle ACh receptors expressed in oocytes, the blockade by Hex did not depend strongly on voltage. Neither Hex nor QX222 produced appreciable use-dependent block on muscle ACh receptors. 8. Of the four conditions studied (neuronal and muscle receptors, Hex and QX222), only the blockade of the neuronal AChR by Hex is characterized by a residence time longer than the normal open time. 9. It is concluded that the modest differences in primary amino acid sequence between muscle and neuronal receptors lead to profound changes in their interactions with channels blockers.

The nicotinic acetylcholine receptor can be expressed in Xenopus oocytes by injection of in vitro synthesized RNA for the alpha, beta, gamma, and delta mouse muscle subunits. However, detectable responses can also be obtained by injection of alpha, beta, and delta subunit RNA only. The receptors expressed in this case (gamma-less receptors) share many of the properties of the normal receptor, including relaxation time constants, Hill slope, and relative permeability for Na+, K+, Cs+, and Tris+. The major single-channel conductances of alpha beta gamma delta and alpha beta delta receptors are similar (34.2 +/- 2.9 and 38.5 +/- 0.6 pS, respectively) but clearly different from the major conductances seen after the combined injection of alpha beta delta mouse subunit RNA and Xenopus gamma subunit RNA. Mutations in the second transmembrane segment of the alpha and beta subunits, known to affect open time and blockade by QX-222, are equally effective in the gamma-less receptor. These data strongly suggest that the gamma-less receptor has the same pore diameter as the normal receptor and that alpha, beta, and delta subunits participate in its formation. Injection of alpha beta gamma delta well as alpha beta delta RNA produced additional subconductance states of around 25 pS. The low conductance state was sensitive to mutations introduced in the alpha or beta subunits with or without the gamma subunit, indicating that this channel did not need the gamma subunits but required at least the alpha and beta subunits to be produced. Injection of alpha beta delta and the adult-type epsilon subunit RNA gave rise to channels with conductances of 35 and 55 pS when the stoichiometry of the injection was 2:1:1:1, but only the 55-pS channel was recorded when the epsilon subunit RNA concentration was increased by 10-fold (stoichiometry of 2:1:1:10). The gamma-less receptor can thus be expressed even when the adult epsilon subunit is present. Whether gamma-less receptors are expressed at normal adult neuromuscular junctions remains unknown.

Tris+/Na+ permeability ratios were measured from shifts in the biionic reversal potentials of the macroscopic ACh-induced currents for 3 wild-type (WT), 1 hybrid, 2 subunit-deficient, and 25 mutant nicotinic receptors expressed in Xenopus oocytes. At two positions near the putative intracellular end of M2, 2' (alpha Thr244, beta Gly255, gamma Thr253, delta Ser258) and -1', point mutations reduced the relative Tris+ permeability of the mouse receptor as much as threefold. Comparable mutations at several other positions had no effects on relative Tris+ permeability. Mutations in delta had a greater effect on relative Tris+ permeability than did comparable mutations in gamma; omission of the mouse delta subunit (delta 0 receptor) or replacement of mouse delta with Xenopus delta dramatically reduced relative Tris+ permeability. The WT mouse muscle receptor (alpha beta gamma delta) had a higher relative permeability to Tris+ than the wild-type Torpedo receptor. Analysis of the data show that (a) changes in the Tris+/Na+ permeability ratio produced by mutations correlate better with the hydrophobicity of the amino acid residues in M2 than with their volume; and (b) the mole-fraction dependence of the reversal potential in mixed Na+/Tris+ solutions is approximately consistent with the Goldman-Hodgkin-Katz voltage equation. The results suggest that the main ion selectivity filter for large monovalent cations in the ACh receptor channel is the region delimited by positions -1' and 2' near the intracellular end of the M2 helix.

We measured the permeability ratios (PX/PNa) of 3 wild-type, 1 hybrid, 2 subunit-deficient, and 22 mutant nicotinic receptors expressed in Xenopus oocytes for alkali metal and organic cations using shifts in the bi-ionic reversal potential of the macroscopic current. Mutations at three positions (2', 6', 10') in M2 affected ion selectivity. Mutations at position 2' (alpha Thr244, beta Gly255, gamma Thr253, delta Ser258) near the intracellular end of M2 changed the organic cation permeability ratios as much as twofold and reduced PCs/PNa and PK/PNa by 16-18%. Mutations at positions 6' and 10' increased the glycine ethyl ester/Na+ and glycine methyl ester/Na+ permeability ratios. Two subunit alterations also affected selectivity: omission of the delta subunit reduced PCs/PNa by 16%, and substitution of Xenopus delta for mouse delta increased Pguanidinium/PNa more than twofold and reduced PCs/PNa by 34% and PLi/PNa by 20%. The wild-type mouse receptor displayed a surprising interaction with the primary ammonium cations; relative permeability peaked at a chain length equal to four carbons. Analysis of the organic permeability ratios for the wild-type mouse receptor shows that (a) the diameter of the narrowest part of the pore is 8.4 A; (b) the mouse receptor departs significantly from size selectivity for monovalent organic cations; and (c) lowering the temperature reduces Pguanidinium/PNa by 38% and Pbutylammonium/PNa more than twofold. The results reinforce present views that positions -1' and 2' are the narrowest part of the pore and suggest that positions 6' and 10' align some permeant organic cations in the pore in an interaction similar to that with channel blocker, QX-222.

