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It has been almost 20 years since the discovery and crystallization of a structural surrogate, the Lymnaea stagnalis acetylcholine binding protein (Ls-AChBP), comprising the extracellular domain of the nicotinic acetylcholine receptors (nAChRs). Structural characterization of this soluble protein has increased our understanding of the requirements for agonist and antagonist interactions at the ligand recognition site of the nAChRs. Application can be extended to orthologs in the pentameric ligand-gated ion channel superfamily, encompassing receptors that depolarize or hyperpolarize upon neurotransmitter association. Despite the lack of transmembrane and intracellular motifs, the highly conserved binding or recognition loci have made these soluble binding proteins, and mutants derived from them, prototypic tools for molecular recognition and structural studies of pentameric ligand-gated ion channels. Targeting nAChRs has been a major goal as this family is associated with neurodegenerative diseases and disorders. Accordingly, the ligand binding site has played a key role to the development of selective ligands for modulation of this transmembrane proteins. In this review article, we cover both the potential and limitations of soluble surrogates, termed the AChBP family, in drug development. This article is part of the special issue on 'Contemporary Advances in Nicotine Neuropharmacology'., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Nicotine is a highly addictive drug found in tobacco that drives its continued use despite the harmful consequences. The initiation of nicotine abuse involves the mesolimbic dopamine system, which contributes to the rewarding sensory stimuli and associative learning processes in the beginning stages of addiction. Nicotine binds to neuronal nicotinic acetylcholine receptors (nAChRs), which come in a diverse collection of subtypes. The nAChRs that contain the α4 and β2 subunits, often in combination with the α6 subunit, are particularly important for nicotine's ability to increase midbrain dopamine neuron firing rates and phasic burst firing. Chronic nicotine exposure results in numerous neuroadaptations, including the upregulation of particular nAChR subtypes associated with long-term desensitization of the receptors. When nicotine is no longer present, for example during attempts to quit smoking, a withdrawal syndrome develops. The expression of physical withdrawal symptoms depends mainly on the α2, α3, α5, and β4 nicotinic subunits in the epithalamic habenular complex and its target regions. Thus, nicotine affects diverse neural systems and an array of nAChR subtypes to mediate the overall addiction process. This article is part of the special issue on 'Contemporary Advances in Nicotine Neuropharmacology'., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Nicotinic acetylcholine receptors (nAChR) are homo- or hetero-pentameric ligand-gated ion channels of the Cys-loop superfamily and play important roles in the nervous system and muscles. Studies on nAChR benefit from in silico modeling due to the lack of high-resolution structures for most receptor subtypes and challenges in experiments addressing the complex mechanism of activation involving allosteric sites. Although there is myriad of computational modeling studies on nAChR, the multitude of the methods and parameters used in these studies makes modeling nAChR a daunting task, particularly for the non-experts in the field. To address this problem, the modeling literature on Torpedo nAChR and α7 nAChR were focused on as examples of heteromeric and homomeric nAChR, and the key in silico modeling studies between the years 1995-2019 were concisely reviewed. This was followed by a critical analysis of these studies by comparing the findings with each other and with the emerging experimental and computational data on nAChR. Based on these critical analyses, suggestions were made to guide the future researchers in the field of in silico modeling of nAChR. This article is part of the special issue on 'Contemporary Advances in Nicotine Neuropharmacology'., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Here we begin by briefly reviewing landmark structural studies on the nicotinic acetylcholine receptor. We highlight challenges that had to be overcome to push through resolution barriers, then focus on what has been gleaned in the past few years from crystallographic and single particle cryo-EM studies of different nicotinic receptor subunit assemblies and ligand complexes. We discuss insights into ligand recognition, ion permeation, and allosteric gating. We then highlight some foundational aspects of nicotinic receptor structural biology that remain unresolved and are areas ripe for future exploration. This article is part of the special issue on 'Contemporary Advances in Nicotine Neuropharmacology'., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Postsynaptic nAChRs in the peripheral nervous system are critical for neuromuscular and autonomic neurotransmission. Pre- and peri-synaptic nAChRs in the brain modulate neurotransmission and are responsible for the addictive effects of nicotine. Subtypes of nAChRs in lymphocytes and non-synaptic locations may modulate inflammation and other cellular functions. All AChRs that function as ligand-gated ion channels are formed from five homologous subunits organized to form a central cation channel whose opening is regulated by ACh bound at extracellular subunit interfaces. nAChR subtype subunit composition can range from α7 homomers to α4β2α6β2β3 heteromers. Subtypes differ in affinities for ACh and other agonists like nicotine and in efficiencies with which their channels are opened and desensitized. Subtypes also differ in affinities for antagonists and for positive and negative allosteric modulators. Some agonists are "silent" with respect to channel opening, and AChRs may be able to signal metabotropic pathways by releasing G-proteins independent of channel opening. Electrophysiological studies that can resolve single-channel openings and molecular genetic approaches have allowed characterization of the structures of ligand binding sites, the cation channel, and the linkages between them, as well as the organization of AChR subunits and their contributions to function. Crystallography and cryo-electron-microscopy are providing increasing insights into the structures and functions of AChRs. However, much remains to be learned about both AChR structure and function, the in vivo functional roles of some AChR subtypes, and the development of better pharmacological tools directed at AChRs to treat addiction, pain, inflammation, and other medically important issues. This article is part of the special issue on 'Contemporary Advances in Nicotine Neuropharmacology'., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The ten types of nicotinic acetylcholine receptor α-subunits show substantial sequence homology, yet some types confer high affinity for α-bungarotoxin, whereas others confer negligible affinity. Combining sequence alignments with structural data reveals three residues unique to α-toxin-refractory α-subunits that coalesce within the 3D structure of the α4β2 receptor and are predicted to fit between loops I and II of α-bungarotoxin. Mutating any one of these residues, Lys189, Ile196 or Lys153, to the α-toxin-permissive counterpart fails to confer α-bungarotoxin binding. However, mutating both Lys189 and Ile196 affords α-bungarotoxin binding with an apparent dissociation constant of 104 nM, while combining mutation of Lys153 reduces the dissociation constant to 22 nM. Analogous residue substitutions also confer high affinity α-bungarotoxin binding upon α-toxin-refractory α2 and α3 subunits. α4β2 receptors engineered to bind α-bungarotoxin exhibit slow rates of α-toxin association and dissociation, and competition by cholinergic ligands typical of muscle nicotinic receptors. Receptors engineered to bind α-bungarotoxin co-sediment with muscle nicotinic receptors on sucrose gradients, and mirror single channel signatures of their α-toxin-refractory counterparts. Thus the inability of α-bungarotoxin to bind to neuronal nicotinic receptors arises from three unique and interdependent residues that coalesce within the receptor's 3D structure., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

α6-containing (α6*) nicotinic acetylcholine receptors (nAChRs) are expressed throughout the periphery and the central nervous system and constitute putative therapeutic targets in pain, addiction and movement disorders. The α6β2* nAChRs are relatively well studied, in part due to the availability of target specific α-conotoxins (α-Ctxs). In contrast, all native α-Ctxs identified that potently block α6β4 nAChRs exhibit higher potencies for the closely related α6β2β3 and/or α3β4 subtypes. In this study, we have identified a novel peptide from Conus ventricosus with pronounced selectivity for the α6β4 nAChR. The peptide-encoding gene was cloned from genomic DNA and the predicted mature peptide, α-Ctx VnIB, was synthesized. The functional properties of VnIB were characterized at rat and human nAChRs expressed in Xenopus oocytes by two-electrode voltage clamp electrophysiology. VnIB potently inhibited ACh-evoked currents at rα6β4 and rα6/α3β4 nAChRs, displayed ∼20-fold and ∼250-fold lower potencies at rα3β4 and rα6/α3β2β3 receptors, respectively, and exhibited negligible effects at eight other nAChR subtypes. Interestingly, even higher degrees of selectivity were observed for hα6/α3β4 over hα6/α3β2β3 and hα3β4 receptors. Finally, VnIB displayed fast binding kinetics at rα6/α3β4 (on-rate t ½  = 0.87 min -1 , off-rate t ½  = 2.7 min -1 ). The overall preference of VnIB for β4* over β2* nAChRs is similar to the selectivity profiles of other 4/6 α-Ctxs. However, in contrast to previously identified native α-Ctxs targeting α6* nAChRs, VnIB displays pronounced selectivity for α6β4 nAChRs over both α3β4 and α6β2β3 receptors. VnIB thus represents a novel molecular probe for elucidating the physiological role and therapeutic properties of α6β4* nAChRs., (Published by Elsevier Ltd.)

