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Parkinson's disease (PD) is a neurodegenerative disorder that is clinically assessed by motor symptoms associated with the loss of midbrain dopaminergic neurons affecting the quality of life for over 8.5 million people worldwide. The neurotoxin 6-hydroxydopamine (6-OHDA) has been used to chemically induce a PD-like state in zebrafish larvae by several laboratories; however, highly variable concentration, methodology, and reagents have resulted in conflicting results suggesting a need to investigate these issues of reproducibility. We propose a protocol that addresses the differences in methodology and induces changes in 6 days postfertilization (dpf) larvae utilizing a 24-h exposure at 3 dpf with 30 μM 6-OHDA. Despite ∼50% lethality, no morphological or development differences in surviving fish are observed. Definition of our model is defined by downregulation of the expression of th1 by reverse transcriptase-quantitative polymerase chain reaction, a marker for dopaminergic neurons and a reduction in movement. Additionally, we observed a downregulation of pink1 and an upregulation of sod1 and sod2 , indicators of mitochondrial dysfunction and response to reactive oxygen species, respectively.

Background: L-theanine, 2-amino-4-(ethylcarbamoyl) butyric acid, an amino acid detected in green tea leaves, is used as a dietary supplement to attenuate stress and enhance mood and cognition. Furthermore, L-theanine induces anxiolytic effects in humans. Recently, L-theanine was reported to reduce morphine physical dependence in primates, suggesting the potential usefulness of L-theanine for drug dependence intervention., Objective: The aim of this study is to determine whether L-theanine attenuates nicotine-withdrawal (somatic and affective signs) and nicotine reward in mice. We also investigated the effects of L-theanine on nicotinic receptors binding and function., Methods: ICR male mice rendered dependent to nicotine through implanted subcutaneous osmotic minipumps for 14 days undertook precipitated nicotine withdrawal by mecamylamine on day 15. Anxiety-like behaviors using LDB, somatic signs observation and hot plate latency were assessed consecutively after treatment with L-theanine. Furthermore, we examined the effect of L-theanine on acute nicotine responses and nicotine conditioned reward in mice and on expressed nicotinic receptors in oocytes., Key Findings: L-theanine reduced in a dose-dependent manner anxiety-like behavior, hyperalgesia and somatic signs during nicotine withdrawal. Also, L-theanine decreased the nicotine CPP, but it did not affect the acute responses of nicotine. Finally, L-theanine did not alter the binding or the function of expressed α4β2 and α7 nAChRs., Conclusion: Our results support the potential of L-theanine as a promising candidate for treating nicotine dependence., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

A series of racemic benzofurans bearing N-methyl-2-pyrrolidinyl residue at C(2) or C(3) has been synthesized and tested for affinity at the α4β2 and α3β4 nicotine acetylcholine receptors (nAChRs). As previously reported for the benzodioxane based analogues, hydroxylation at proper position of benzene ring results in high α4β2 nAChR affinity and α4β2 vs. α3β4 nAChR selectivity. 7-Hydroxy-N-methyl-2-pyrrolidinyl-1,4-benzodioxane (2) and its 7- and 5-amino benzodioxane analogues 3 and 4, which are all α4β2 nAChR partial agonists, and 2-(N-methyl-2-pyrrolidinyl)-6-hydroxybenzofuran (12) were selected for functional characterization at the two α4β2 stoichiometries, the high sensitivity (α4) 2 (β2) 3 and the low sensitivity (α4) 3 (β2) 2 . The benzene pattern substitution, which had previously been found to control α4β2 partial agonist activity and α4β2 vs. α3β4 selectivity, proved to be also involved in stoichiometry-selectivity. The 7-hydroxybenzodioxane derivative 2 selectively activates (α4) 2 (β2) 3 nAChR, which cannot be activated by its 5-amino analogue 4. A marginal structural modification, not altering the base pyrrolidinyl benzodioxane scaffold, resulted in opposite activity profiles at the two α4β2 nAChR isoforms providing an interesting novel case study., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The extracellular domain of the nicotinic acetylcholine receptor isoforms formed by three α4 and two β2 subunits ((α4)3(β2)2 nAChR) harbors two high-affinity "canonical" acetylcholine (ACh)-binding sites located in the two α4:β2 intersubunit interfaces and a low-affinity "noncanonical" ACh-binding site located in the α4:α4 intersubunit interface. In this study, we used ACh, cytisine, and nicotine (which bind at both the α4:α4 and α4:β2 interfaces), TC-2559 (which binds at the α4:β2 but not at the α4:α4 interface), and 3-(2-chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)-1H-pyrrazol-4-yl)isoxazole (CMPI, which binds at the α4:α4 but not at the α4:β2 interface), to investigate the binding and gating properties of CMPI at the α4:α4 interface. We recorded whole-cell currents from Xenopus laevis oocytes expressing (α4)3(β2)2 nAChR in response to applications of these ligands, alone or in combination. The electrophysiological data were analyzed in the framework of a modified Monod-Wyman-Changeux allosteric activation model. We show that CMPI is a high-affinity, high-efficacy agonist at the α4:α4 binding site and that its weak direct activating effect is accounted for by its inability to productively interact with the α4:β2 sites. The data presented here enhance our understanding of the functional contributions of ligand binding at the α4:α4 subunit interface to (α4)3(β2)2 nAChR-channel gating. These findings support the potential use of α4:α4 specific ligands to increase the efficacy of the neurotransmitter ACh in conditions associated with decline in nAChRs activity in the brain., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

(1) Background: The lung cholinergic pathway is important for controlling pulmonary inflammation in acute lung injury, a condition that is characterized by a sudden onset and intense inflammation. This study investigated changes in the expression levels of nicotinic and muscarinic acetylcholine receptors (nAChR and mAChR) in the lung during acute lung injury. (2) Methods: acute lung injury (ALI) was induced in wild-type and cholinergic-deficient (VAChT-KD HOM ) mice using intratracheal lipopolysaccharide (LPS) instillation with or without concurrent treatment with nicotinic ligands. Bronchoalveolar lavage fluid was collected to evaluate markers of inflammation, and then the lung was removed and processed for isolation of membrane fraction and determination of acetylcholine receptors level using radioligand binding assays. (3) Results: LPS-induced increase in lung inflammatory markers (e.g., neutrophils and IL-1β) was significantly higher in VAChT-KD HOM than wild-type mice. In contrast, LPS treatment resulted in a significant increase in lung's α7 nicotinic receptor level in wild-type, but not in VAChT-KD HOM mice. However, treatment with PNU 282987, a selective α7 nicotinic receptor agonist, restored VAChT-KD HOM mice's ability to increase α7 nicotinic receptor levels in response to LPS-induced acute lung injury and reduced lung inflammation. LPS also increased muscarinic receptors level in VAChT-KD HOM mice, and PNU 282987 treatment reduced this response. (4) Conclusions: Our data indicate that the anti-inflammatory effects of the lung cholinergic system involve an increase in the level of α7 nicotinic receptors. Pharmacological agents that increase the expression or the function of lung α7 nicotinic receptors have potential clinical uses for treating acute lung injury.