Fifteen chimeric nicotinic receptor beta subunits were constructed consisting of N-terminal neuronal beta 4 sequences and C-terminal beta 2 sequences. Responses to cytisine, nicotine, or tetramethylammonium were compared to acetylcholine responses for these subunits expressed in Xenopus oocytes with alpha 3 subunits. The results show that (i) two residues in the extracellular domain of chimeric beta 4.beta 2 subunits (108 beta 2F/beta 4V, 110 beta 2S/beta 4T) account for much of the relative cytisine sensitivity; and (ii) four extracellular residues of chimeric beta 4.beta 2 subunits (112 beta 2A/beta 4V, 113 beta 2V/beta 4I and 115 beta 2S/beta 4R, 116 beta 2Y/beta 4S) account for most of the relative tetramethylammonium sensitivity. The data did not permit localization of nicotine sensitivity to any particular region.

In cardiac atrial cells, muscarinic acetylcholine receptors activate a K+ current directly via a guanine nucleotide-binding protein (G protein). Serotonin type 1A receptors may activate a similar pathway in hippocampal neurons. To develop a system in which receptor/G protein/K+ channel coupling can be experimentally manipulated, we have used a highly efficient recombinant vaccinia virus vector system to express human serotonin 1A receptors in primary cultures of rat atrial myocytes. The expressed 1A receptors activated the inwardly rectifying K+ conductance that is normally activated by the endogenous muscarinic acetylcholine receptors. Maximal responses to either agonist occluded further activation by the other agonist. The average activation time constants for serotonin were about 5 times slower than for acetylcholine. The data support suggestions that the intracellular signaling pathway from seven-helix receptors to G proteins and directly to ion channels is widespread in excitable cells. After a fraction of the G proteins are activated irreversibly by guanosine 5'-[gamma-thio]triphosphate, subsequent transduction proceeds more efficiently. One possible interpretation is that multiple G-protein molecules are required to activate each channel. Vaccinia virus expression vectors are thus useful for expressing seven-helix receptors in primary cultures of postmitotic cells and have provided a heterologous expression system for the signaling pathway from seven-helix receptors to G proteins and directly to ion channels.

We have been examining the interaction of a local anesthetic derivative, QX-222, with the ion channel pore of the muscle AChR, using a combination of mutagenesis, oocyte expression, and electrophysiology. Single channel recording, together with macroscopic voltage-jump relaxations, provides a measure of the residence time of the open channel blocker within the pore. We have found systematic changes in the apparent affinity of the open channel for QX-222 following amino acid substitutions in the proposed M2 transmembrane helix of each of the four subunits of the AChR. Assigning the number 1' to the residue at the cytoplasmic end of the M2 helix, positions 2',6',10',14', and 18' are modeled as forming the lining of the pore. Polar to nonpolar substitutions at 6' decrease QX-222 residence time, while the opposite effect is seen at position 10'. Nonpolar to polar substitutions have the converse effect. The distance between the aromatic and quaternary amine moieties of QX-222 corresponds almost exactly to the repeat distance of an alpha helix. This structural feature is common to many local anesthetic drugs. We propose a model for the binding of QX-222 within the ion channel of the AChR that is consistent with these observations.

Serotonin 5-HT1c and acetylcholine M1 receptors activate phosphoinositidase, resulting in an increased formation of IP3 and 1,2 diacylglycerol. In Xenopus oocytes injected with mRNA encoding either of these receptors, Ca2+ released from intracellular stores in response to IP3 then opens Ca(2+)-gated Cl-channels. In the present experiments, oocytes expressing a transcript from a cloned mouse serotonin 5-HT1c receptor were exposed to identical 15-s pulses of agonist, administered 2 min apart; the second current response was two to three times that of the first. However, in those oocytes coinjected with the 5-HT1c receptor transcript and a low molecular weight fraction (0.3-1.5 kb) of rat brain mRNA, the second current response was approximately 50% of the first. Thus, the low molecular weight RNA encodes a protein (or proteins) that causes desensitization. Experiments using fura-2 or a Ca(2+)-free superfusate indicated that desensitization of the 5-HT1c receptor response does not result from a sustained elevation of intracellular Ca2+ level or require the entry of extracellular Ca2+. Photolysis of caged IP3 demonstrated that an increase in IP3 and a subsequent rise in Ca2+ do not produce desensitization of either the IP3 or 5-HT1c peak current responses. Furthermore, in oocytes coinjected with the low molecular weight RNA and a transcript from the rat M1 acetylcholine receptor, the M1 current response was greatly attenuated. Our data suggest that the proteins involved in attenuation of the M1 current response and desensitization of the 5-HT1c current response may be the same.

The cytoplasmic injection of mRNA synthesized in vitro into Xenopus oocytes is widely used for heterologous expression of ion channels and neurotransmitter receptors. We report two new methods for expression of ion channels and receptors in oocytes using vaccinia virus (VV). 1) A recombinant VV carrying the Shaker H4 K+ channel cDNA driven by the VV P7.5 early promoter was injected into oocytes. 2) A recombinant VV containing the bacteriophage T7 RNA polymerase driven by the P7.5 promoter was coinjected along with plasmids containing a T7 promoter and cDNAs for channels and receptors. The functionally expressed proteins include a) voltage-gated ion channels: the Shaker H4 K+ channel and the rat brain IIA Na+ channel, b) a ligand-gated ion channel: the mouse muscle nicotinic acetylcholine receptor (AChR), and c) a G protein-coupled receptor: the rat brain 5HT1C receptor. After virus/cDNA injection into oocytes, these channels and receptors generally showed characteristics and expression levels similar to those observed in mRNA-injected oocytes. However, the AChR expressed at lower levels in virus/cDNA-injected oocytes than in mRNA-injected oocytes. Because our methods bypass mRNA synthesis, they are more rapid and convenient than the mRNA injection method. Potential applications to structure-function studies and expression cloning are discussed.

