Neuronal Nicotinic Acetylcholine Receptors Research Articles

Differential efficacies of the nicotinic α4β2 desensitizing agents in reducing nicotine self-administration in female rats.

Desensitization of neuronal nicotinic acetylcholine receptors holds promise as an effective treatment of tobacco addiction. Previously, we found that sazetidine-A (Saz-A), which selectively desensitizes α4β2 nicotinic receptors, significantly decreased intravenous (IV) nicotine self-administration (SA) in rats with an effective dose of 3mg/kg in acute and repeated injection studies. We also found that chronic infusions of Saz-A at doses of 2 and 6mg/kg/day significantly reduced nicotine SA in rats. In continuing studies, we have characterized other Saz-A analogs, YL-2-203 and VMY-2-95, to determine their efficacies in reducing nicotine SA in rats. Young adult female Sprague-Dawley rats were fitted with IV catheters and were trained for nicotine SA (0.03mg/kg/infusion) on a fixed ratio 1 schedule for ten sessions. The same rats were also implanted subcutaneously with osmotic minipumps to continually deliver 2 or 6mg/kg body weight YL-2-203, VMY-2-95, or saline for four consecutive weeks. Chronic administration of VMY-2-95 at doses of 2 and 6mg/kg/day caused significant (p<0.01) decreases in nicotine SA over the 2weeks of continued nicotine SA and for the 1-week period of resumed access after a week of enforced abstinence, whereas chronic administration of YL-2-203 at the same doses was not found to be effective. These studies, together with our previous studies of Saz-A, revealed a spectrum of efficacies for these α4β2 nicotinic receptor desensitizing agents and provide a path forward for the most effective compounds to be further developed as possible aids to smoking cessation.

Levamisole: A Positive Allosteric Modulator for the α3β4 Nicotinic Acetylcholine Receptors Prevents Weight Gain in the CD-1 Mice on a High Fat Diet.

Neuronal nicotinic acetylcholine receptors (nAChRs) regulate the function of multiple neurotransmitter pathways throughout the central nervous system. This includes nAChRs found on the proopiomelanocortin neurons in the hypothalamus. Activation of these nAChRs by nicotine causes a decrease in the consumption of food in rodents. This study tested the effect of subtype selective allosteric modulators for nAChRs on the body weight of CD-1 mice. Levamisole, an allosteric modulator for the α3β4 subtype of nAChRs, prevented weight gain in mice that were fed a high fat diet. PNU-120596 and desformylflustrabromine were observed to be selective PAMs for the α7 and α4β2 nAChR, respectively. Both of these compounds failed to prevent weight gain in the CD-1 mice. These results suggest that the modulation of hypothalamic α3β4 nAChRs is an important factor in regulating food intake, and the PAMs for these receptors need further investigation as potential therapeutic agents for controlling weight gain.

Tumor suppressor menin is required for subunit-specific nAChR \u03b15 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

In the central nervous system (CNS), cholinergic transmission induces synaptic plasticity that is required for learning and memory. However, our understanding of the development and maintenance of cholinergic circuits is limited, as the factors regulating the expression and clustering of neuronal nicotinic acetylcholine receptors (nAChRs) remain poorly defined. Recent studies from our group have implicated calpain-dependent proteolytic fragments of menin, the product of the MEN1 tumor suppressor gene, in coordinating the transcription and synaptic clustering of nAChRs in invertebrate central neurons. Here, we sought to determine whether an analogous cholinergic mechanism underlies menin’s synaptogenic function in the vertebrate CNS. Our data from mouse primary hippocampal cultures demonstrate that menin and its calpain-dependent C-terminal fragment (C-menin) regulate the subunit-specific transcription and synaptic clustering of neuronal nAChRs, respectively. MEN1 knockdown decreased nAChR α5 subunit expression, the clustering of α7 subunit-containing nAChRs at glutamatergic presynaptic terminals, and nicotine-induced presynaptic facilitation. Moreover, the number and function of glutamatergic synapses was unaffected by MEN1 knockdown, indicating that the synaptogenic actions of menin are specific to cholinergic regulation. Taken together, our results suggest that the influence of menin on synapse formation and synaptic plasticity occur via modulation of nAChR channel subunit composition and functional clustering.