The low sensitivity (α4)3(β2)2 (LS) and high sensitivity (α4)2(β2)3 (HS) nAChR isoforms may contribute to a variety of brain functions, pathophysiological processes, and pharmacological effects associated with nicotine use. In this study, we examined the contributions of the LS and HS α4β2 nAChR isoforms in nicotine self-administration, withdrawal symptoms, antinociceptive and hypothermic effects. We utilized two nAChR positive allosteric modulators (PAMs): desformylflustrabromine (dFBr), a PAM of both the LS and HS α4β2 nAChRs, and CMPI, a PAM selective for the LS nAChR. We found that dFBr, but not CMPI, decreased intravenous nicotine self-administration in male mice in a dose-dependent manner. Unlike dFBr, which fully reverses somatic and affective symptoms of nicotine withdrawal, CMPI at doses up to 15 mg/kg in male mice only partially reduced nicotine withdrawal-induced somatic signs, anxiety-like behavior and sucrose preference, but had no effects on nicotine withdrawal-induced hyperalgesia. These results indicate that potentiation of HS α4β2 nAChRs is necessary to modulate nicotine's reinforcing properties that underlie nicotine intake and to reverse nicotine withdrawal symptoms that influence nicotine abstinence. In contrast, both dFBr and CMPI enhanced nicotine's hypothermic effect and reduced nicotine's antinociceptive effects in male mice. Therefore, these results indicate a more prevalent role of HS α4β2 nAChR isoforms in mediating various behavioral effects associated with nicotine, whereas the LS α4β2 nAChR isoform has a limited role in mediating body temperature and nociceptive responses. These findings will facilitate the development of more selective, efficacious, and safe nAChR-based therapeutics for nicotine addiction treatment., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Neuronal nicotinic acetylcholine receptor (nAChR)-based therapeutics are sought as a potential alternative strategy to opioids for pain management. In this study, we examine the antinociceptive effects of 3-(2-chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)-1 H -pyrazol-4-yl)isoxazole (CMPI), a novel positive allosteric modulator (PAM), with preferential selectivity to the low agonist sensitivity (α4)3(β2)2 nAChR and desformylflustrabromine (dFBr), a PAM for α4-containing nAChRs. We used hot plate and tail flick tests to measure the effect of dFBr and CMPI on the latency to acute thermal nociceptive responses in rats. Intraperitoneal injection of dFBr, but not CMPI, dose-dependently increased latency in the hot plate test. In the tail flick test, the effect achieved at the highest dFBr or CMPI dose tested was only <20% of the maximum possible effects reported for nicotine and other nicotinic agonists. Moreover, the coadministration of dFBr did not enhance the antinociceptive effect of a low dose of nicotine. Our results show that the direct acute effect of dFBr is superior to that for CMPI, indicating that selectivity to (α4)3(β2)2 nAChR is not advantageous in alleviating responses to acute thermal nociceptive stimulus. However, further studies are necessary to test the suitability of (α4)3(β2)2 nAChR-selective PAMs in chronic pain models.

Receptors containing α4 and β2 subunits are a major neuronal nicotinic acetylcholine receptor (nAChR) subtype in the brain. This receptor plays a critical role in nicotine addiction, with potential smoking cessation therapeutics producing modulation of α4β2 nAChR. In addition, compounds that act as agonists at α4β2 nAChR may be useful for the treatment of pathological pain. Further, as the α4β2 nAChR has been implicated in cognition, therapeutics that act as α4β2 nAChR agonists are also being examined as treatments for cognitive disorders and neurological diseases that impact cognitive function, such as Alzheimer's disease and schizophrenia. This review will cover the molecular in vitro evidence that allosteric modulators of the α4β2 neuronal nAChR provide several advantages over traditional α4β2 nAChR orthosteric ligands. Specifically, we explore the concept that nAChR allosteric modulators allow for greater pharmacological selectivity, while minimizing potential deleterious off-target effects. Further, here we discuss the development and preclinical in vivo behavioral assessment of allosteric modulators at the α4β2 neuronal nAChR as therapeutics for smoking cessation, pathological pain, as well as cognitive disorders and neurological diseases that impact cognitive function. This article is part of the special issue on 'Contemporary Advances in Nicotine Neuropharmacology'., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Disposition of beta-amyloid peptide 1-42 (Aβ1-42) in the space around the synapses and formation of Aβ-containing aggregates known as neuritic or senile plaques are hallmark features of neurodegenerative pathologies associated with Alzheimer's disease (AD). While AD is a multifactorial disease that includes other proteinopathies (e.g., hyperphosphorylated tau aggregates) and neurotransmitter disturbances (e.g., loss of cortical cholinergic innervation), Aβ (soluble or in senile plaques) remains the major undisputed factor that contributes to the pathological and behavior presentation of AD. Overproduction of Aβ and mutations in Aβ precursor (amyloid precursor protein) or enzymes involved in Aβ1-42 production and removal (γ secretase/presenilins) have been shown in cases of early onset of AD and produced AD-like pathologies in animal models. In addition, the level of soluble Aβ1-42 has been shown to correlate with cognitive impairment in animal models before the presence of senile plaques or other histological features of AD. However, much still is unknown about the biochemical processes leading to amyloid formation and its relation to the pathogenesis, neuronal damage/dysfunction, and behavioral changes associated with AD. In this article, we review animal models that have been developed to study AD-like pathologies and then provide detailed methodology to develop an acute rat model of Aβ-induced cognitive impairment. We use this model to examine the cognitive-enhancing effect of novel pharmacological interventions targeting nicotinic acetylcholine receptors.