κ-Hexatoxins (κ-HXTXs) are a family of excitotoxic insect-selective neurotoxins from Australian funnel-web spiders that are lethal to a wide range of insects, but display no toxicity towards vertebrates. The prototypic κ-HXTX-Hv1c selectively blocks native and expressed cockroach large-conductance calcium-activated potassium (BK Ca or K Ca 1.1) channels, but not their mammalian orthologs. Despite this potent and selective action on insect K Ca 1.1 channels, we found that the classical K Ca 1.1 blockers paxilline, charybdotoxin and iberiotoxin, which all block insect K Ca 1.1 channels, are not lethal in crickets. We therefore used whole-cell patch-clamp analysis of cockroach dorsal unpaired median (DUM) neurons to study the effects of κ-HXTX-Hv1c on sodium-activated (K Na ), delayed-rectifier (K DR ) and 'A-type' transient (K A ) K + channels. 1 μM κ-HXTX-Hv1c failed to significantly inhibit cockroach K Na and K DR channels, but did cause a 30 ± 7% saturating inhibition of K A channel currents, possibly via a Kv4 (Shal-like) action. However, this modest action at such a high concentration of κ-HXTX-Hv1c would indicate a different lethal target. Accordingly, we assessed the actions of κ-HXTX-Hv1c on neurotransmitter-gated ion channels in cockroach DUM neurons. We found that κ-HXTX-Hv1c failed to produce any major effects on GABA A or glutamate-Cl receptors but dramatically slowed nicotine-evoked ACh receptor (nAChR) current decay and reversed nAChR desensitization. These actions occurred without any alterations to nAChR current amplitude or the nicotine concentration-response curve, and are consistent with a positive allosteric modulation of nAChRs. κ-HXTX-Hv1c therefore represents the first venom peptide that selectively modulates insect nAChRs with a mode of action similar to the excitotoxic insecticide spinosyn A. This article is part of the Special Issue entitled 'Venom-derived Peptides as Pharmacological Tools.', (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Animal toxins are peptides that often bind with remarkable affinity and selectivity to membrane receptors such as nicotinic acetylcholine receptors (nAChRs). The latter are, for example, targeted by α-conotoxins, a family of peptide toxins produced by venomous cone snails. nAChRs are implicated in numerous physiological processes explaining why the design of new pharmacological tools and the discovery of potential innovative drugs targeting these receptor channels appear so important. This work describes a methodology developed to discover new ligands of nAChRs from complex mixtures of peptides. The methodology was set up by the incubation of Torpedo marmorata electrocyte membranes rich in nAChRs with BSA tryptic digests (>100 peptides) doped by small amounts of known nAChRs ligands (α-conotoxins). Peptides that bind to the receptors were purified and analyzed by MALDI-TOF/TOF mass spectrometry which revealed an enrichment of α-conotoxins in membrane-containing fractions. This result exhibits the binding of α-conotoxins to nAChRs. Negative controls were performed to demonstrate the specificity of the binding. The usefulness and the power of the methodology were also investigated for a discovery issue. The workflow was then applied to the screening of Conus ermineus crude venom, aiming at characterizing new nAChRs ligands from this venom, which has not been extensively investigated to date. The methodology validated our experiments by allowing us to bind two α-conotoxins (α-EI and α-EIIA) which have already been described as nAChRs ligands. Moreover, a new conotoxin, never described to date, was also captured, identified and sequenced from this venom. Classical pharmacology tests by radioligand binding using a synthetic homologue of the toxin confirm the activity of the new peptide, called α-EII B . The K i value of this peptide for Torpedo nicotinic receptors was measured at 2.2 ± 0.7 nM., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Studies with heterologous expression systems have shown that the α4β2 nicotinic acetylcholine receptor (nAChR) subtype can exist in two stoichiometries (with two [(α4)2(β2)3] or three [(α4)3(β2)2] copies of the α subunit in the receptor pentamer) which have different pharmacological and functional properties and are differently regulated by chronic nicotine treatment. However, the effects of nicotine treatment in vivo on native α4β2 nAChR stoichiometry are not well known. We investigated in C57BL/6 mice the in vivo effect of 14-day chronic nicotine treatment and subsequent withdrawal, on the subunit expression and β2/α4 subunit ratio of (3)H-epibatidine labeled α4β2*-nAChR in total homogenates of cortex and thalamus. We found that in basal conditions the ratio of the β2/α4 subunit in the cortex and thalamus is different indicating a higher proportion in receptors with (α4)2(β2)3 subunit stoichiometry in the thalamus. For cortex exposure to chronic nicotine elicited an increase in receptor density measured by (3)H-epibatidine binding, an increase in the α4 and β2 protein levels, and an increase in β2/α4 subunit ratio, that indicates an increased proportion of receptors with the (α4)2(β2)3 stoichiometry. For thalamus we did not find a significant increase in receptor density, α4 and β2 protein levels, or changes in β2/α4 subunit ratio. All the changes elicited by chronic nicotine in cortex were transient and returned to basal levels with an average half-life of 2.8 days following nicotine withdrawal. These data suggest that chronic nicotine exposure in vivo favors increased assembly of α4β2 nAChR containing three β2 subunits. A greater change in stoichiometry was observed for cortex (which has relatively low basal expression of (α4)2(β2)3 nAChR) than in thalamus (which has a relatively high basal expression of (α4)2(β2)3 nAChR)., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