Reflex bronchoconstriction evoked by inhaled nicotine aerosol in guinea pigs: role of the nicotinic acetylcholine receptor

We have previously reported that stimulation of vagal bronchopulmonary C‐fibers by nicotine via its action on neuronal nicotinic acetylcholine receptor (NnAChR) is primarily responsible for the acute bronchoconstriction evoked by inhaled cigarette smoke. Recent studies have reported that nicotine at high concentrations can also activate the transient receptor potential ankyrin 1 receptor (TRPA1) expressed in these sensory nerves. This study was carried out to investigate the mechanisms involved in the bronchoconstrictive effect of inhaled nicotine, and the relative roles of NnAChR and TRPA1 in this effect. Guinea pigs were anesthetized and mechanically ventilated; nicotine aerosol (2% solution; 1 × VT) was diluted by air to 2 × VT and delivered directly into the lung via trachea tube. Our results showed: 1) Delivery of one breath of nicotine aerosol immediately triggered a pronounced airway constriction; the increase in airway resistance (Raw) reached a peak of 588 ± 205 % in 10–40 sec, which gradually returned to baseline after 1–5 min. The response was reproducible in the same animal tested 20 min later. 2) Challenge with aerosolized alkaline solution of the same pH as that in the nicotine solution (10.5) did not cause any change in Raw. 3) Pretreatment with atropine (0.1 mg/kg; i.v.) and mecamylamine (Mec; an antagonist of NnAChR) (2%; 4 breaths of aerosol) almost completely abolished the nicotine‐induced bronchoconstriction: ΔRaw was reduced by 83.1 ± 8.2 % and 92.5 ± 1.9 %, respectively. The Mec pretreatment did not block the bronchoconstriction and bradycardia generated by electrical stimulation of the distal end of one sectioned vagus nerve, indicating its minimal systemic effects. 4) Pretreatment with HC‐030031 (0.5 mg/kg; i.v.), a selective TRPA1 antagonist, abolished the bronchoconstriction induced by allyl isothiocyanate (1.7 mg/kg, i.v.), a TRPA1 agonist, but did not render any attenuation of the nicotine‐evoked bronchoconstriction. In conclusion, inhalation of a single puff of nicotine aerosol evoked acute bronchoconstriction mediated through the cholinergic reflex pathway, which was triggered by activation of NnAChR located in airway sensory nerves. In contrast, TRPA1 receptor did not play a significant role in this response.Support or Funding InformationSupported by NIH grants HL96914, HL107462 and UL1TR001998

NACHO Mediates Nicotinic Acetylcholine Receptor Function throughout the Brain

Neuronal nicotinic acetylcholine receptors (nAChRs) participate in diverse aspects of brain function and mediate behavioral and addictive properties of nicotine. Neuronal nAChRs derive from combinations of αand β subunits, whose assembly is tightly regulated. NACHO was recently identified as a chaperone for α7-type nAChRs. Here, we find NACHO mediates assembly of all major classes of presynaptic and postsynaptic nAChR tested. NACHO acts at early intracellular stages of nAChR subunit assembly and then synergizes with RIC-3 for receptor surface expression. NACHO knockout mice show profound deficits in binding sites for α-bungarotoxin, epibatidine, and conotoxin MII, illustrating essential roles for NACHO in proper assembly of α7-, α4β2-, and α6-containing nAChRs, respectively. By contrast, GABAA receptors are unaffected consistent with NACHO specifically modulating nAChRs. NACHO knockout mice show abnormalities in locomotor and cognitive behaviors compatible with nAChR deficiency and underscore the importance of this chaperone for physiology and disease associated with nAChRs.

Cyclic imine toxins from dinoflagellates: a growing family of potent antagonists of the nicotinic acetylcholine receptors.

We present an overview of the toxicological profile of the fast-acting, lipophilic macrocyclic imine toxins, an emerging family of organic compounds associated with algal blooms, shellfish contamination and neurotoxicity. Worldwide, shellfish contamination incidents are expanding; therefore, the significance of these toxins for the shellfish food industry deserves further study. Emphasis is directed to the dinoflagellate species involved in their production, their chemical structures, and their specific mode of interaction with their principal natural molecular targets, the nicotinic acetylcholine receptors, or with the soluble acetylcholine-binding protein, used as a surrogate receptor model. The dinoflagellates Karenia selliformis and Alexandrium ostenfeldii / A. peruvianum have been implicated in the biosynthesis of gymnodimines and spirolides, while Vulcanodinium rugosum is the producer of pinnatoxins and portimine. The cyclic imine toxins are characterized by a macrocyclic skeleton comprising 14-27 carbon atoms, flanked by two conserved moieties, the cyclic imine and the spiroketal ring system. These phycotoxins generally display high affinity and broad specificity for the muscle type and neuronal nicotinic acetylcholine receptors, a feature consistent with their binding site at the receptor subunit interfaces, composed of residues highly conserved among all nAChRs, and explaining the diverse toxicity among animal species. This is an article for thespecial issue XVth International Symposium on Cholinergic Mechanisms.