Introduction: Nicotine withdrawal symptoms are important factors in determining the relapse rate to tobacco smoking and drugs that diminish these symptoms would potentially have a higher success rate as smoking cessation aids. Unlike US Food and Drug administration approved smoke cessation aids (nicotine and varenicline) which act as nicotinic acetylcholine receptors (nAChRs) agonists, desformylflustrabromine (dFBr) acts as a nAChR positive allosteric modulator with higher selectivity to the α4β2 nAChR. In animal studies, dFBr was well tolerated and reduced intravenous nicotine self-administration. In this study, we use behavioral test in mouse model of spontaneous nicotine withdrawal to assess the effect of dFBr on nicotine withdrawal symptoms., Methods: Spontaneous nicotine withdrawal in nicotine-dependent ICR male mice was established 18-24 h after termination (minipump removal) of 14 days infusion of nicotine. After that (day 15), spontaneous signs of nicotine withdrawal were examined in the following order: anxiety-like behaviors, somatic signs, and then hyperalgesia using previously published behavioral protocols. Fifteen minutes before withdrawal signs testing, mice received a subcutaneous acute injection of vehicle or dFBr at the doses of 0.02, 0.1, and 1 mg/kg to determine the effect of dFBr on nicotine withdrawal symptoms., Results: dFBr produced dose-dependent reversal of nicotine withdrawal signs in mouse model of spontaneous nicotine withdrawal., Implications: Positive allosteric modulators of nAChR such as dFBr reduce nicotine withdrawal symptoms supporting the potential clinical use of this novel class of nAChR-based therapeutics as smoking cessation aid.

Positive allosteric modulators (PAMs) of nicotinic acetylcholine receptors (nAChRs) have potential therapeutic application in neuropathologies associated with decrease in function or loss of nAChRs. In this study, we characterize the pharmacological interactions of the nAChRs PAM, LY2087101, with the α4β2 nAChR using mutational and computational analyses. LY2087101 potentiated ACh-induced currents of low-sensitivity (α4)3(β2)2 and high-sensitivity (α4)2(β2)3 nAChRs with similar potencies albeit to a different maximum potentiation (potentiation I max  = ~840 and 450%, respectively). Amino acid substitutions within the α4 subunit transmembrane domain [e.g. α4Leu256 and α4Leu260 within the transmembrane helix 1 (TM1); α4Phe316 within the TM3; and α4Gly613 within TM4] significantly reduced LY2087101 potentiation of (α4)3(β2)2 nAChR. The locations of these amino acid residues and LY2087101 computational docking analyses identify two LY2087101 binding sites: an intrasubunit binding site within the transmembrane helix bundle of α4 subunit at the level of α4Leu260/α4Phe316 and intersubunit binding site at the α4:α4 subunit interface at the level of α4Leu256/α4Ile315 with both sites extending toward the extracellular end of the transmembrane domain. We also show that desformylflustrabromine (dFBr) binds to these two sites identified for LY2087101. These results provide structural information that are pertinent to structure-based design of nAChR allosteric modulators.

Neuronal nicotinic acetylcholine receptors (nAChRs) are promising drug targets to manage several neurological disorders and nicotine addiction. Growing evidence indicates that positive allosteric modulators of nAChRs improve pharmacological specificity by binding to unique sites present only in a subpopulation of nAChRs. Furthermore, nAChR positive allosteric modulators such as NS9283 and CMPI have been shown to potentiate responses of (α4)3(β2)2 but not (α4)2(β2)3 nAChR isoforms. This selective potentiation underlines that the α4:α4 interface, which is present only in the (α4)3(β2)2 nAChR, is an important and promising drug target. In this report we used site-directed mutagenesis to substitute specific amino acid residues and computational analyses to elucidate CMPI's binding mode at the α4:α4 subunit extracellular interface and identified a unique set of amino acid residues that determined its affinity. We found that amino acid residues α4Gly-41, α4Lys-64, and α4Thr-66 were critical for (α4)3(β2)2 nAChR potentiation by CMPI, but not by NS9283, whereas amino acid substitution at α4His-116, a known determinant of NS9283 and of agonist binding at the α4:α4 subunit interface, did not reduce CMPI potentiation. In contrast, substitutions at α4Gln-124 and α4Thr-126 reduced potentiation by CMPI and NS9283, indicating that their binding sites partially overlap. These results delineate the role of amino acid residues contributing to the α4:α4 subunit extracellular interface in nAChR potentiation. These findings also provide structural information that will facilitate the structure-based design of novel therapeutics that target selectively the (α4)3(β2)2 nAChR., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Alzheimer's disease (AD) is a progressive neurodegenerative disorder and currently there is no efficient treatment. The classic drug-design strategy based on the "one-molecule-one-target" paradigm was found to be ineffective in the case of multifactorial diseases like AD. A novel multi-target-directed ligand strategy based on the assumption that a single compound consisting of two or more distinct pharmacophores is able to hit multiple targets has been proposed as promising. Herein, we investigated 7-methoxytacrine - memantine heterodimers developed with respect to the multi-target-directed ligand theory. The spectroscopic, microscopic and cell culture methods were used for systematic investigation of the interference of the heterodimers with β-secretase (BACE1) activity, Aβ peptide amyloid fibrillization (amyloid theory) and interaction with M1 subtype of muscarinic (mAChRs), nicotinic (nAChRs) acetylcholine receptors (cholinergic theory) and N-methyl-d-aspartate receptors (NMDA) (glutamatergic theory). The drug-like properties of selected compounds have been evaluated from the point of view of blood-brain barrier penetration and cell proliferation. We have confirmed the multipotent effect of novel series of compounds. They inhibited effectively Aβ peptide amyloid fibrillization and affected the BACE1 activity. Moreover, they have AChE inhibitory potency but they could not potentiate cholinergic transmission via direct interaction with cholinergic receptors. All compounds were reported to act as an antagonist of both M1 muscarinic and muscle-type nicotinic receptors. We have found that 7-methoxytacrine - memantine heterodimers are able to hit multiple targets associated with Alzheimer's disease and thus, have a potential clinical impact for slowing or blocking the neurodegenerative process related to this disease., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Photoaffinity labeling techniques have been used for decades to identify drug binding sites and to study the structural biology of allosteric transitions in transmembrane proteins including pentameric ligand-gated ion channels (pLGIC). In a typical photoaffinity labeling experiment, to identify drug binding sites, UV light is used to introduce a covalent bond between a photoreactive ligand (which upon irradiation at the appropriate wavelength converts to a reactive intermediate) and amino acid residues that lie within its binding site. Then protein chemistry and peptide microsequencing techniques are used to identify these amino acids within the protein primary sequence. These amino acid residues are located within homology models of the receptor to identify the binding site of the photoreactive probe. Molecular modeling techniques are then used to model the binding of the photoreactive probe within the binding site using docking protocols. Photoaffinity labeling directly identifies amino acids that contribute to drug binding sites regardless of their location within the protein structure and distinguishes them from amino acids that are only involved in the transduction of the conformational changes mediated by the drug, but may not be part of its binding site (such as those identified by mutational studies). Major limitations of photoaffinity labeling include the availability of photoreactive ligands that faithfully mimic the properties of the parent molecule and protein preparations that supply large enough quantities suitable for photoaffinity labeling experiments. When the ligand of interest is not intrinsically photoreactive, chemical modifications to add a photoreactive group to the parent drug, and pharmacological evaluation of these chemical modifications become necessary. With few exceptions, expression and affinity-purification of proteins are required prior to photolabeling. Methods to isolate milligram quantities of highly enriched pLGIC suitable for photoaffinity labeling experiments have been developed. In this chapter, we discuss practical aspects of experimental strategies to identify allosteric modulator binding sites in pLGIC using photoaffinity labeling.