This report analyzes the contribution of individual nicotinic acetylcholine receptor (AChR) subunits to the single-channel properties of the AChR ion channel. By in vitro synthesis of mRNA from cDNA clones encoding each AChR subunit (alpha, beta, gamma, and delta) from mouse BC3H-1 cells and Torpedo electric organ and microinjection of appropriate mRNA combinations into Xenopus oocytes, we studied the single-channel properties of both 'homologous' (all subunits from the same species) and 'hybrid' (subunits from both species) AChRs as they were expressed in the oocyte membrane. AChR expression was determined by surface binding of 125I-labeled alpha-bungarotoxin to intact oocytes, and those with binding sites of 1 fmol/cell or more were chosen for patch-clamp studies. Our results indicate the following: (1) Species difference in single-channel conductance can be explained largely by the charge distribution flanking the M2 transmembrane domain. (2) The alpha and delta subunits from mouse AChR independently lengthen the channel open time, in some cases by 10-fold; the beta subunit from mouse shortens the channel open time; the mouse gamma subunit lengthens open time less dramatically. (3) Voltage sensitivity, as measured by the ratio of channel open times at -60 mV and +60 mV, is influenced by the beta and delta subunits, in agreement with our previous study by two-electrode voltage-clamp recording. We conclude that single-channel properties of the AChR are governed by multiple elements located on different AChR subunits.

The binding site for an open-channel blocker, QX-222, at mouse muscle nicotinic acetylcholine receptors was probed using site-directed mutagenesis, oocyte expression, and electrophysiological analysis. The proposed cytoplasmic end of the M2 transmembrane helix is termed position 1'. At position 10' (alpha S252, beta T263, gamma A261, delta A266), Ala residues yield stronger and longer binding of QX-222 than Ser or Thr residues. These effects are opposite and roughly equal (30%-50% per mutation) to previously reported effects at position 6'. The polar end of an anesthetic molecule seems to bind to the position 6' OH groups, which provide a water-like region; the nonpolar moiety is near position 10' and binds more strongly in a nonpolar environment. Interactions with adjacent OH-rich turns of an amphiphilic helix may explain the widespread blocking effects of local anesthetics at the conduction pore of ion channels.

The STE2 gene of the yeast Saccharomyces cerevisiae encodes a 431-residue polypeptide that has been shown by chemical cross-linking and genetic studies to be a component of the receptor for the peptide mating pheromone, alpha-factor. To demonstrate directly that the ligand binding site of the alpha-factor receptor is comprised solely of the STE2 gene product, the STE2 protein was expressed in Xenopus oocytes. Oocytes microinjected with synthetic STE2 mRNA displayed specific surface binding for 35S-labeled alpha-factor (up to 40 sites/micron2/ng RNA). Oocytes injected with either STE2 antisense RNA or heterologous receptor mRNA (nicotinic acetylcholine receptor alpha, beta, gamma, and delta subunit mRNAs) showed no binding activity (indistinguishable from uninjected control oocytes). The apparent KD (7 nM) of the alpha-factor binding sites expressed on the oocyte surface, determined by competition binding studies, agreed with the values reported for intact yeast cells and yeast plasma membrane fractions. These findings demonstrate that the STE2 gene product is the only yeast polypeptide required for biogenesis of a functional alpha-factor receptor. Electrophysiological measurements indicated that the membrane conductance of oocytes injected with STE2 mRNA, or with both STE2 and GPA1 (encoding a yeast G protein alpha-subunit) mRNAs, did not change and was not affected by pheromone binding. Thus, the alpha-factor receptor, like mammalian G protein-coupled receptors, apparently lacks activity as an intrinsic or ligand-gated ion channel. This report is the first instance in which a membrane-bound receptor from a unicellular eukaryote has been expressed in a vertebrate cell.

Site-directed mutagenesis and expression in Xenopus oocytes were used to study acetylcholine receptors in which serine residues (i) were replaced by alanines (alpha, delta subunits) or (ii) replaced a phenylalanine (beta subunit) at a postulated polar site within the M2 transmembrane helix. As the number of serines decreased, there were decreases in the residence time and consequently the equilibrium binding affinity of QX-222, a quaternary ammonium anesthetic derivative thought to bind within the open channel. Receptors with three serine-to-alanine mutations also displayed a selective decrease in outward single-channel currents. Both the direction of this rectification and the voltage dependence of QX-222 blockade suggest that the residues mutated are within the aqueous pore of the receptor and near its cytoplasmic (inner) surface.

In this study, in vitro synthesized mRNA encoding mouse and Torpedo nicotinic acetylcholine receptor subunits was injected into Xenopus oocytes, followed by assays for assembly onto the oocyte surface (using [125I]alpha-bungarotoxin binding) and for acetylcholine-induced conductances (using voltage clamp). We constructed hybrid acetylcholine receptors in Xenopus oocytes by injecting all 8 possible combinations of 4 subunit-specific mRNAs in which a single subunit is derived from the other species. For each hybrid combination, there is detectable assembly and conductance. We also constructed cDNA clones that encode chimeric acetylcholine receptor subunits in which part of the gamma subunit from Torpedo was replaced by the homologous region of the delta subunit from mouse. None of the chimeric subunits was able to replace the Torpedo gamma, mouse delta, or Torpedo delta subunit with regard to assembly or function. We therefore conclude that widely spaced (and unknown) parts of the protein chain are required for the intersubunit interactions that eventually lead to functional assembly of the receptor.