Probing for and Quantifying Agonist Hydrogen Bonds in α6β2 Nicotinic Acetylcholine Receptors.

Designing subtype-selective agonists for neuronal nicotinic acetylcholine receptors is a challenging and significant goal aided by intricate knowledge of each subtype's binding patterns. We previously reported that in α6β2 receptors, acetylcholine makes a functional cation-π interaction with Trp149, but nicotine and TC299423 do not, suggesting a distinctive binding site. This work explores hydrogen binding at the backbone carbonyl associated with α6β2 Trp149. Substituting residue i + 1, Thr150, with its α-hydroxy analogue (Tah) attenuates the carbonyl's hydrogen bond accepting ability. At α6(T150Tah)β2, nicotine shows a 24-fold loss of function, TC299423 shows a modest loss, and acetylcholine shows no effect. Nicotine was further analyzed via a double-mutant cycle analysis utilizing N'-methylnicotinium, which indicated a hydrogen bond in α6β2 with a ΔΔG of 2.6 kcal/mol. Thus, even though nicotine does not make the conserved cation-π interaction with Trp149, it still makes a functional hydrogen bond to its associated backbone carbonyl.

BMS-933043, a Selective α7 nAChR Partial Agonist for the Treatment of Cognitive Deficits Associated with Schizophrenia

The therapeutic treatment of negative symptoms and cognitive dysfunction associated with schizophrenia is a significant unmet medical need. Preclinical literature indicates that α7 neuronal nicotinic acetylcholine (nACh) receptor agonists may provide an effective approach to treating cognitive dysfunction in schizophrenia. We report herein the discovery and evaluation of 1c (BMS-933043), a novel and potent α7 nACh receptor partial agonist with high selectivity against other nicotinic acetylcholine receptor subtypes (>100-fold) and the 5-HT3A receptor (>300-fold). In vivo activity was demonstrated in a preclinical model of cognitive impairment, mouse novel object recognition. BMS-933043 has completed Phase I clinical trials.

Conjugates of α-Cobratoxin with CdSe Quantum Dots: Preparation and Biological Activity

α-Cobratoxin is the main neurotoxin in the cobra Naja kaouthia venom; it binds efficiently and selectively with neuronal α7 and muscle type nicotinic acetylcholine receptor and can be used for specific labeling and visualization of these receptors in organs and tissues. For these applications we have prepared conjugates of α-cobratoxin with CdSe quantum dots which have many benefits as compared to organic fluorescent labels. To prepare the conjugate, CdSe quantum dots with ZnS shell were functionalized using a tripeptide glutathione and coupled to toxin using water soluble carbodiimide. The conjugate was purified by gel-filtration chromatography and tested for biological activity. It was found that conjugate preserved the capacity to interact with both neuronal α7 and muscle type nicotinic acetylcholine receptor. Its cytotoxicity to mammalian cells was not higher than that of functionalized quantum dots.

Molecular mechanism of action and safety of 5-(3-chlorophenyl)-4-hexyl-2,4-dihydro-3H-1,2,4-triazole-3-thione - a novel anticonvulsant drug candidate.

Previously, it was found that 5-(3-chlorophenyl)-4-hexyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (TP-315) effectively protects mice from maximal electroshock-induced seizures. The aim of this study was to determine possible interactions between TP-315 and different molecular targets, i.e. GABAA receptors, voltage-gated sodium channels, and human neuronal α7 and α4β2 nicotinic acetylcholine receptors. The influence of TP-315 on the viability of human hepatic HepG2 cells was also established using PrestoBlue and ToxiLight assays. It was found that the anticonvulsant activity of TP-315 results (at least partially) from its influence on voltage-gated sodium channels (VGSCs). Moreover, the title compound slightly affected the viability of human hepatic cells.

N-methyl serotonin analogues from the Bufo bufo toad venom interact efficiently with the α7 nicotinic acetylcholine receptors.

Two low-molecular-weight compounds were isolated from the parotid gland secret of the toad Bufo bufo, which by absorption spectra and HPLC-MS/MS chromatography data correspond to di- and trimethyl derivatives of serotonin (5-hydorxytryptamine): bufotenine (confirmed by counter synthesis) and bufotenidine (5-HTQ). In experiments on competitive radioligand binding, these compounds showed a higher affinity and selectivity for neuronal α7 nicotinic acetylcholine receptors compared with the muscular cholinergic receptors. The most efficient compound in terms of binding value was bufotenine, the efficiency of 5-HTQ was an order of magnitude lower, and the minimal activity was exhibited by serotonin.