Positive allosteric modulators (PAMs) of nicotinic acetylcholine (ACh) receptors (nAChRs) have potential clinical applications in the treatment of nicotine dependence and many neuropsychiatric conditions associated with decreased brain cholinergic activity, and 3-(2-chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)-1H-pyrrazol-4-yl)isoxazole (CMPI) has been identified as a PAM selective for neuronal nAChRs containing theα4 subunit. In this report, we compare CMPI interactions with low-sensitivity (α4)3(β2)2 and high-sensitivity (α4)2(β2)3 nAChRs, and with muscle-type nAChRs. In addition, we use the intrinsic reactivity of [(3)H]CMPI upon photolysis at 312 nm to identify its binding sites inTorpedonAChRs. Recording fromXenopusoocytes, we found that CMPI potentiated maximally the responses of (α4)3(β2)2nAChR to 10μM ACh (EC10) by 400% and with anEC50of ∼1µM. CMPI produced a left shift of the ACh concentration-response curve without altering ACh efficacy. In contrast, CMPI inhibited (∼35% at 10µM) ACh responses of (α4)2(β2)3nAChRs and fully inhibited human muscle andTorpedonAChRs with IC50values of ∼0.5µM. Upon irradiation at 312 nm, [(3)H]CMPI photoincorporated into eachTorpedo[(α1)2β1γδ] nAChR subunit. Sequencing of peptide fragments isolated from [(3)H]CMPI-photolabeled nAChR subunits established photolabeling of amino acids contributing to the ACh binding sites (αTyr(190),αTyr(198),γTrp(55),γTyr(111),γTyr(117),δTrp(57)) that was fully inhibitable by agonist and lower-efficiency, state-dependent [(3)H]CMPI photolabeling within the ion channel. Our results establish that CMPI is a potent potentiator of nAChRs containing anα4:α4 subunit interface, and that its intrinsic photoreactivy makes it of potential use to identify its binding sites in the (α4)3(β2)2nAChR., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Nicotine addiction, the result of tobacco use, leads to over six million premature deaths world-wide per year, a number that is expected to increase by a third within the next two decades. While more than half of smokers want and attempt to quit, only a small percentage of smokers are able to quit without pharmacological interventions. Therefore, over the past decades, researchers in academia and the pharmaceutical industry have focused their attention on the development of more effective smoking cessation therapies, which is now a growing 1.9 billion dollar market. Because the role of neuronal nicotinic acetylcholine receptors (nAChR) in nicotine addiction is well established, nAChR based therapeutics remain the leading strategy for smoking cessation. However, the development of neuronal nAChR drugs that are selective for a nAChR subpopulation is challenging, and only few neuronal nAChR drugs are clinically available. Among the many neuronal nAChR subtypes that have been identified in the brain, the α4β2 subtype is the most abundant and plays a critical role in nicotine addiction. Here, we review the role of neuronal nAChRs, especially the α4β2 subtype, in the development and treatment of nicotine addiction. We also compare available smoking cessation medications and other nAChR orthosteric and allosteric ligands that have been developed with emphasis on the difficulties faced in the development of clinically useful compounds with high nAChR subtype selectivity.

Desformylflustrabromine (dFBr) is a positive allosteric modulator (PAM) of α4β2 and α2β2 nAChRs that, at concentrations >1 µM, also inhibits these receptors and α7 nAChRs. However, its interactions with muscle-type nAChRs have not been characterized, and the locations of its binding site(s) in any nAChR are not known. We report here that dFBr inhibits human muscle (αβεδ) and Torpedo (αβγδ) nAChR expressed in Xenopus oocytes with IC50 values of ∼ 1 μM. dFBr also inhibited the equilibrium binding of ion channel blockers to Torpedo nAChRs with higher affinity in the nAChR desensitized state ([(3)H]phencyclidine; IC50 = 4 μM) than in the resting state ([(3)H]tetracaine; IC50 = 60 μM), whereas it bound with only very low affinity to the ACh binding sites ([(3)H]ACh, IC50 = 1 mM). Upon irradiation at 312 nm, [(3)H]dFBr photoincorporated into amino acids within the Torpedo nAChR ion channel with the efficiency of photoincorporation enhanced in the presence of agonist and the agonist-enhanced photolabeling inhibitable by phencyclidine. In the presence of agonist, [(3)H]dFBr also photolabeled amino acids in the nAChR extracellular domain within binding pockets identified previously for the nonselective nAChR PAMs galantamine and physostigmine at the canonical α-γ interface containing the transmitter binding sites and at the noncanonical δ-β subunit interface. These results establish that dFBr inhibits muscle-type nAChR by binding in the ion channel and that [(3)H]dFBr is a photoaffinity probe with broad amino acid side chain reactivity., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

For almost 30 years, photoaffinity labeling and protein microsequencing techniques have been providing novel insights about the structure of nicotinic acetylcholine receptors (nAChR) and the diversity of nAChR drug binding sites. Photoaffinity labeling allows direct identification of amino acid residues contributing to a drug binding site without prior knowledge of the location of the binding site within the nAChR or the orientation of the ligand within the binding site. It also distinguishes amino acids that contribute to allosteric binding sites from those involved in allosteric modulation of gating. While photoaffinity labeling was used initially to identify amino acids contributing to the agonist binding sites and the ion channel, it has been used recently to identify binding sites for allosteric modulators at subunit interfaces in the extracellular and the transmembrane domains, and within a subunit's transmembrane helix bundle. In this article, we review the different types of photoaffinity probes that have been used and the various binding sites that have been identified within the structure of nAChR, with emphasis on our recent studies of allosteric modulator binding sites.