This study used messenger RNA encoding each subunit (alpha, beta, gamma and delta) of the nicotinic acetylcholine (ACh) receptor from mouse BC3H-1 cells and from Torpedo electric organ. The mRNA was synthesized in vitro by transcription with SP6 polymerase from cDNA clones. All 16 possible combinations that include one mRNA for each of alpha, beta, gamma, and delta were injected into oocytes. After allowing 2-3 d for translation and assembly, we assayed each oocyte for (a) receptor assembly, measured by the binding of [125I]alpha-bungarotoxin to the oocyte surface, and (b) ACh-induced conductance, measured under voltage clamp at various membrane potentials. All combinations yielded detectable assembly (30-fold range among different combinations) and ACh-induced conductances (greater than 1,000-fold range at 1 microM). On double-logarithmic coordinates, the dose-response relations all had a slope near 2 for low concentrations of ACh. Data were corrected for variations in efficiency of translation among identically injected oocytes by expressing ACh-induced conductance per femtomole of alpha-bungarotoxin-binding sites. Five combinations were tested for d-tubocurarine inhibition by the dose-ratio method; the apparent dissociation constant ranged from 0.08 to 0.27 microM. Matched responses and geometric means are used for describing the effects of changing a particular subunit (mouse vs. Torpedo) while maintaining the identity of the other subunits. A dramatic subunit-specific effect is that of the beta subunit on voltage sensitivity of the response: gACh(-90 mV)/gACh(+30 mV) is always at least 1, but this ratio increases by an average of 3.5-fold if beta M replaces beta T. Also, combinations including gamma T or delta M usually produce greater receptor assembly than combinations including the homologous subunit from the other species. Finally, EACh is defined as the concentration of ACh inducing 1 microS/fmol at -60 mV; EACh is consistently lower for alpha M. We conclude that receptor assembly, voltage sensitivity, and EACh are governed by different properties.

Whole-cell or single-channel currents through acetylcholine (ACh) receptor channels were studied in voltage-clamped rat myoballs or in excised membrane patches from myoballs. The recording pipette contained CsCl to suppress outward currents, and tetrodotoxin was used to help suppress Na+ currents. To minimize problems associated with bath applied agonists, myoballs were bathed in a solution containing the inactive (cis) isomer of the photo-isomerizable azobenzene derivative, Bis-Q. Calibrated light flashes of varying intensity were presented to produce concentration jumps of agonist, trans-Bis-Q. The resulting whole-cell current relaxations through ACh channels approach a steady state along an exponential time course, then decline as the newly created agonist diffuses away over the next few seconds. The dose-response relationship was inferred from Hill (double-log) plots for myoballs bathed in 500 nM-cis-Bis-Q at three membrane potentials. At low agonist concentrations (less than 300 nM-trans-Bis-Q), the slope of the Hill plot averaged 1.62 at -150 mV, 1.89 at -100 mV, and 2.05 at +80 mV. These results are consistent with an apparent agonist affinity constant that decreases with membrane depolarization and shifts the responses further down on the dose-response curve. When the myoballs were bathed in higher concentrations of cis-Bis-Q (1.5-20 microM), the slope of the Hill plot was reduced at all membrane potentials, although it was still closer to two at positive potentials. This is expected from the known sigmoid shape of the dose-response relation. The shallow dependence of the Hill slope on agonist concentration suggests the presence of negative cooperativity in the over-all binding of agonist molecules. Following treatment of the membrane with dithiothreitol to reduce disulphide groups, the Hill slope for the reversibly bound agonist, trans-Bis-Q, remained near two. The kinetics of currents at hyperpolarized membrane potentials became complicated at higher agonist concentrations in a manner that was consistent with open-channel block by trans-Bis-Q; the currents showed a slow secondary increase in conductance. Averaged single-channel recordings at higher agonist concentrations resemble macroscopic relaxations under comparable conditions. Furthermore, those recordings also suggested that open channels are blocked by trans-Bis-Q at concentrations greater than 2 microM; the block depends strongly on membrane potential and increases with hyperpolarization. Currents at positive membrane potentials showed no evidence of open-channel block.

Kinetic and equilibrium aspects of receptor activation by two irreversibly bound ('tethered') agonists, QBr and bromoacetylcholine (BrACh), were examined in cultured embryonic rat muscle. Myoballs were treated with dithiothretitol (2 mM), washed, exposed to BrACh or QBr, and then washed again. Voltage-clamp recordings were made both in the whole-cell mode and with excised outside-out patches at 15 degrees C. Whole-cell voltage-jump relaxations resembled those observed with reversibly bound agonists. The relaxation time constants were 5 ms for tethered QBr and 10 ms for tethered BrACh (-100 mV, 15 degrees C). At more positive membrane potentials, the relaxation rate constants increased and the conductance decreased. Whole-cell light-flash relaxations with tethered QBr were also studied. The conductance was increased and decreased, respectively, by cis----trans and trans----cis photoisomerizations. The relaxation time constants equalled those for voltage jumps. The functional stoicheiometry of tethered QBr was investigated by studying the relaxations in response to light flashes that produced known changes in the mole fractions of the two isomers. It is concluded that the open state of each receptor channel is controlled by the isomeric state of a single tethered QBr molecule. In single-channel recordings, tethered agonists opened channels with the same conductance as reversibly bound agonists (30 pS at 15 degrees C and -100 mV). More than 80% of the conductance was contributed by a population of openings with an average burst duration (lifetime) of 5 ms for QBr and 10 ms for BrACh. Thus the single-channel and macroscopic currents seem to be dominated by the same type of channel; these are presumably monoliganded receptors. About 30% of the openings belonged to a population with an average lifetime of about 0.5 ms. This population contributed less than 5% of the conductance. There were also more long openings (greater than 50 ms) than expected from a simple exponential distribution. A few patches from BrACh-treated cells showed openings with a conductance of 45 pS (-100 mV) and an average duration of approximately 2 ms. These data allow one to assess whether the agonist-receptor binding step plays a role in generating the brief openings. The main population of openings (burst durations 5 ms with QBr and 10 ms with BrACh) seem to be contributed by monoliganded receptors. One can therefore rule out the hypothesis that the brief channels arise exclusively from mono- and biliganded receptors, respectively.