How does an agonist activate a receptor? In this article I consider the activation process in muscle nicotinic acetylcholine receptors (AChRs), a prototype for understanding the energetics of binding and gating in other ligand-gated ion channels. Just as movements that generate gating currents activate voltage-gated ion channels, movements at binding sites that generate an increase in affinity for the agonist activate ligand-gated ion channels. The main topics are: i) the schemes and intermediate states of AChR activation, ii) the energy changes of each of the steps, iii) the sources of the energies, iv) the three kinds of AChR agonist binding site and v) the correlations between binding and gating energies. The binding process is summarized as sketches of different conformations of an agonist site. The results suggest that agonists lower the free energy of the active conformation of the protein in stages by establishing favorable, local interactions at each binding site, independently. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Pentameric ligand-gated ion channels (pLGICs) play a central role in intercellular communications in the nervous system by converting the binding of a chemical messenger - a neurotransmitter - into an ion flux through the postsynaptic membrane. Here, we present an overview of the most recent advances on the signal transduction mechanism boosted by X-ray crystallography of both prokaryotic and eukaryotic homologues of the nicotinic acetylcholine receptor (nAChR) in conjunction with time-resolved analyses based on single-channel electrophysiology and Molecular Dynamics simulations. The available data consistently point to a global mechanism of gating that involves a large reorganization of the receptor mediated by two distinct quaternary transitions: a global twisting and a radial expansion/contraction of the extracellular domain. These transitions profoundly modify the organization of the interface between subunits, which host several sites for orthosteric and allosteric modulatory ligands. The same mechanism may thus mediate both positive and negative allosteric modulations of pLGICs ligand binding at topographically distinct sites. The emerging picture of signal transduction is expected to pave the way to new pharmacological strategies for the development of allosteric modulators of nAChR and pLGICs in general. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Neuronal nicotinic acetylcholine receptors (nAChRs) are a heterogeneous family of pentameric ligand-gated cation channels that are expressed throughout the brain and involved in a wide range of physiological and pathophysiological processes. The nAChR subtypes share a common basic structure, but their biophysical and pharmacological properties depend on their subunit composition, which is therefore central to understanding their function in the nervous system and discovering new subtype selective drugs. The development of new technologies and the generation of mice carrying deletions or the expression of gain-of-function nAChR subunits, or GFP-tagged receptor genes has allowed the in vivo identification of complex subtypes and to study the role of individual subtypes in specific cells and complex neurobiological systems but much less is known about which native nAChR subtypes are involved in specific physiological functions and pathophysiological conditions in human brain. We briefly review some recent findings concerning the structure and function of native nAChRs, focussing on the subtypes identified in the rodent habenulo-interpeduncular pathway, a pathway involved in nicotine reinforcement and withdrawal. We also discuss recent findings concerning the expression of native subtypes in primate brain. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

General anesthetics are a heterogeneous group of small amphiphilic ligands that interact weakly at multiple allosteric sites on many pentameric ligand gated ion channels (pLGICs), resulting in either inhibition, potentiation of channel activity, or both. Allosteric principles imply that modulator sites must change configuration and ligand affinity during receptor state transitions. Thus, general anesthetics and related compounds are useful both as state-dependent probes of receptor structure and as potentially selective modulators of pLGIC functions. This review focuses on general anesthetic sites in nicotinic acetylcholine receptors, which were among the first anesthetic-sensitive pLGIC experimental models studied, with particular focus on sites formed by transmembrane domain elements. Structural models place many of these sites at interfaces between two or more pLGIC transmembrane helices both within subunits and between adjacent subunits, and between transmembrane helices and either lipids (the lipid-protein interface) or water (i.e. the ion channel). A single general anesthetic may bind at multiple allosteric sites in pLGICs, producing a net effect of either inhibition (e.g. blocking the ion channel) or enhanced channel gating (e.g. inter-subunit sites). Other general anesthetic sites identified by photolabeling or crystallography are tentatively linked to functional effects, including intra-subunit helix bundle sites and the lipid-protein interface. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Nicotinic acetylcholine receptors (nAChRs) are highly conserved between humans and non-human primates. Conservation exists at the level of genomic structure, protein structure and epigenetics. Overall homology of nAChRs at the protein level is 98% in macaques versus 89% in mice, which is highly relevant for evaluating subtype-specific ligands that have different affinities in humans versus rodents. In addition to conservation at the protein level, there is high conservation of genomic structure in terms of intron and exon size and placement of CpG sites that play a key role in epigenetic regulation. Analysis of single nucleotide polymorphisms (SNPs) shows that while the majority of SNPs are not conserved between humans and macaques, some functional polymorphisms are. Most significantly, cynomolgus monkeys express a similar α5 nAChR Asp398Asn polymorphism to the human α5 Asp398Asn polymorphism that has been linked to greater nicotine addiction and smoking related disease. Monkeys can be trained to readily self-administer nicotine, and in an initial study we have demonstrated that cynomolgus monkeys bearing the α5 D398N polymorphism show a reduced behavioral sensitivity to oral nicotine and tend to consume it in a different pattern when compared to wild-type monkeys. Thus the combination of highly homologous nAChR, higher cortical functions and capacity for complex training makes non-human primates a unique model to study in vivo functions of nicotinic receptors. In particular, primate studies on nicotine addiction and evaluation of therapies to prevent or overcome nicotine addiction are likely to be highly predictive of treatment outcomes in humans. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The current study compares the antidepressant-like effect elicited by nicotine between wild-type (β4+/+) and knockout (β4-/-) mice, and subsequently, the effect of 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2), a positive allosteric modulator of α7 nicotinic receptors, on the previously determined activity of nicotine. Mice from each sex were injected daily with nicotine base (0.2 mg/kg; s.c.) or co-administered with PAM-2 (1.0 mg/kg; i.p.) for 3 weeks. Forced swim tests were performed to determine the acute (day 1), subchronic (day 7), and chronic (days 14 and 21) effects of the drugs, as well as their residual effects after treatment cessation (days 28 and 35). Our results indicate that nicotine mediates antidepressant-like activity after acute, subchronic, and chronic treatments in β4+/+, but not β4-/-, mice, and that these effects are not mediated by unspecific locomotor stimulation. Nicotine co-administered with PAM-2 produces antidepressant-like activity in both β4+/+ and β4-/- mice, except after the acute treatment of β4-/- mice, and decreases locomotor activity. This suggests that although the β4 subunit regulates the antidepressant-like activity of nicotine it does not affect the activity elicited by PAM-2 when is co-administered with nicotine. The residual antidepressant-like activity of PAM-2 + nicotine was observed only in female mice, suggesting gender-specific differences. Our findings clearly indicate that β4-containing nAChRs play an important role in the antidepressant-like activity elicited by nicotine but they are not essential for the modulatory activity of PAM-2. In fact, PAM-2 inhibits α4β4 and α3β4 AChRs at higher concentration ranges compared to that for the PAM activity previously found at the α7 AChR., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Honey badgers (Mellivora capensis) prey upon and survive bites from venomous snakes (Family: Elapidae), but the molecular basis of their venom resistance is unknown. The muscular nicotinic cholinergic receptor (nAChR), targeted by snake α-neurotoxins, has evolved in some venom-resistant mammals to no longer bind these toxins. Through phylogenetic analysis of mammalian nAChR sequences, we show that honey badgers, hedgehogs, and pigs have independently acquired functionally equivalent amino acid replacements in the toxin-binding site of this receptor. These convergent amino acid changes impede toxin binding by introducing a positively charged amino acid in place of an uncharged aromatic residue. In venom-resistant mongooses, different replacements at these same sites are glycosylated, which is thought to disrupt binding through steric effects. Thus, it appears that resistance to snake venom α-neurotoxin has evolved at least four times among mammals through two distinct biochemical mechanisms operating at the same sites on the same receptor., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Neuronal nicotinic acetylcholine receptors (nAChRs) are widely distributed in the nervous system and are implicated in many normal and pathological processes. The structural determinants of allostery in nAChRs are not well understood. One class of nAChR allosteric modulators, including the small molecule morantel (Mor), acts from a site that is structurally homologous to the canonical agonist site but exists in the β(+)/α(-) subunit interface. We hypothesized that all nAChR subunits move with respect to each other during channel activation and allosteric modulation. We therefore studied five pairs of residues predicted to span the interfaces of α3β2 receptors, one at the agonist interface and four at the modulator interface. Substituting cysteines in these positions, we used disulfide trapping to perturb receptor function. The pair α3Y168-β2D190, involving the C loop region of the β2 subunit, mediates modulation and agonist activation, because evoked currents were reduced up to 50% following oxidation (H2O2) treatment. The pair α3S125-β2Q39, below the canonical site, is also involved in channel activation, in accord with previous studies of the muscle-type receptor; however, the pair is differentially sensitive to ACh activation and Mor modulation (currents decreased 60% and 80%, respectively). The pairs α3Q37-β2A127 and α3E173-β2R46, both in the non-canonical interface, showed increased currents following oxidation, suggesting that subunit movements are not symmetrical. Together, our results from disulfide trapping and further mutation analysis indicate that subunit interface movement is important for allosteric modulation of nAChRs, but that the two types of interfaces contribute unequally to receptor activation., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