At concentrations that produce anesthesia, many barbituric acid derivatives act as positive allosteric modulators of inhibitory GABAA receptors (GABAARs) and inhibitors of excitatory nicotinic acetylcholine receptors (nAChRs). Recent research on [(3)H]R-mTFD-MPAB ([(3)H]R-5-allyl-1-methyl-5-(m-trifluoromethyldiazirinylphenyl)barbituric acid), a photoreactive barbiturate that is a potent and stereoselective anesthetic and GABAAR potentiator, has identified a second class of intersubunit binding sites for general anesthetics in the α1β3γ2 GABAAR transmembrane domain. We now characterize mTFD-MPAB interactions with the Torpedo (muscle-type) nAChR. For nAChRs expressed in Xenopus oocytes, S- and R-mTFD-MPAB inhibited ACh-induced currents with IC50 values of 5 and 10 µM, respectively. Racemic mTFD-MPAB enhanced the equilibrium binding of [(3)H]ACh to nAChR-rich membranes (EC50 = 9 µM) and inhibited binding of the ion channel blocker [(3)H]tenocyclidine in the nAChR desensitized and resting states with IC50 values of 2 and 170 µM, respectively. Photoaffinity labeling identified two binding sites for [(3)H]R-mTFD-MPAB in the nAChR transmembrane domain: 1) a site within the ion channel, identified by photolabeling in the nAChR desensitized state of amino acids within the M2 helices of each nAChR subunit; and 2) a site at the γ-α subunit interface, identified by photolabeling of γMet299 within the γM3 helix at similar efficiency in the resting and desensitized states. These results establish that mTFD-MPAB is a potent nAChR inhibitor that binds in the ion channel preferentially in the desensitized state and binds with lower affinity to a site at the γ-α subunit interface where etomidate analogs bind that act as positive and negative nAChR modulators.

Galanthamine and physostigmine are clinically used cholinomimetics that both inhibit acetylcholinesterase and also interact directly with and potentiate nAChRs. As with most nAChR-positive allosteric modulators, the location and number of their binding site(s) within nAChRs are unknown. In this study, we use the intrinsic photoreactivities of [(3)H]physostigmine and [(3)H]galanthamine upon irradiation at 312 nm to directly identify amino acids contributing to their binding sites in the Torpedo californica nAChR. Protein sequencing of fragments isolated from proteolytic digests of [(3)H]physostigmine- or [(3)H]galanthamine-photolabeled nAChR establish that, in the presence of agonist (carbamylcholine), both drugs photolabeled amino acids on the complementary (non-α) surface of the transmitter binding sites (γTyr-111/γTyr-117/δTyr172). They also photolabeled δTyr-212 at the δ-β subunit interface and γTyr-105 in the vestibule of the ion channel, with photolabeling of both residues enhanced in the presence of agonist. Furthermore, [(3)H]physostigmine photolabeling of γTyr-111, γTyr-117, δTyr-212, and γTyr-105 was inhibited in the presence of nonradioactive galanthamine. The locations of the photolabeled amino acids in the nAChR structure and the results of computational docking studies provide evidence that, in the presence of agonist, physostigmine and galanthamine bind to at least three distinct sites in the nAChR extracellular domain: at the α-γ interface (1) in the entry to the transmitter binding site and (2) in the vestibule of the ion channel near the level of the transmitter binding site, and at the δ-β interface (3) in a location equivalent to the benzodiazepine binding site in GABA(A) receptors.

Bupropion, a clinically used antidepressant and smoking-cessation drug, acts as a noncompetitive antagonist of nicotinic acetylcholine receptors (nAChRs). To identify its binding site(s) in nAChRs, we developed a photoreactive bupropion analogue, (±)-2-(N-tert-butylamino)-3'-[(125)I]-iodo-4'-azidopropiophenone (SADU-3-72). Based on inhibition of [(125)I]SADU-3-72 binding, SADU-3-72 binds with high affinity (IC(50) = 0.8 μM) to the Torpedo nAChR in the resting (closed channel) state and in the agonist-induced desensitized state, and bupropion binds to that site with 3-fold higher affinity in the desensitized (IC(50) = 1.2 μM) than in the resting state. Photolabeling of Torpedo nAChRs with [(125)I]SADU-3-72 followed by limited in-gel digestion of nAChR subunits with endoproteinase Glu-C established the presence of [(125)I]SADU-3-72 photoincorporation within nAChR subunit fragments containing M1-M2-M3 helices (αV8-20K, βV8-22/23K, and γV8-24K) or M1-M2 helices (δV8-14). Photolabeling within βV8-22/23K, γV8-24K, and δV8-14 was reduced in the desensitized state and inhibited by ion channel blockers selective for the resting (tetracaine) or desensitized (thienycyclohexylpiperidine (TCP)) state, and this pharmacologically specific photolabeling was localized to the M2-9 leucine ring (δLeu(265), βLeu(257)) within the ion channel. In contrast, photolabeling within the αV8-20K was enhanced in the desensitized state and not inhibited by TCP but was inhibited by bupropion. This agonist-enhanced photolabeling was localized to αTyr(213) in αM1. These results establish the presence of two distinct bupropion binding sites within the Torpedo nAChR transmembrane domain: a high affinity site at the middle (M2-9) of the ion channel and a second site near the extracellular end of αM1 within a previously described halothane (general anesthetic) binding pocket.

Photoreactive derivatives of the general anesthetic etomidate have been developed to identify their binding sites in γ-aminobutyric acid, type A and nicotinic acetylcholine receptors. One such drug, [(3)H]TDBzl-etomidate (4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl-[(3)H]1-(1-phenylethyl)-1H-imidazole-5-carboxylate), acts as a positive allosteric potentiator of Torpedo nACh receptor (nAChR) and binds to a novel site in the transmembrane domain at the γ-α subunit interface. To extend our understanding of the locations of allosteric modulator binding sites in the nAChR, we now characterize the interactions of a second aryl diazirine etomidate derivative, TFD-etomidate (ethyl-1-(1-(4-(3-trifluoromethyl)-3H-diazirin-3-yl)phenylethyl)-1H-imidazole-5-carboxylate). TFD-etomidate inhibited acetylcholine-induced currents with an IC(50) = 4 μM, whereas it inhibited the binding of [(3)H]phencyclidine to the Torpedo nAChR ion channel in the resting and desensitized states with IC(50) values of 2.5 and 0.7 mm, respectively. Similar to [(3)H]TDBzl-etomidate, [(3)H]TFD-etomidate bound to a site at the γ-α subunit interface, photolabeling αM2-10 (αSer-252) and γMet-295 and γMet-299 within γM3, and to a site in the ion channel, photolabeling amino acids within each subunit M2 helix that line the lumen of the ion channel. In addition, [(3)H]TFD-etomidate photolabeled in an agonist-dependent manner amino acids within the δ subunit M2-M3 loop (δIle-288) and the δ subunit transmembrane helix bundle (δPhe-232 and δCys-236 within δM1). The fact that TFD-etomidate does not compete with ion channel blockers at concentrations that inhibit acetylcholine responses indicates that binding to sites at the γ-α subunit interface and/or within δ subunit helix bundle mediates the TFD-etomidate inhibitory effect. These results also suggest that the γ-α subunit interface is a binding site for Torpedo nAChR negative allosteric modulators (TFD-etomidate) and for positive modulators (TDBzl-etomidate).