Injection of poly(A)+ RNA from rat brain into Xenopus oocytes caused the appearance of Cl currents in response to serotonin (5-HT) and acetylcholine (ACh). Both neurotransmitters evoked two-component currents similar in their time course to the oocyte's endogenous cholinergic muscarinic response, which was shown in previous studies to be mediated by IP3 synthesis leading to Ca release from intracellular stores. The responses to ACh and 5-HT exhibited self- and cross-desensitization, i.e., application of either ACh or 5-HT inhibited the subsequent response to either one of the two transmitters. Intracellular injection of guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) mimicked the 5-HT and ACh response, and also completely suppressed the response to the subsequent application of either ACh or 5-HT. Treatment of the oocytes with pertussis toxin (PTX) caused a 50% attenuation of ACh and 5-HT responses. In the membranes of both control and mRNA-injected oocytes, PTX catalyzed the ADP-ribosylation of a single Mr = approximately 40,000 protein. Injection of the purified beta gamma-subunits of transducin enhanced the 5-HT response. The 5-HT and GTP-gamma-S responses were inhibited by intracellular injection of the Ca2+ chelator, EGTA, as previously shown for the ACh response. These data suggest that ACh and 5-HT receptors, synthesized in the oocytes on the template of brain mRNA, act through a common pathway that involves (a) a guanine nucleotide binding protein and (b) IP3 production leading to Ca mobilization.

Calcium ions flow into cells through several distinct classes of voltage-dependent calcium-selective channels. Such fluxes play important roles in electrical signaling at the cell membrane and in chemical signaling within cells. Further information about calcium channels was obtained by injecting RNA isolated from rat brain, heart and skeletal muscle into Xenopus oocytes. Macroscopic currents through voltage-operated calcium channels were resolved when the endogenous calcium-dependent chloride current was blocked by replacing external calcium with barium and chloride with methanesulfonate. The resulting barium current was insensitive to tetrodotoxin but was completely blocked by cadmium or cobalt. With both heart and brain RNA at least two distinct types of calcium ion conductance were found, distinguishable by their time course and inactivation properties. In oocytes injected with heart RNA, the slowly inactivating component was selectively blocked by the calcium-channel antagonist nifedipine. Barium ion currents induced by heart RNA were modulated by isoproterenol, cyclic adenosine monophosphate, and acetylcholine.

The photochemical properties of the azobenzene derivative, Bis-Q, were exploited to carry out an agonist concentration jump followed by a molecular rearrangement of bound agonist molecules at acetylcholine (ACh) receptor channels of voltage-clamped rat myoballs. Myoballs were bathed in solutions containing low concentrations of cis-Bis-Q, the inactive isomer. Whole-cell current relaxations were studied following a light flash that produced a concentration jump of agonist, trans-Bis-Q, followed by a second flash that produced net trans----cis photoisomerizations of Bis-Q molecules. The concentration-jump relaxation provided a measure of the mean burst duration for ACh receptor channels occupied by trans-Bis-Q (7.7 ms, 22 degrees C). The second current relaxation was a more rapid conductance decrease (phase 1, tau = 0.8 ms). Phase 1 may represent either the burst duration for receptors initially occupied by a single cis- and a single trans-Bis-Q molecule or that for unliganded receptors. Single-channel current recordings from excised outside-out membrane patches showed that single channels open following an agonist concentration jump comparable to that used in the whole-cell experiments; when many such records were averaged, a synthetic macroscopic relaxation was produced. Individual open channels closed faster following a flash that promoted trans----cis photoisomerizations of the bound ligand, thus confirming the whole-cell observations of phase 1.

In Xenopus laevis oocytes, adenosine and other purinergic agonists induce a K+-conductance increase that is fully mimicked by intracellular application of cAMP. Acetylcholine suppresses the K+-conductance increase caused by adenosine, by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, or by intracellular injection of cAMP. This effect of acetylcholine is not mimicked by intracellular injection of Ca2+ or of the Ca-mobilizing agent inositol 1,4,5-trisphosphate. However, adenosine and cAMP responses are inhibited by 4 beta-phorbol 12,13-dibutyrate and 4 beta-phorbol 12-myristate 13-acetate. These results suggest that, in Xenopus oocytes, the muscarinic inhibition of purinergic and cAMP responses is mediated through the activation of the phospholipid-dependent, Ca-activated protein kinase (protein kinase C).