To develop antigen-specific immunotherapies for autoimmune diseases, knowledge of the molecular structure of targeted immunological hotspots will guide the production of reagents to inhibit and halt production of antigen specific attack agents. To this end we have identified three noncontiguous segments of the Torpedo nicotinic acetylcholine receptor (AChR) α-subunit that contribute to the conformationally sensitive immunological hotspot on the AChR termed the main immunogenic region (MIR): α(1-12), α(65-79), and α(110-115). This region is the target of greater than 50% of the anti-AChR Abs in serum from patients with myasthenia gravis (MG) and animals with experimental autoimmune myasthenia gravis (EAMG). Many monoclonal antibodies (mAbs) raised in one species against an electric organ AChR cross react with the neuromuscular AChR MIR in several species. Probing the Torpedo AChR α-subunit with mAb 132A, a disease inducing anti-MIR mAb raised against the Torpedo AChR, we have determined that two of the three MIR segments, α(1-12) and α(65-79), form a complex providing the signature components recognized by mAb 132A. These two segments straddle a third, α(110-115), that seems not to contribute specific side chains for 132A recognition, but is necessary for optimum antibody binding. This third segment appears to form a foundation upon which the three-dimensional 132A epitope is anchored., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Recent genetic and pharmacological studies have implicated the α3, β4 and α5 subunits of the nicotinic acetylcholine receptor (nAChR) in dependence to nicotine and other abused drugs and nicotine withdrawal. The α3β4* nAChR subtype has been shown to co-assemble with the α5 or β3 nAChR subunits, and is found mainly in the autonomic ganglia and select brain regions. It has been difficult to study the α3β4 nAChR because there have been no selective nonpeptidic ligands available to independently examine its pharmacology. We recently reported the synthesis of a [(125)I]-radiolabeled analog of a high affinity, selective small-molecule α3β4 nAChR ligand, AT-1012. We report here the vitro characterization of this radioligand in receptor binding and in vitro autoradiographic studies targeting the α3β4* nAChR. Binding of [(125)I]AT-1012 was characterized at the rat α3β4 and α4β2 nAChR transfected into HEK cells, as well as at the human α3β4α5 nAChR in HEK cells. Binding affinity of [(125)I]AT-1012 at the rat α3β4 nAChR was 1.4 nM, with a B(max) of 10.3 pmol/mg protein, similar to what was determined for unlabeled AT-1012 using [(3)H]epibatidine. Saturation isotherms suggested that [(125)I]AT-1012 binds to a single site on the α3β4 nAChR. Similar high binding affinity was also observed for [(125)I]AT-1012 at the human α3β4α5 nAChR transfected into HEK cells. [(125)I]AT-1012 did not bind with high affinity to membranes from α4β2 nAChR-transfected HEK cells. Binding studies with [(3)H]epibatidine further confirmed that AT-1012 had over 100-fold binding selectivity for α3β4 over α4β2 nAChR. K(i) values determined for known nAChR compounds using [(125)I]AT-1012 as radioligand were comparable to those obtained with [(3)H]epibatidine. [(125)I]AT-1012 was also used to label α3β4 nAChR in rat brain slices in vitro using autoradiography, which showed highly localized binding of the radioligand in brain regions consistent with the discreet localization of the α3β4 nAChR. We demonstrate that [(125)I]AT-1012 is an excellent tool for labeling the α3β4 nAChR in the presence of other nAChR subtypes., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Considerable evidence indicates that adenosine A(2A) receptors (A(2A)Rs) modulate cholinergic neurotransmission, nicotinic acetylcholine receptor (nAChR) function, and nicotine-induced behavioural effects. To explore the interaction between A(2A) and nAChRs, we examined if the complete genetic deletion of adenosine A(2A)Rs in mice induces compensatory alterations in the binding of different nAChR subtypes, and whether the long-term effects of nicotine on nAChR regulation are altered in the absence of the A(2A)R gene. Quantitative autoradiography was used to measure cytisine-sensitive [¹²⁵I]epibatidine and [¹²⁵I]α-bungarotoxin binding to α4β2* and α7 nAChRs, respectively, in brain sections of drug-naïve (n = 6) or nicotine treated (n = 5-7), wild-type and adenosine A(2A)R knockout mice. Saline or nicotine (7.8 mg/kg/day; free-base weight) were administered to male CD1 mice via subcutaneous osmotic minipumps for a period of 14 days. Blood plasma levels of nicotine and cotinine were measured at the end of treatment. There were no compensatory developmental alterations in nAChR subtype distribution or density in drug-naïve A(2A)R knockout mice. In nicotine treated wild-type mice, both α4β2* and α7 nAChR binding sites were increased compared with saline treated controls. The genetic ablation of adenosine A(2A)Rs prevented nicotine-induced upregulation of α7 nAChRs, without affecting α4β2* receptor upregulation. This selective effect was observed at plasma levels of nicotine that were within the range reported for smokers (10-50 ng ml⁻¹). Our data highlight the involvement of adenosine A(2A)Rs in the mechanisms of nicotine-induced α7 nAChR upregulation, and identify A(2A)Rs as novel pharmacological targets for modulating the long-term effects of nicotine on α7 receptors., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