The Cys-loop receptor super-family of neurotransmitter-gated ion channels mediates fast synaptic transmission throughout the human nervous system. These receptors exhibit widely varying pharmacologies, yet their structural characterization has relied heavily on their homology with the naturally abundant muscle-type Torpedo nicotinic acetylcholine receptor. Here we examine for the first time the structure of a human α4β2 neuronal nicotinic acetylcholine receptor. We show that human α4β2 nicotinic receptors adopt a secondary/tertiary fold similar to that of the Torpedo nicotinic receptor with a large proportion of both α-helix and β-sheet, but exhibit a substantially increased thermal stability. Both receptors bind agonist, but with different patterns of agonist recognition - particularly in the nature of the interactions between aromatic residues and the agonist quaternary amine functional group. By comparing α4β2 and Torpedo receptors, we begin to delineate their structural similarities and differences., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We synthesized the R- and S-enantiomers of ethyl 1-(1-(4-(3-((trifluoromethyl)-3H-diazirin-3-yl)phenyl)ethyl)-1H-imidazole-5-carboxylate (trifluoromethyldiazirinyl-etomidate), or TFD-etomidate, a novel photoactivable derivative of the stereoselective general anesthetic etomidate (R-(2-ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate)). Anesthetic potency was similar to etomidate's, but stereoselectivity was reversed and attenuated. Relative to etomidate, TFD-etomidate was a more potent inhibitor of the excitatory receptors, nAChR (nicotinic acetylcholine receptor) ((alpha1)(2)beta1delta1gamma1) and 5-HT(3A)R (serotonin type 3A receptor), causing significant inhibition at anesthetic concentrations. S- but not R-TFD-etomidate enhanced currents elicited from inhibitory alpha1beta2gamma2L GABA(A)Rs by low concentrations of GABA, but with a lower efficacy than R-etomidate, and site-directed mutagenesis suggests they act at different sites. [(3)H]TFD-etomidate photolabeled the alpha-subunit of the nAChR in a manner allosterically regulated by agonists and noncompetitive inhibitors. TFD-etomidate's novel pharmacology is unlike that of etomidate derivatives with photoactivable groups in the ester position, which behave like etomidate, suggesting that it will further enhance our understanding of anesthetic mechanisms.

Chlorpromazine (CPZ), a potent nicotinic acetylcholine receptor (nAChR) noncompetitive antagonist, binds with higher affinity in the ion channel in the desensitized state than in the closed channel state and with low affinity to additional sites in nAChR-rich membranes. For nAChR equilibrated with agonist, we confirm previous reports that [(3)H]CPZ occupies a site near the cytoplasmic end of the M2 ion channel domain, photolabeling positions M2-2, M2-6, and/or M2-9 in each subunit. We find that [(3)H]CPZ also binds at the extracellular end of the channel, photolabeling amino acids at positions M2-16 (alpha,gamma), M2-17 (alpha,beta,delta), and M2-20 (alpha,beta,delta). The photolabeling at the cytoplasmic end of the channel is fully inhibitable by phencyclidine or proadifen, whereas neither drug inhibits [(3)H]CPZ photolabeling at the extracellular end, establishing that positively charged drugs can bind simultaneously at the cytoplasmic and extracellular ends of the ion channel. [(3)H]CPZ photolabeling is not detected in the transmembrane domain outside the ion channel, but it photolabels alphaMet-386 and alphaSer-393 in the cytoplasmic alphaMA helix. In the nAChR equilibrated with alpha-bungarotoxin to stabilize the nAChR in a closed state, [(3)H]CPZ photolabels amino acids at M2-5 (alpha), M2-6 (alpha,beta,delta), and M2-9 (beta,delta), with no labeling at M2-2. These results provide novel information about the modes of drug binding within the nAChR ion channel and indicate that within the nAChR transmembrane domain, the binding of cationic aromatic amine antagonists can be restricted to the ion channel domain, in contrast to the uncharged, allosteric potentiators and inhibitors that also bind within the delta subunit helix bundle and at subunit interfaces.

A HEK-293 cell line that stably expresses mouse 5-HT(3A)Rs containing a C-terminal extension that confers high-affinity binding of alpha-bungarotoxin (alphaBgTx) was established (alphaBgTx-5-HT(3A)Rs) and used to purify alphaBgTx-5-HT(3A)Rs in a lipid environment for use in structural studies using photoaffinity labeling. alphaBgTx-5-HT(3A)Rs were expressed robustly (60 pmol of [(3)H]BRL-43694 binding sites (approximately 3 microg of receptor) per milligram of protein) and displayed the same functional properties as wild-type receptors (serotonin EC(50) = 5.3 +/- 0.04 microM). While [(125)I]alphaBgTx bound to the alphaBgTx-5-HT(3A)Rs with high affinity (K(d) = 11 nM), application of nonradioactive alphaBgTx (up to 300 microM) had no effect on serotonin-induced current responses. alphaBgTx-5-HT(3A)Rs were purified on an alphaBgTx-derivatized affinity column from detergent extracts in milligram quantities and at approximately 25% purity. The hydrophobic photolabel 3-trifluoromethyl-3-(m-[(125)I]iodophenyl)diazirine ([(125)I]TID) was used to identify the amino acids at the lipid-protein interface of purified and lipid-reconstituted alphaBgTx-5-HT(3A)Rs. [(125)I]TID photoincorporation into the alphaBgTx-5-HT(3A)R subunit was initially mapped to subunit proteolytic fragments of 8 kDa, containing the M4 transmembrane segment and approximately 60% of incorporated (125)I, and 17 kDa, containing the M1-M3 transmembrane segments. Within the M4 segment, [(125)I]TID labeled Ser(451), equivalent to the [(125)I]TID-labeled residue Thr(422) at the lipid-exposed face of the Torpedo nicotinic acetylcholine receptor (nAChR) alpha1M4 alpha-helix. These results provide a first definition of the surface of the 5-HT(3A)R M4 helix that is exposed to lipid and establish that this surface is equivalent to the surface exposed to lipid in the Torpedo nAChR.