Voltage-jump and light-flash experiments have been performed on isolated Electrophorus electroplaques exposed simultaneously to nicotinic agonists and to the photoisomerizable compound 2,2'-bis-[alpha-(trimethylammonium)methyl]-azobenzene (2BQ). Dose-response curves are shifted to the right in a nearly parallel fashion by 2BQ, which suggests competitive antagonism; dose-ratio analyses show apparent dissociation constants of 0.3 and 1 microM for the cis and trans isomers, respectively. Flash-induced trans----cis concentration jumps produce the expected decrease in agonist-induced conductance; the time constant is several tens of milliseconds. From the concentration dependence of these rates, we conclude that the association and dissociation rate constants for the cis-2BQ-receptor binding are approximately 10(8) M-1 s-1 and 60 s-1 at 20 degrees C; the Q10 is 3. Flash-induced cis----trans photoisomerizations produce molecular rearrangements of the ligand-receptor complex, but the resulting relaxations probably reflect the kinetics of buffered diffusion rather than of the interaction between trans-2BQ and the receptor. Antagonists seem to bind about an order of magnitude more slowly than agonists at nicotinic receptors.

The nicotinic acetylcholine (AcCho) receptor (AcChoR) is a multisubunit protein complex of stoichiometry alpha 2 beta gamma delta. The several subunits show homology with each other within a given species; in addition, homology is found between analogous subunits between species. We have used the phage SP6 RNA polymerase transcription system to produce single-species RNA in vitro for various AcChoR subunits from cDNAs. Injection of an equimolar mixture of RNA for the alpha, beta, gamma, and delta subunits of Torpedo californica AcChoR into Xenopus oocytes results in the appearance of functional receptors in the oocyte membrane. No response to AcCho is detected when the beta or gamma subunit RNA is omitted, and a small response is seen when the delta subunit RNA is omitted. Replacement of Torpedo delta subunit RNA by the mouse BC3H-1 cell line AcChoR delta subunit RNA leads to the formation of functional receptors that show a 3-4-fold greater response to AcCho than does the full Torpedo complex. No response is seen when the mouse delta RNA replaces Torpedo gamma RNA. By amino acid homology profile comparisons, the mouse delta subunit appears to be moderately but not highly similar to the Torpedo delta subunit; the apparent similarity to the Torpedo gamma subunit is only slightly less. Therefore, the features of the primary sequence that determine the functional delta character of the mouse polypeptide are not revealed by simple homology comparisons.

The cis and trans isomers of the photoisomerizable compound, 3,3'-bis-[alpha-(trimethylammonium)methyl]azobenzene (Bis-Q), were purified by high-performance liquid chromatography using the ion-pair partitioning technique on a reverse-phase column. Solutions of cis-Bis-Q are stable at -20 degrees; at 25 degrees, thermal isomerization proceeds at a rate of 0.65%/day. cis-Bis-Q is less than 1% as potent a nicotinic agonist as the trans configuration. At concentrations of 1.5 microM or less, cis-Bis-Q exerts little or no blockade of the conductances induced by agonists. In voltage-clamped Electrophorus electroplaques exposed to cis-Bis-Q, laser flashes induce cis leads to trans photoisomerizations and increase the agonist-induced current by a factor of 20 within a few milliseconds.

Lysed Torpedo synaptosomes or washed synaptosomal membranes were incubated with [32P]NAD+ and subjected to electrophoresis on SDS-polyacrylamide gels. More than eight membrane proteins were ADP-ribosylated. The most intensely labeled proteins were those of Mr = 62,000 and 82,000. Radiolabeling was more intense in synaptosomes than in other subcellular fractions. Cholera toxin caused ribosylation of additional synaptosomal proteins with Mr = 42,000 and (in some preparations) 49,000. Neither endogenous nor cholera toxin-catalyzed ADP-ribosylation required added guanyl nucleotides. Cholera toxin increased the adenylate cyclase activity of synaptosomal membranes, suggesting that the cholera toxin substrates are regulatory components of adenylate cyclase in these synaptosomes.

These experiments employ the photoisomerizable compound, 3,3'-bis-[alpha-(trimethylammonium)methyl]azobenzene (Bis-Q), to study the response to muscarinic agents in frog myocardium. In homogenates from the heart, trans-Bis-Q blocks the binding of [3H]-N-methylscopolamine to muscarinic receptors. In voltage-clamped atrial trabeculae, trans-Bis-Q blocks the agonist-induced potassium conductance. The equilibrium dose-response curve for carbachol is shifted to the right, suggesting competitive blockade. Both the biochemical and electrophysiological data yield a dissociation constant of 4-5 microM for trans-Bis-Q; the cis configuration is severalfold less potent as a muscarinic blocker. Voltage-clamped preparations were exposed simultaneously to carbachol and Bis-Q and were subjected to appropriately filtered flashes (less than 1 ms duration) from a xenon flashlamp. Trans leads to cis and cis leads to trans photoisomerizations cause small (less than 20%) increases and decreases, respectively, in the agonist-induced current. The relaxation follows an S-shaped time course, including an initial delay or period of zero slope. The entire waveform is described by [1 - exp(-kt)]n. At 23 degrees C, k is approximately 3 s-1 and n is 2. Neither k nor n is affected when: (a) [Bis-Q] is varied between 5 and 100 microM; (b) [carbachol] is varied between 1 and 50 microM; (c) carbachol is replaced by other agonists (muscarine, acetylcholine, or acetyl-beta-methylcholine); or (d) the voltage is varied between the normal resting potential and a depolarization of 80 mV. However, in the range of 13-30 degrees C, k increases with temperature; the Q10 is between 2 and 2.5. In the same range, n does not change significantly. Like other investigators, we conclude that the activation kinetics of the muscarinic K+ conductance are not determined by ligand-receptor binding, but rather by a subsequent sequence of two (or more) steps with a high activation energy.