L-Dopa-induced dyskinesias are a serious long-term side effect of dopamine replacement therapy for Parkinson's disease for which there are few treatment options. Our previous studies showed that nicotine decreased l-dopa-induced abnormal involuntary movements (AIMs). Subsequent work with knockout mice demonstrated that α6β2* nicotinic receptors (nAChRs) play a key role. The present experiments were done to determine if α4β2* nAChRs are also involved in l-dopa-induced dyskinesias. To approach this, we took advantage of the finding that α6β2* nAChRs are predominantly present on striatal dopaminergic nerve terminals, while a significant population of α4β2* nAChRs are located on other neurons. Thus, a severe dopaminergic lesion would cause a major loss in α6β2*, but not α4β2* nAChRs. Experiments were therefore done in which rats were unilaterally lesioned with 6-hydroxydopamine, at a dose that led to severe nigrostriatal damage. The dopamine transporter, a dopamine nerve terminal marker, was decreased by >99%. This lesion also decreased striatal α6β2* nAChRs by 97%, while α4β2* nAChRs were reduced by only 12% compared to control. A series of β2* nAChR compounds, including TC-2696, TI-10165, TC-8831, TC-10600 and sazetidine reduced l-dopa-induced AIMs in these rats by 23-32%. TC-2696, TI-10165, TC-8831 were also tested for parkinsonism, with no effect on this behavior. Tolerance did not develop with up to 3 months of treatment. Since α4α5β2 nAChRs are also predominantly on striatal dopamine terminals, these data suggest that drugs targeting α4β2 nAChRs may reduce l-dopa-induced dyskinesias in late stage Parkinson's disease., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The cognitive deficits associated with schizophrenia are recognized as a core component of the disorder, yet there remain no available therapeutics to treat these symptoms of the disease. As a result, there is a need for establishing predictive preclinical models to identify the therapeutic potential of novel compounds. In the present study, rhesus monkeys were trained in the object retrieval-detour task, which is dependent on the prefrontal cortex, a brain region implicated in the cognitive deficits associated with schizophrenia. The NMDA receptor antagonist ketamine significantly impaired performance without affecting measures of motor or visuospatial abilities. Pre-treatment with the nicotinic α7 agonist GTS-21 (0.03 mg/kg) significantly attenuated the ketamine-induced impairment, consistent with reports from clinical trials suggesting that nicotinic α7 receptor agonism has pro-cognitive potential in clinical populations. In contrast, pretreatment with the acetylcholinesterase inhibitor donepezil failed to reverse the ketamine-induced impairment, consistent with studies showing a lack of pro-cognitive effects in patients with schizophrenia. These data suggest that the ketamine-impaired object retrieval-detour task could provide a model with improved predictive validity for drug development, and confirm the need for additional efforts in back-translation. This article is part of a Special Issue entitled 'Cognitive Enhancers'., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Cognitive deficits in schizophrenia are critically important predictors of long-term psychosocial outcome and are not significantly ameliorated by currently available medications. Cognitive remediation training has shown promise for alleviating cognitive symptoms of schizophrenia, but the clinical significance has often been limited by small effect sizes. Approaches that achieve larger improvement involve time requirements that can be cost-prohibitive within the current clinical care system. This mini-review evaluates the theoretical potential of a pharmacological enhancement strategy of cognitive remediation training with nicotinic acetylcholine receptor (nAChR) agonists. nAChR agonists can facilitate sensory processing, alertness, attention, learning and memory. While these effects may be too subtle and short-lasting to be of clinical relevance as a primary treatment of cognitive deficits, they constitute an ideal effects profile for enhancing training benefits. Several mechanisms are described through which repeated coupling of cognitive training challenges with nAChR stimulation may enhance and accelerate cognitive remediation training effects, advancing such interventions into more effective and practicable treatments of some of the most debilitating symptoms of schizophrenia. This article is part of a Special Issue entitled 'Cognitive Enhancers'., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In this multicenter, double-blind, placebo-controlled, randomized, four way cross-over proof-of-mechanism study, we tested the effect of the positive allosteric α7 nicotinic acetylcholine receptor (nAChR) modulator JNJ-39393406 in a key translational assay (sensory P50 gating) in 39 regularly smoking male patients with schizophrenia. All patients were clinically stable and JNJ-39393406 was administered as an adjunct treatment to antipsychotics. No indication was found that JNJ-39393406 has the potential to reverse basic deficits of information processing in schizophrenia (sensory P50 gating) or has a significant effect on other tested electrophysiological markers (MMN, P300 and quantitative resting EEG). Sensitivity analyses including severity of disease, baseline P50 gating, medication and gene variants of the CHRNA7 gene did not reveal any subgroups with consistent significant effects. It is discussed that potential positive effects in subgroups not present or not large enough in the current study or upon chronic dosing are possible, but unlikely to be developed. This article is part of a Special Issue entitled 'Cognitive Enhancers'., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A large number of studies in both humans and experimental animals have demonstrated nicotine-induced improvements in various aspects of cognitive function, including attention and memory. The prefrontal cortex (PFC) is thought to be critically involved in the modulation of executive function and these attentional processes are enhanced by nicotine acting at nicotinic acetylcholine receptors. The involvement of nicotinic processes on cognitive flexibility in particular has not been specifically investigated. The effects of nicotine on attentional flexibility were therefore evaluated using the rodent attentional set shifting task in rats. Nicotine injected both acutely and following repeated pre-exposure significantly improved both intradimensional and extradimensional set shifting performance in the task. Further investigation of the acute effects of nicotine demonstrated this improvement in attentional flexibility to be dose-dependent. These results implicate the nicotinic receptor system in the mediation of processes underlying cognitive flexibility and suggest that nicotine improves attentional flexibility in rats, both within and between perceptual dimensions of a compound stimulus. Nicotine-induced alterations in prefrontal circuitry may underlie these effects on cognitive flexibility. This article is part of a Special Issue entitled 'Cognitive Enhancers'., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Substantial progress in the identification of genes encoding for a large number of proteins responsible for various aspects of neurotransmitter release, postsynaptic detection and downstream signaling, has advanced our understanding of the mechanisms by which neurons communicate and interact. Nicotinic acetylcholine receptors represent a large and well-characterized family of ligand-gated ion channels that is expressed broadly throughout the central and peripheral nervous system, and in non-neuronal cells. With 16 mammalian genes identified that encode for nicotinic receptors and the ability of the subunits to form heteromeric or homomeric receptors, the repertoire of conceivable receptor subtype combinations is enormous and offers unique possibilities for the design and development of new therapeutics that target nicotinic acetylcholine receptors. The aim of this review is to provide the reader with recent insights in nicotinic acetylcholine receptors from genes, structure and function to diseases, and with the latest findings on the pharmacology of these receptors. Although so far only a few nicotinic drugs have been marketed or are in late stage development, much progress has been made in the design of novel chemical entities that are being explored for the treatment of various diseases, including addiction, depression, ADHD, cognitive deficits in schizophrenia and Alzheimer's disease, pain and inflammation. A pharmacological analysis of these compounds, including those that were discontinued, can improve our understanding of the pharmacodynamic and pharmacokinetic requirements for nicotinic 'drug-like' molecules and will reveal if hypotheses on therapies based on targeting specific nicotinic receptor subtypes have been adequately tested in the clinic., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Cocaine use is associated with impaired cognitive function, which may negatively impact treatment outcomes. One pharmacological strategy to improve cognition involves nicotinic acetylcholine receptor (nAChR) stimulation. However, the effects of chronic cocaine exposure on nAChR distribution and function have not been characterized. Thus, one goal of this study was to examine nAChR availability in rhesus monkeys with an extensive cocaine self-administration history (n = 4; ~6 years, mean intake, 1463 mg/kg) compared to age-matched cocaine-naive control monkeys (n = 5). Using [¹¹C]-nicotine and positron emission tomography (PET) imaging, cocaine-experienced monkeys showed significantly higher receptor availability in the hippocampus compared to cocaine-naive monkeys. A second goal was to examine the effects of nAChR agonists on multiple domains of cognitive performance in these same monkeys. For these studies, working memory was assessed using a delayed match-to-sample (DMS) task, associative learning and behavioral flexibility using stimulus discrimination and reversal learning tasks. When administered acutely, the nonselective high-efficacy agonist nicotine, the low-efficacy α4β2* subtype-selective agonist varenicline and the high-efficacy α7 subtype-selective agonist, PNU-282987 significantly improved DMS performance in both cocaine-naive and cocaine-experienced monkeys. Individual doses of nicotine and varenicline that engendered maximum cognitive enhancing effects on working memory did not affect discrimination or reversal learning, while PNU-282987 disrupted reversal learning in the cocaine-naive monkeys. These findings indicate that a cocaine self-administration history influenced nAChR distribution and the effects of nAChR agonists on cognitive performance, including a reduced sensitivity to the disrupting effects on reversal learning. The cognitive enhancing effects of nAChR agonists may be beneficial in combination with behavioral treatments for cocaine addiction. This article is part of a Special Issue entitled 'Cognitive Enhancers'., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The pharmacological properties of (±)-2-(N-tert-butylamino)-3'-iodo-4'-azidopropiophenone [(±)-SADU-3-72], a photoreactive analog of bupropion (BP), were characterized at different muscle nicotinic acetylcholine receptors (AChRs) by functional and structural approaches. Ca²⁺ influx results indicate that (±)-SADU-3-72 is 17- and 6-fold more potent than BP in inhibiting human (h) embryonic (hα1β1γδ) and adult (hα1β1εδ) muscle AChRs, respectively. (±)-SADU-3-72 binds with high affinity to the [³H]TCP site within the resting or desensitized Torpedo AChR ion channel, whereas BP has higher affinity for desensitized AChRs. Molecular docking results indicate that both SADU-3-72 enantiomers interact with the valine (position 13') and serine (position 6') rings. However, an additional domain, between the outer (position 20') and valine rings, is observed in Torpedo AChR ion channels. Our results indicate that the azido group of (±)-SADU-3-72 may enhance its interaction with polar groups and the formation of hydrogen bonds at AChRs, thus supporting the observed higher potency and affinity of (±)-SADU-3-72 compared to BP. Collectively our results are consistent with a model where BP/SADU-3-72 and TCP bind to overlapping sites within the lumen of muscle AChR ion channels. Based on these results, we believe that (±)-SADU-3-72 is a promising photoprobe for mapping the BP binding site, especially within the resting AChR ion channel., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Here we provide functional and immunocytochemical evidence supporting the presence on Nucleus Accumbens (NAc) dopaminergic terminals of cyclothiazide-sensitive, alfa-amino-3-hydroxy-5-methyl-4-isoxazolone propionate (AMPA) receptors, which activation causes Ca²⁺-dependent [³H]dopamine ([³H]DA) exocytosis. These AMPA receptors cross-talk with co-localized nicotinic receptors (nAChRs), as suggested by the finding that in vitro short-term pre-exposure of synaptosomes to 30 μM nicotine caused a significant reduction of both the 30 μM nicotine and the 100 μM AMPA-evoked [³H]DA overflow. Entrapping pep2-SVKI, a peptide known to compete for the binding of GluA2 subunit to scaffolding proteins involved in AMPA receptor endocytosis, in NAC synaptosomes prevented the nicotine-induced reduction of AMPA-mediated [³H]DA exocytosis, while pep2-SVKE, used as negative control, was inefficacious. Immunocytochemical studies showed that a significant percentage of NAc terminals were dopaminergic and that most of these terminals also posses GluA2 receptor subunits. Western blot analysis of GluA2 immunoreactivity showed that presynaptic GluA2 proteins in NAc terminals were reduced in nicotine-pretreated synaptosomes when compared to the control. The nACh-AMPA receptor-receptor interaction was not limited to dopaminergic terminals since nicotine pre-exposure also affected the presynaptic AMPA receptors controlling hippocampal noradrenaline release, but not the presynaptic AMPA receptors controlling GABA and acetylcholine release. These observations could be relevant to the comprehension of the molecular mechanisms at the basis of nicotine rewarding., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In the current study, we investigated the effect of the activation of the alpha-7 nicotinic acetylcholine receptor (α7 nAchR) on dextran sulphate sodium (DSS)-induced colitis and referred mechanical hyperalgesia in mice. Colitis was induced in CD1 male mice through the intake of 4% DSS in tap water for 7 days. Control mice received unadulterated water. Referred mechanical hyperalgesia was evaluated for 7 days after the beginning of 4% DSS intake. Referred mechanical hyperalgesia started within 1 day after beginning DSS drinking, peaked at 3 days and persisted for 7 days. This time course profile perfectly matched with the appearance of signs of colitis. Both acute and chronic oral treatments with nicotine (0.1-1.0 mg/kg, p.o.) were effective in inhibiting the established referred mechanical hyperalgesia. The antinociceptive effect of nicotine was completely abrogated by cotreatment with the selective α7 nAchR antagonist methyllycaconitine (MLA) (1.0 mg/kg). Consistent with these results, i.p. treatment with the selective α7 nAchR agonist PNU 282987 (0.1-1.0 mg/kg) reduced referred mechanical hyperalgesia at all periods of evaluation. Despite their antinociceptive effects, nicotinic agonists did not affect DSS-induced colonic damage or inflammation. Taken together, the data generated in the present study show the potential relevance of using α7 nAchR agonists to treat referred pain and discomfort associated with inflammatory bowel diseases., (Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.)