Nicotinic acetylcholine receptor (nAChR) agonists, such as epibatidine and its molecular derivatives, are potential therapeutic agents for a variety of neurological disorders. In order to identify determinants for subtype-selective agonist binding, it is important to determine whether an agonist binds in a common orientation in different nAChR subtypes. To compare the mode of binding of epibatidine in a muscle and a neuronal nAChR, we photolabeled Torpedo alpha(2)betagammadelta and expressed human alpha4beta2 nAChRs with [(3)H]epibatidine and identified by Edman degradation the photolabeled amino acids. Irradiation at 254 nm resulted in photolabeling of alphaTyr(198) in agonist binding site Segment C of the principal (+) face in both alpha subunits and of gammaLeu(109) and gammaTyr(117) in Segment E of the complementary (-) face, with no labeling detected in the delta subunit. For affinity-purified alpha4beta2 nAChRs, [(3)H]epibatidine photolabeled alpha4Tyr(195) (equivalent to Torpedo alphaTyr(190)) in Segment C as well as beta2Val(111) and beta2Ser(113) in Segment E (equivalent to Torpedo gammaLeu(109) and gammaTyr(111), respectively). Consideration of the location of the photolabeled amino acids in homology models of the nAChRs based upon the acetylcholine-binding protein structure and the results of ligand docking simulations suggests that epibatidine binds in a single preferred orientation within the alpha-gamma transmitter binding site, whereas it binds in two distinct orientations in the alpha4beta2 nAChR.

The development of nicotinic acetylcholine receptor (nAChR) agonists, particularly those that discriminate between neuronal nAChR subtypes, holds promise as potential therapeutic agents for many neurological diseases and disorders. To this end, we photoaffinity labeled human alpha4beta2 and rat alpha4beta4 nAChRs affinity-purified from stably transfected HEK-293 cells, with the agonists [(125)I]epibatidine and 5[(125)I]A-85380. Our results show that both agonists photoincorporated into the beta4 subunit with little or no labeling of the beta2 and alpha4 subunits respectively. [(125)I]epibatidine labeling in the beta4 subunit was mapped to two overlapping proteolytic fragments that begin at beta4V102 and contain Loop E (beta4I109-P120) of the agonist binding site. We were unable to identify labeled amino acid(s) in Loop E by protein sequencing, but we were able to demonstrate that beta4Q117 in Loop E is the principal site of [(125)I]epibatidine labeling. This was accomplished by substituting residues in the beta2 subunit with the beta4 homologs and finding [(125)I]epibatidine labeling in beta4 and beta2F119Q subunits with little, if any, labeling in alpha4, beta2, or beta2S113R subunits. Finally, functional studies established that the beta2F119/beta4Q117 position is an important determinant of the receptor subtype-selectivity of the agonist 5I-A-85380, affecting both binding affinity and channel activation.

The Torpedo nicotinic acetylcholine receptor (nAChR) is the only member of the Cys-loop superfamily of ligand-gated ion channels (LGICs) that is available in high abundance in a native membrane preparation. To study the structure of the other LGICs using biochemical and biophysical techniques, detergent solubilization, purification, and lipid reconstitution are usually required. To assess the effects of purification on receptor structure, we used the hydrophobic photoreactive probe 3-trifluoromethyl-3-(m-[(125)I]iodophenyl)diazirine ([(125)I]TID) to compare the state-dependent photolabeling of the Torpedo nAChR before and after purification and reincorporation into lipid. For the purified nAChR, the agonist-sensitive photolabeling within the M2 ion channel domain of positions M2-6, M2-9, and M2-13, the agonist-enhanced labeling of deltaThr274 (deltaM2-18) within the delta subunit helix bundle, and the labeling at the lipid-protein interface (alphaMu4) were the same as for the nAChR in native membranes. However, addition of agonist did not enhance [(125)I]TID photolabeling of deltaIle288 within the deltaM2-M3 loop. These results indicate that after purification and reconstitution of the Torpedo nAChR, the difference in structure between the resting and desensitized states within the M2 ion channel domain was preserved, but not the agonist-dependent change of structure of the deltaM2-M3 loop. To further characterize the pharmacology of [(125)I]TID binding sites in the nAChR in the desensitized state, we examined the effect of phencyclidine (PCP) on [(125)I]TID photolabeling. PCP inhibited [(125)I]TID labeling of amino acids at the cytoplasmic end of the ion channel (M2-2 and M2-6) while potentiating labeling at M2-9 and M2-13 and allosterically modulating the labeling of amino acids within the delta subunit helix bundle.

Radioligand binding, photoaffinity labeling, and docking and molecular dynamics were used to characterize the tricyclic antidepressant (TCA) binding sites in the nicotinic acetylcholine receptor (nAChR). Competition experiments indicate that the noncompetitive antagonist phencyclidine (PCP) inhibits [3H]imipramine binding to resting (closed) and desensitized nAChRs. [3H]2-azidoimipramine photoincorporates into each subunit from the desensitized nAChR with approximately 25% of the labeling specifically inhibited by TCP (a PCP analog), whereas no TCP-inhibitable labeling was observed in the resting (closed) state. For the desensitized nAChR and within the alpha subunit, the majority of specific [3H]2-azidoimipramine labeling mapped to a approximately 20 kDa Staphylococcus aureus V8 protease fragment (alphaV8-20; Ser173-Glu338). To further map the labeling site, the alphaV8-20 fragment was further digested with endoproteinase Lys-C and resolved by Tricine SDS-PAGE. The principal labeled fragment (11 kDa) was further purified by rpHPLC and subjected to N-terminal sequencing. Based on the amino terminus (alphaMet243) and apparent molecular weight, the 11 kDa fragment contains the channel lining M2 segment. Finally, docking and molecular dynamics results indicate that imipramine and PCP interact preferably with the M2 transmembrane segments in the middle of the ion channel. Collectively, these results are consistent with a model where PCP and TCA bind to overlapping sites within the lumen of the Torpedo nAChR ion channel.