These experiments examine changes in the agonist-induced conductance that occur when the agonist-receptor complex is perturbed. Voltage-clamped Electrophorus electroplaques are exposed to the photoisomerizable agonist trans-Bis-Q. A 1-microsecond laser flash photoisomerizes some trans-Bis-Q molecules bound to receptors; because the cis configuration is not an agonist, receptor channels close within a few hundred microseconds. This effect is called phase 1. We compare (a) the fraction of channels that close during phase 1 with (b) the fraction of trans-Bis-Q molecules that undergo trans leads to cis photoisomerization. Parameter a is measured as the fractional diminution in voltage-clamp currents during phase 1. Parameter b is measured by changes in the optical spectra of Bis-Q solutions caused by flashes. At low flash intensities, a is twice b, which shows that the channel can be closed by photoisomerizing either of two bound agonist molecules. Conventional dose-response studies with trans-Bis-Q also give a Hill coefficient of two. As a partial control for changes in the photochemistry caused by binding of Bis-Q to receptors, spectral measurements are performed on the photoisomerizable agonist QBr, covalently bound to solubilized acetylcholine receptors from Torpedo. The bound and free agonist molecules have the same photoisomerization properties. These results verify the concept that the open state of the acetylcholine receptor channel is much more likely to be associated with the presence of two bound agonist molecules than with a single such molecule.

After disulphide bonds are reduced with dithiothreitol, trans-3- (alpha-bromomethyl)-3'-[alpha- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this "tethered agonist" shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 muM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to "open-channel blockade" bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3',bis-[alpha-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel's activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules.

1. The kinetics of tubocurarine inhibition were studied at the post-synaptic membrane of frog skeletal muscle fibres. Acetylcholine (ACh) and (+)-tubocurarine were ionophoresed from twin-barrel micropipettes, and the membrane potential of the muscle fibre was recorded intracellularly. Tubocurarine-receptor binding was measured by decreases in the response to identical pulses of ACh. 2. The responses to both ACh and tubocurarine had brief latencies and reached their maxima rapidly. It is suggested that under these conditions the kinetics of tubocurarine action are not slowed by diffusion in the space outside the synaptic cleft. 3. After a pulse of tubocurarine, recovery from inhibition proceeds along a roughly exponential time course with a rate constant, 1/tau off approximately equal to 0.5 sec-1. This rate constant does not depend on the maximal level of inhibition and varies only slightly with temperature (Q10 = 1.25). 4. After a sudden maintained increase in tubocurarine release, the ACh responses decrease and eventually reach a new steady-state level. Inhibition develops exponentially with time and the apparent rate constant, 1/tau on, is greater than 1/tau off. When the steady-state inhibition reduces the ACh response to 1/n of its original level, the data are summarized by the relation, 1/tau on = n(1/tau off). 5. When the ACh sensitivity is reduced with cobra toxin, both 1/tau on and 1/tau off increase. Thus, the kinetics of tubocurarine inhibition depend on the density of ACh receptors in the synaptic cleft. 6. After treatment with collagenase, part of the nerve terminal is displaced and the post-synaptic membrane is exposed directly to the external solution. Under these circumstances, 1/tau off increases more than tenfold. 7. Bath-applied tubocurarine competitively inhibits the responses to brief ionophoretic ACh pulses with an apparent equilibrium dissociation constant, K = 0.5 microM. 8. In denervated frog muscle fibres, extrasynaptic receptors have a lower apparent affinity for tubocurarine. After a pulse of tubocurarine, inhibition decays tenfold more rapidly at these extrasynaptic sites than at the synapse. 9. It is suggested that each tubocurarine molecule binds repeatedly to several ACh receptors before escaping from the synaptic from the synaptic cleft and that the probability of this repetitive binding is enhanced because the nerve terminal presents a physical barrier to diffusion out of the cleft. Consequently, the receptor transiently buffer the concentration of tubocurarine in the cleft, and the macroscopic kinetics of inhibition are much slower than the molecular binding rates for tubocurarine.

To test our present quantitative knowledge of nicotinic transmission, we reconstruct the postsynaptic conductance change that results after a presynaptic nerve terminal liberates a quantum of acetylcholine (ACh) into the synaptic cleft. The theory assumes that ACh appears suddenly in the cleft and that is subsequent fate is determined by radial diffusion, by enzymatic hydrolysis, and by binding to receptors. Each receptor has one channel and two ACh binding sites; the channel opens when both sites are occupied and the rate-limiting step id the binding and dissociation of the second ACh molecule. The calculations reproduce the experimentally measured growth phase (200 microseconds), peak number of open channels (2,000), and exponential decay phase. The time constant of the decay phase exceeds the channel duration by approximately equal to 20%. The normal event is highly localized: at the peak, two-thirds of the open channels are within an area of 0.15 micrometer 2. This represents 75% of the available channels within this area. The model also simulates voltage and temperature dependence and effects of inactivating esterase and receptors. The calculations show that in the absence of esterase, transmitter is buffered by binding to receptors and the postsynaptic response can be potentiated.