Drug development for nicotinic acetylcholine receptors (nAChR) is challenged by subtype diversity arising from variations in subunit composition. On-target activity for neuronal heteromeric receptors is typically associated with CNS receptors that contain α4 and other subunits, while off-target activity could be associated with ganglionic-type receptors containing α3β4 binding sites and other subunits, including β4, β2, α5, or α3 as a structural subunit in the pentamer. Additional interest in α3 β4 α5-containing receptors arises from genome-wide association studies linking these genes, and a single nucleotide polymorphism (SNP) in α5 in particular, to lung cancer and heavy smoking. While α3 and β4 readily form receptors in expression system such as the Xenopus oocyte, since α5 is not required for function, simple co-expression approaches may under-represent α5-containing receptors. We used a concatamer of human α3 and β4 subunits to form ligand-binding domains, and show that we can force the insertions of alternative structural subunits into the functional pentamers. These α3β4 variants differ in sensitivity to ACh, nicotine, varenicline, and cytisine. Our data indicated lower efficacy for varenicline and cytisine than expected for β4-containing receptors, based on previous studies of rodent receptors. We confirm that these therapeutically important α4 receptor partial agonists may present different autonomic-based side-effect profiles in humans than will be seen in rodent models, with varenicline being more potent for human than rat receptors and cytisine less potent. Our initial characterizations failed to find functional effects of the α5 SNP. However, our data validate this approach for further investigations., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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., ((c) 2010 Elsevier Ltd. All rights reserved.)

The interaction of tricyclic antidepressants (TCAs) with the human (h)alpha3beta4 nicotinic acetylcholine receptor (AChR) in different conformational states was compared with that for mecamylamine by using functional and structural approaches including, Ca(2+) influx, radioligand binding, and molecular docking. The results established that: (a) [(3)H]imipramine binds to a single site with relatively high affinity (K(d) = 0.41 +/- 0.04 microM), (b) imipramine inhibits [(3)H]imipramine binding to the resting/kappa-bungarotoxin-bound AChR (K(i) = 0.68 +/- 0.08 microM) with practically the same affinity as to the desensitized/epibatidine-bound AChR (K(i) = 0.83 +/- 0.08 microM), suggesting that TCAs do not discriminate between these conformational states, and (c) although TCAs (IC(50) approximately 1.8-2.7 microM) and mecamylamine (IC(50) = 3.3 +/- 0.4 microM) inhibit (+/-)-epibatidine-induced Ca(2+) influx with potencies in the same concentration range, TCAs (K(i) approximately 1-3.6 microM), but not mecamylamine (apparent IC(50) approximately 0.2 mM), inhibit [(3)H]imipramine binding to halpha3beta4 AChRs in different conformational states. This is explained by our docking results where imipramine, in the neutral and protonated states, interacts with the leucine (position 9') and valine/phenylalanine (position 13') rings, whereas protonated mecamylamine (>99% at physiological pH) interacts with the outer ring (position 20'). Our data indicate that TCAs bind to overlapping sites located between the serine and valine/phenylalanine rings in the halpha3beta4 AChR ion channel, whereas protonated mecamylamine can be attracted to the channel mouth before blocking ion flux by interacting with a luminal site in its neutral state., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Neonicotinoid insecticides are widely used for crop protection based on their selective actions on insect nicotinic acetylcholine receptors (insect nAChRs). Loops C and D in insect nAChRs have been shown to possess structural features favorable for neonicotinoid-nAChR interactions. However, it remains to be resolved whether such features serve either co-operatively, or independently, to enhance neonicotinoid sensitivity of nAChRs. We therefore examined using voltage-clamp electrophysiology the effects on the response to imidacloprid of combinatorial substitutions of residues in loops C and D of the chicken alpha4beta2 nAChR by those present in insect nAChRs. The E219P mutation in loop C of the alpha4 subunit resulted in enhanced responses to imidacloprid of alpha4beta2, whereas E219S and E219T mutations barely influenced its actions. On the other hand, mutations in loop D (T77R; E79V and T77N; E79R) alone shifted the imidacloprid concentration-response curve to the left (lower concentrations). Interestingly, all three mutations did, however, further enhance the agonist efficacy of imidacloprid when combined with the mutations in loop D. Such synergistic effects of the two loops on the interactions with imidaclprid were observed irrespective of subunit stoichiometry. Computational modeling of the ligand binding domain of the wild-type and mutant alpha4beta2 nAChRs using the crystal structure of the acetylcholine binding protein from Lymnaea stagnalis also indicated that interactions with loop F of loops C and D may contribute to determining the response to imidacloprid.