Using an acetylcholine-derivatized affinity column, we have purified human alpha4beta2 neuronal nicotinic acetylcholine receptors (nAChRs) from a stably transfected HEK-293 cell line. Both the quantity and the quality of the purified receptor are suitable for applying biochemical methods to directly study the structure of the alpha4beta2 nAChR. In this first study, the lipid-protein interface of purified and lipid-reconstituted alpha4beta2 nAChRs was directly examined using photoaffinity labeling with the hydrophobic probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID). [125I]TID photoincorporated into both alpha4 and beta2 subunits, and for each subunit the labeling was initially mapped to fragments containing the M4 and M1-M3 transmembrane segments. For both the alpha4 and beta2 subunits, approximately 60% of the total labeling was localized within fragments that contain the M4 segment, which suggests that the M4 segment has the greatest exposure to lipid. Within M4 segments, [125I]TID labeled homologous amino acids alpha4-Cys582/beta2-Cys445, which are also homologous to the [125I]TID-labeled residues alpha1-Cys418 and beta1-Cys447 in the lipid-exposed face of Torpedo nAChR alpha1M4 and beta1M4, respectively. Within the alpha4M1 segment, [125I]TID labeled residues Cys226 and Cys231, which correspond to the [125I]TID-labeled residues Cys222 and Phe227 at the lipid-exposed face of the Torpedo alpha1M1 segment. In beta2M1, [125I]TID labeled beta2-Cys220, which is homologous to alpha4-Cys226. We conclude from these studies that the alpha4beta2 nAChR can be purified from stably transfected HEK-293 cells in sufficient quantity and purity for structural studies and that the lipid-protein interfaces of the neuronal alpha4beta2 nAChR and the Torpedo nAChR display a high degree of structural homology.

Interactions of benzophenone (BP) with the Torpedo nicotinic acetylcholine receptor (nAChR) were characterized by electrophysiological analyses, radioligand binding assays, and photolabeling of nAChR-rich membranes with [3H]BP to identify the amino acids contributing to its binding sites. BP acted as a low potency noncompetitive antagonist, reversibly inhibiting the ACh responses of nAChRs expressed in Xenopus oocytes (IC50 = 600 microM) and the binding of the noncompetitive antagonist [3H]tetracaine to nAChR-rich membranes (IC50 = 150 microM). UV irradiation at 365 nm resulted in covalent incorporation of [3H]BP into the nAChR subunits (delta > alpha approximately beta > gamma), with photoincorporation limited to the nAChR transmembrane domain. Comparison of nAChR photolabeling in the closed state (absence of agonist) and desensitized state (equilibrated with agonist) revealed selective desensitized state labeling in the delta subunit of deltaPhe-232 in deltaM1 and deltaPro-286/deltaIle-288 near the beginning of deltaM3 that are within a pocket at the interface between the transmembrane and extracellular domains. There was labeling in the closed state within the ion channel at position M2-13 (alphaVal-255, betaVal-261, and deltaVal-269) that was reduced by 90% upon desensitization and labeling in the transmembrane M3 helices of the beta and gamma subunits (betaMet-285, betaMet-288, and gammaMet-291) that was reduced by 50-80% in the desensitized state. Labeling at the lipid interface (alphaMet-415 in alphaM4) was unaffected by agonist. These results provide a further definition of the regions in the nAChR transmembrane domain that differ in structure between the closed and desensitized states.

The lipid requirements of the Torpedo californica nicotinic acetylcholine receptor (nAChR) were assessed by reconstituting purified receptors into lipid vesicles of defined composition and by using photolabeling with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine ([125I]TID) to determine functionality. Earlier studies demonstrated that nAChRs reconstituted into membranes containing phosphatidylcholine (PC), the anionic lipid phosphatidic acid (PA), and cholesterol (CH) are particularly effective at stabilizing the nAChR in the resting (closed) state that is capable of undergoing agonist-induced conformational transitions (i.e., functionality). The present studies demonstrate that (1) there is no obligatory requirement for PC, (2) increasing the CH content serves to increase the degree to which nAChRs are stabilized in the resting state, and this effect saturates at approximately 35 mol % (molar lipid percentage), and (3) the effect of increasing levels of PA saturates at approximately 12 mol % and in the absence of PA nAChRs are stabilized in the desensitized state (i.e., nonfunctional). Native Torpedo membranes contain approximately 35 mol % CH but less than 1 mol % PA, suggesting that other anionic lipids may substitute for PA. We report that (1) phosphatidylserine (PS) and phosphatidylinositol (PI), anionic lipids that are abundant in native Torpedo membranes, also stabilize the receptor in the resting state although with reduced efficacy (approximately 50-60%) compared to PA, and (2) for nAChRs reconstituted into PA/CH membranes at different lipid-protein molar ratios, receptor functionality decreases rapidly below approximately 65 lipids per receptor. Collectively, these results are consistent with a functional requirement of a single shell of lipids surrounding the nAChR and specific anionic lipid- and sterol (CH)-protein interactions.

The photoactivatable sterol probe [3alpha-(3)H]6-Azi-5alpha-cholestan-3beta-ol ([3H]Azicholesterol) was used to identify domains in the Torpedo californica nicotinic acetylcholine receptor (nAChR) that interact with cholesterol. [3H]Azicholesterol partitioned into nAChR-enriched membranes very efficiently (>98%), photoincorporated into nAChR subunits on an equal molar basis, and neither the pattern nor the extent of labeling was affected by the presence of the agonist carbamylcholine, consistent with photoincorporation at the nAChR lipid-protein interface. Sites of [3H]Azicholesterol incorporation in each nAChR subunit were initially mapped by Staphylococcus aureus V8 protease digestion to two relatively large homologous fragments that contain either the transmembrane segments M1-M2-M3 (e.g., alphaV8-20) or M4 (e.g., alphaV8-10). The distribution of [3H]Azicholesterol labeling between these two fragments (e.g., alphaV8-20, 29%; alphaV8-10, 71%), suggests that the M4 segment has the greatest interaction with membrane cholesterol. Photolabeled amino acid residues in each M4 segment were identified by Edman degradation of isolated tryptic fragments and generally correspond to acidic residues located at either end of each transmembrane helix (e.g., alphaAsp-407). [3H]Azicholesterol labeling was also mapped to peptides that contain either the M3 or M1 segment of each nAChR subunit. These results establish that cholesterol likely interacts with the M4, M3, and M1 segments of each subunit, and therefore, the cholesterol binding domain fully overlaps the lipid-protein interface of the nAChR.