Cholinergic agonists cause an increase in the membrane permeability of Na and K at the innervated face of Electrophorus electroplaques. Therefore, sustained exposure to agonist reduces Na and K concentration gradients. There gradients are monitored with voltage-clamp sequences and pharmacological treatments that selectively measure the Nernst potentials for individual ions. EK is normally near--90 mV but moves toward zero during bath application of agonist. Depolarizations by bath-applied agonist measure primarily this shift of EK, not short-circuiting of EK by the agonist-induced conductance. After a rapid jump of agonist concentration, there is a fast (millisecond) depolarization due to the conductance increase, followed by a much slower additional "creep" due to the shift in EK. Sodium replaces the lost intracellular potassium: ENa, normally very positive, also moves toward zero. The shifts in EK and ENa are normally reversible but become permanent after blockade of the Na-K pump. In the presence of agonist, the shifts can be driven further by passing current of the appropriate polarity. Similar ion redistribution occurs with other drugs, such as batrachotoxin and nystatin, which induce prolonged increases in Na permeability. The redistributions cause little net change in the reversal potential of the neurally evoked postsynaptic current.

This paper compares the conductance induced by bath-applied acetyl-choline (ACh) and by the same transmitter released from nerve terminals at Electrophorus electroplaques. For the former case, dose-response relations are characterized by the maximal agonist-induced conductance, rgamma (130 mmho/cm2), and by the concentration which induces half this conductance; this concentration is termed Kapp and equals 50 micron at -85 mV. For the latter case, neurally evoked postsynaptic currents (PSCs) are characterized by the peak conductance during strongly facilitated release, gPSC, and by the rate constant for decay, alpha. Since gPSC roughly equals rgamma, it is concluded that the PSC activates nearly all available receptor channels. These and other data agree with recent estimates that during the growth phase of the quantal response, (a) the ACh concentration is at least several hundred micromolar; and (b) most nearby channels are activated. However both alpha and Kapp increase during depolarization, at a rate of about e-fold per 86 mV. These observations on voltage sensitivity suggest that a suprathreshold synaptic event is rapidly terminated because the action potential abruptly releases ACh molecules from receptors.

In these experiments, agonist-induced conductance is measured while a sudden perturbation is produced at the agonist-receptor binding site. A voltage-clamped Electrophorus electroplaque is exposed to trans-Bis-Q, a potent agonist. Some channels are open; these receptors have bound agonist molecules. A light flash isomerizes 3(-35)% of the trans-Bis-Q molecules to their cis form, a far poorer agonist. This causes a rapid decrease of membrane conductance (phase 1), followed by a slower increase (phase 2). Phase 1 has the amplitude and wavelength dependence expected if the channel closes within 100 mus after a single bound trans-Bis-Q is isomerized, and if the photochemistry of bound Bis-Q resembles that in solution. Therefore, the receptor channel responds rapidly, and with a hundred-fold greater closing rate, after this change in the structure of a bound ligand. Phase 2 (the conductance increase) seems to represent the relaxation back toward equilibrium after phase 1, because (a) phase 2 has the same time constant (1(-5) ms) as a voltage- or concentration-jump relaxation under identical conditions; and (b) phase 2 is smaller if the flash has led to a net decrease in (trans-Bis-Q). Still slower signals follow: phase 3, a decrease of conductance (time constant 5(-10 ms); and phase 4, an equal and opposite increase (several seconds). Phase 3 is abolished by curare and does not depend on the history of the membrane voltage. We consider several mechanisms for phases 3 and 4.

Kinetic measurements are employed to reconstruct the steady-state activation of acetylcholine [Ach] receptor channels in electrophorus electroplaques. Neurally evoked postsynaptic currents (PSCs) decay exponentially; at 15 degrees C the rate constant, alpha, equals 1.2 ms(-1) at 0 mV and decreases e-fold for every 86 mV as the membrane voltage is made more negative. Voltage-jump relaxations have been measured with bath-applied ACh, decamethonium, carbachol, or suberylcholine. We interpret the reciprocal relaxation time 1/tau as the sum of the rate constant alpha for channel closing and a first-order rate constant for channel opening. Where measureable, the opening rate increases linearly with [agonist] and does not vary with voltage. The voltage sensitivity of small steady-state conductances (e- fold for 86 mV) equals that of the closing rate alpha, confirming that the opening rate has little or no additional voltage sensitivity. Exposure to alpha-bungarotoxin irreversibly decreases the agonist-induced conductance but does not affect the relaxation kinetics. Tubocurarine reversibly reduces both the conductance and the opening rate. In the simultaneous presence of two agonist species, voltage-jump relaxations have at least two exponential components. The data are fit by a model in which (a) the channel opens as the receptor binds the second in a sequence of two agonist molecules, with a forward rate constant to 10(7) to 2x10(8) M(-1)s(-1); and (b) the channel then closes as either agonist molecule dissociates, with a voltage-dependent rate constant of 10(2) to 3x10(3)s(-1).

In Electrophorus electroplaques, the agonist-induced postsynaptic conductance depends on membrane potential. During steady exposure to agonists, after a voltage step the conductance relaxes on a millisecond time scale, exponentially approaching a new equilibrium value. The relaxation rate constant k is an instantaneous function of voltage, insensitive to the past or present conductance. Two components sum to form k. A concentration-sensitive component increases linearly with agonist concentration and decreases during desensitization or exposure to curare. Thus this component reflects the average frequency at which acetylcholine receptors are opening. The voltage-sensitive component, obtained by extrapolating k to zero agonist concentration, increases at more positive potentials. For acetylcholine, the voltage-sensitive component equals the rate constant for the exponential decay of postsynaptic currents; it thus seems to be the closing rate for active receptors. The voltage-sensitive component has the relative amplitudes acetylcholine less than carbamoylcholine less than decamethonium, and for each agonist equals the closing rate determined from "noise" measurements at neuromuscular junctions. The kinetic data explain several aspects of the steady-state conductance induced by agonists, but shed no light on apparent cooperative effects.