Neuronal nicotinic receptors (NAChRs) form a heterogeneous family of ion channels that are differently expressed in many regions of the central nervous system (CNS) and peripheral nervous system. These different receptor subtypes, which have characteristic pharmacological and biophysical properties, have a pentameric structure consisting of the homomeric or heteromeric combination of 12 different subunits (alpha2-alpha10, beta2-beta4). By responding to the endogenous neurotransmitter acetylcholine, NAChRs contribute to a wide range of brain activities and influence a number of physiological functions. Furthermore, it is becoming evident that the perturbation of cholinergic nicotinic neurotransmission can lead to various diseases involving nAChR dysfunction during development, adulthood and ageing. In recent years, it has been discovered that NAChRs are present in a number of non-neuronal cells where they play a significant functional role and are the pathogenetic targets in several diseases. NAChRs are also the target of natural ligands and toxins including nicotine (Nic), the most widespread drug of abuse. This review will attempt to survey the major achievements reached in the study of the structure and function of NAChRs by examining their regional and cellular localisation and the molecular basis of their functional diversity mainly in pharmacological and biochemical terms. The recent availability of mice with the genetic ablation of single or double nicotinic subunits or point mutations have shed light on the role of nAChRs in major physiological functions, and we will here discuss recent data relating to their behavioural phenotypes. Finally, the role of NAChRs in disease will be considered in some details.

Binding of nicotine to nicotinic acetylcholine receptors (nAChRs) elicits a series of dose-dependent behaviors that go from altered exploration, sedation, and tremors, to seizures and death. nAChRs are pentameric ion channels usually composed of alpha and beta subunits. A gene cluster comprises the alpha3, alpha5 and beta4 subunits, which coassemble to form functional receptors. We examined the role of the beta4 subunits in nicotine-induced seizures and hypolocomotion in beta4 homozygous null (beta4 -/-) and alpha3 heterozygous (+/-) mice. beta4 -/- mice were less sensitive to the effects of nicotine both at low doses, measured as decreased exploration in an open field, and at high doses, measured as sensitivity to nicotine-induced seizures. Using in situ hybridization probes for the alpha3 and alpha5 subunits, we showed that alpha5 mRNA levels are unchanged, whereas alpha3 mRNA levels are selectively decreased in the mitral cell layer of the olfactory bulb, and the inferior and the superior colliculus of beta4 -/- brains. alpha3 +/- mice were partially resistant to nicotine-induced seizures when compared to wild-type littermates. mRNA levels for the alpha5 and the beta4 subunits were unchanged in alpha3 +/- brains. Together, these results suggest that the beta4 and the alpha3 subunits are mediators of nicotine-induced seizures and hypolocomotion.

The modulatory effects exerted by the glucocorticoid hydrocortisone (HC) on the nicotinic acetylcholine receptor (AChR) were studied in mutants of the alpha subunit M4 transmembrane region. Based on the photoaffinity labeling of alpha M4 412 with the steroid promegestone this position was mutated to different residues to explore the properties of side-chain volume, hydrophobicity, and charge on AChR-steroid interactions. All mutants showed channel kinetics indistinguishable from those of the wild-type AChR, both in the absence and presence of HC (200 and 400 microM), in single-channel recordings at different acetylcholine (ACh) concentrations. An alanine-substituted quadruple mutant of four putative lipid-exposed residues in alpha M4 (L411, M415, C418 and T422) exhibited less inhibition by HC than that observed in wild-type AChR. When we dissected the quadruple mutant into four individual alanine-substituted receptors, we found that the T422 mutant AChR behaved like the quadruple mutant, whereas the other three were indistinguishable from the wild-type. We conclude that T422, a residue close to the extracellular-facing membrane hemilayer in alpha M4, has direct bearing on the changes in HC sensitivity and propose its involvement in the steroid-AChR interaction site.

No published study on synaptogenesis in masseter muscle has focused on the shift of nicotinic acetylcholine receptors (nAChRs) from the embryonic type (alpha(2)-, beta-, gamma- and delta-subunits) to the adult-type (alpha(2)-, beta-, epsilon- and delta-subunits) and the elimination of nAChRs outside the neuromuscular junction. To identify the time course of the nAChR transitions in rat masseter muscle between 1 and 63 days of age, the expression of delta-, epsilon- and gamma-subunit mRNAs was analysed by competitive polymerase chain reaction in combination with reverse transcription. The expression of the delta-subunit was high between 1 and 7 days of age, then decreased by 95% (P<0.0001) between 7 and 28 days, suggesting that the nAChR elimination occurs during this period. The quantity of the epsilon-subunit increased by approximately 600% (P<0.0001) between 1 and 21 days of age, whereas the quantity of the gamma-subunit decreased by 85% (P<0.0001) during the same period. This result indicates that the nAChR type shift is terminated at 21 days of age. The feeding behaviour of the rats inevitably changed from suckling to biting after 19 days of age, because they were weaned at that age. As the nAChR type shift was terminated soon after weaning, the termination could be related to the change in feeding behaviour. However, it might also be the case that nAChR elimination is not directly related to the change in feeding behaviour, as the elimination continued at the same rate for 9 days after weaning (from 19 to 28 days of age).

Identification of all residues involved in the recognition and binding of cholinergic ligands (e.g. agonists, competitive antagonists, and noncompetitive agonists) is a primary objective to understand which structural components are related to the physiological function of the nicotinic acetylcholine receptor (AChR). The picture for the localization of the agonist/competitive antagonist binding sites is now clearer in the light of newer and better experimental evidence. These sites are located mainly on both alpha subunits in a pocket approximately 30-35 A above the surface membrane. Since both alpha subunits are identical, the observed high and low affinity for different ligands on the receptor is conditioned by the interaction of the alpha subunit with other non-alpha subunits. This molecular interaction takes place at the interface formed by the different subunits. For example, the high-affinity acetylcholine (ACh) binding site of the muscle-type AChR is located on the alphadelta subunit interface, whereas the low-affinity ACh binding site is located on the alphagamma subunit interface. Regarding homomeric AChRs (e.g. alpha7, alpha8, and alpha9), up to five binding sites may be located on the alphaalpha subunit interfaces. From the point of view of subunit arrangement, the gamma subunit is in between both alpha subunits and the delta subunit follows the alpha aligned in a clockwise manner from the gamma. Although some competitive antagonists such as lophotoxin and alpha-bungarotoxin bind to the same high- and low-affinity sites as ACh, other cholinergic drugs may bind with opposite specificity. For instance, the location of the high- and the low-affinity binding site for curare-related drugs as well as for agonists such as the alkaloid nicotine and the potent analgesic epibatidine (only when the AChR is in the desensitized state) is determined by the alphagamma and the alphadelta subunit interface, respectively. The case of alpha-conotoxins (alpha-CoTxs) is unique since each alpha-CoTx from different species is recognized by a specific AChR type. In addition, the specificity of alpha-CoTxs for each subunit interface is species-dependent. In general terms we may state that both alpha subunits carry the principal component for the agonist/competitive antagonist binding sites, whereas the non-alpha subunits bear the complementary component. Concerning homomeric AChRs, both the principal and the complementary component exist on the alpha subunit. The principal component on the muscle-type AChR involves three loops-forming binding domains (loops A-C). Loop A (from mouse sequence) is mainly formed by residue Y(93), loop B is molded by amino acids W(149), Y(152), and probably G(153), while loop C is shaped by residues Y(190), C(192), C(193), and Y(198). The complementary component corresponding to each non-alpha subunit probably contributes with at least four loops. More specifically, the loops at the gamma subunit are: loop D which is formed by residue K(34), loop E that is designed by W(55) and E(57), loop F which is built by a stretch of amino acids comprising L(109), S(111), C(115), I(116), and Y(117), and finally loop G that is shaped by F(172) and by the negatively-charged amino acids D(174) and E(183). The complementary component on the delta subunit, which corresponds to the high-affinity ACh binding site, is formed by homologous loops. Regarding alpha-neurotoxins, several snake and alpha-CoTxs bear specific residues that are energetically coupled with their corresponding pairs on the AChR binding site. The principal component for snake alpha-neurotoxins is located on the residue sequence alpha1W(184)-D(200), which includes loop C. In addition, amino acid sequence 55-74 from the alpha1 subunit (which includes loop E), and residues gammaL(119) (close to loop F) and gammaE(176) (close to loop G) at the low-affinity binding site, or deltaL(121) (close to the homologous region of loop G) at the high-affinity binding site, are i

We have previously shown that two histidine residues of the nicotinic acetylcholine receptor are relevant for alpha-bungarotoxin binding. This paper studies: (1) the interaction between alpha-bungarotoxin and the peptide alpha173-202--synthesized according to the sequence of the Torpedo californica receptor alpha subunit--and between the toxin and the same peptide containing His186 modified with ethoxyformic anhydride or substituted by Ala; (2) the influence of the presence of Cys192-Cys193 disulfide bridge on such interactions. Solid-phase and in-solution competition assays were performed: ethoxyformylation of His186 or its substitution by Ala led to a significant drop in the toxin binding capacity only for peptides containing the bridge. Circular dichroism and fourth derivate spectra of all peptides were also analyzed. Results strongly indicate the involvement of His186 in the toxin binding to those peptides with the bridge--also present in the native receptor molecules--but not to their reduced forms; on the other hand, they give further support to the already established premise that, though the bridge does not participate directly in receptor-toxin binding, its presence is relevant to define the appropriate conformation of the interaction area.

Infrared (IR) spectroscopy is a useful technique in the study of protein conformation and dynamics. The possibilities of the technique become apparent specially when applied to large proteins in turbid suspensions, as is often the case with membrane proteins. The present review describes the applications of IR spectroscopy to the study of membrane proteins, with an emphasis on recent work and on spectra recorded in the transmission mode, rather than using reflectance techniques. Data treatment procedures are discussed, including band analysis and difference spectroscopy methods. A technique for the analysis of protein secondary and tertiary structures that combines band analysis by curve-fitting of original spectra with protein thermal denaturation is described in detail. The assignment of IR protein bands in H2O and in D2O, one of the more difficult points in protein IR spectroscopy, is also reviewed, including some cases of unclear assignments such as loops, beta-hairpins, or 3(10)-helices. The review includes monographic studies of some membrane proteins whose structure and function have been analysed in detail by IR spectroscopy. Special emphasis has been made on the role of subunit III in cytochrome c oxidase structure, and the proton pathways across this molecule, on the topology and functional cycle of sarcoplasmic reticulum Ca(2+)-ATPase, and on the role of lipids in determining the structure of the nicotinic acetylcholine receptor. In addition, shorter descriptions of retinal proteins and references to other membrane proteins that have been studied less extensively are also included.

The present investigation utilised monoclonal antibodies directed against subunits of the nicotinic acetylcholine receptor in immunoblot and immunoprecipitation studies, which failed to demonstrate that the native 5-hydroxytryptamine3 (5-HT3) receptor complex purified from porcine brain contains the alpha1, alpha3, alpha4, alpha5, alpha7 or beta2 subunits of the nicotinic acetylcholine receptor.

Nicotine is a very widely used drug of abuse, which exerts a number of neurovegetative, behavioural and psychological effects by interacting with neuronal nicotinic acetylcholine receptors (NAChRs). These receptors are distributed widely in human brain and ganglia, and form a family of ACh-gated ion channels of different subtypes, each of which has a specific pharmacology and physiology. As human NAChRs have been implicated in a number of human central nervous system disorders (including the neurodegenerative Alzheimer's disease, schizophrenia and epilepsy), they are suitable potential targets for rational drug therapy. Much of our current knowledge about the structure and function of NAChRs comes from studies carried out in other species, such as rodents and chicks, and information concerning human nicotinic receptors is still incomplete and scattered in the literature. Nevertheless, it is already evident that there are a number of differences in the anatomical distribution, physiology, pharmacology, and expression regulation of certain subtypes between the nicotinic systems of humans and other species. This review will attempt to survey the major achievements reached in the study of the structure and function of NAChRs by examining the molecular basis of their functional diversity viewed mainly from pharmacological and biochemical perspectives. It will also summarize our current knowledge concerning the structure and function of the NAChRs expressed by other species, and the newly discovered drugs used to classify their numerous subtypes. Finally, the role of NAChRs in behaviour and pathology will be considered.

Reactivity of histidine residues of the Discopyge tschudii nicotinic acetylcholine receptor was studied by reaction with DEP and the influence of their modification on functional properties of the receptor was evaluated. Determination of two kinetically distinguishable classes was achieved. The fast-reacting class is composed of 7 histidine residues with an apparent velocity constant k1 = 0.0248 +/- 0.0031 min-1. The second includes--at least--21 histidine residues with a velocity constant k2 = 0.0016 +/- 0.0009 min-1. The circular dichroism spectra of the native receptor and the most DEP-derivative indicate no significant modifications in the alpha-helix content, and fourth derivative spectroscopy analyses show that the environment around the aromatic amino acids remains unchanged. DEP treatment of the receptor results in a time- and reagent concentration-dependent loss of its alpha-bungarotoxin binding ability; these results agree with those obtained with the membrane-bound receptor. The decrease in the neurotoxin binding capacity was correlated with the DEP-reaction extent of the slow groups. Incorporation of 1.93 +/- 0.23 mol of DEP accounted for the maximal binding capacity drop, thus indicating the involvement of two histidine residues per alpha-bungarotoxin binding site. Neither amino groups nor tyrosine residues were modified during the reaction with DEP, indicating that the derivatization of histidine residues is responsible for the observed effect. Faster-reacting residues appear to be involved in agonist-induced ion flux through the nAChR channel. These results strongly support the connection between histidine residues and the receptor functional activity and lead us to infer that the changes observed in alpha-bungarotoxin binding and ionic channel capacity are the consequence of independent events induced by reaction with DEP.