Possible involvement of neuronal nicotinic acetylcholine receptors in compensatory brain mechanisms at early stages of Parkinson's disease

A role of nicotinic acetylcholine receptors (nAChR) in the development of Parkinson's disease (PD) has been investigated using two mouse models corresponding to the presymptomatic stage and the early symptomatic stage of PD. Quantitative determination of nAChR in the striatum and substantia nigra (SN) was performed using the radioactive derivatives of epibatidine, -conotoxin MII, and -bungarotoxin as ligands. The number of ligand-binding sites changed differently depending on their location in the brain, the stage of the disease and the receptor subtype. Epibatidine binding decreased in the striatum to 66% and 70% at the presymptomatic and early symptomatic stages, respectively, whereas in SN a 160% increase was registered at the presymptomatic stage. The -conotoxin MII binding on striatal dopaminergic axonal terminals at the presymptomatic stage decreased by 20% and at the symptomatic stage it demonstrated a further decrease. The increase in -bungarotoxin binding at the presymptomatic stage and a decrease at the early symptomatic stage was observed in the striatum. In SN, the level of -bungarotoxin binding decreased at the presymptomatic stage and kept constant at the symptomatic stage. The significant decrease in the expression of Chrna4 and Chrna6 genes encoding 4 and 6 nAChR subunits was observed in SN at the early symptomatic stage, while a 13-fold increase in expression of the Chrna7 gene encoding the 7 nAChR subunit was detected at the presymptomatic stage. The data obtained suggest possible involvement of nAChR in compensatory mechanisms at early PD stages.

Development of spiroguanidine-derived α7 neuronal nicotinic receptor partial agonists

We describe the synthesis of quinuclidine-containing spiroguanidines and their utility as α7 neuronal nicotinic acetylcholine receptor (nAChR) partial agonists. The convergent synthetic route developed for this study allowed for rapid SAR investigation and provided access to a structurally diverse set of analogs. A potent and selective α7 nAChR partial agonist, N‐(6‐methyl‐1,3‐benzoxazol‐2‐yl)‐3′,5′‐dihydro‐4‐azaspiro[bicyclo[2.2.2]octane‐2,4′‐imidazole]‐2′‐amine (BMS-910731, 16), was identified. This compound induced immediate early genes c-fos and Arc in a preclinical rodent model of α7 nAChR-derived cellular activation and plasticity. Importantly, the ability to incorporate selectivity for the α7 nACh receptor over the 5-HT3A receptor in this series suggested a significant difference in steric requirements between the two receptors.

Human polymorphisms in nicotinic receptors: a functional analysis in iPS-derived dopaminergic neurons.

Tobacco smoking is a public health problem, with ∼5 million deaths per year, representing a heavy burden for many countries. No effective therapeutic strategies are currently available for nicotine addiction, and it is therefore crucial to understand the etiological and pathophysiological factors contributing to this addiction. The neuronal α5 nicotinic acetylcholine receptor (nAChR) subunit is critically involved in nicotine dependence. In particular, the human polymorphism α5D398N corresponds to the strongest correlation with nicotine dependence risk found to date in occidental populations, according to meta-analysis of genome-wide association studies. To understand the specific contribution of this subunit in the context of nicotine addiction, an efficient screening system for native human nAChRs is needed. We have differentiated human induced pluripotent stem (iPS) cells into midbrain dopaminergic (DA) neurons and obtained a comprehensive characterization of these neurons by quantitative RT-PCR. The functional properties of nAChRs expressed in these human DA neurons, with or without the polymorphism in the α5 subunit, were studied with the patch-clamp electrophysiological technique. Our results in human DA neurons carrying the polymorphism in the α5 subunit showed an increase in EC50, indicating that, in the presence of the polymorphism, more nicotine or acetylcholine chloride is necessary to obtain the same effect. This human cell culturing system can now be used in drug discovery approaches to screen for compounds that interact specifically with human native and polymorphic nAChRs.-Deflorio, C., Blanchard, S., Carisì, M. C., Bohl, D., Maskos, U. Human polymorphisms in nicotinic receptors: a functional analysis in iPS-derived dopaminergic neurons.

Nicotine-Induced Effects on Nicotinic Acetylcholine Receptors (nAChRs), Ca2+ and Brain-Derived Neurotrophic Factor (BDNF) in STC-1 Cells.

In addition to the T2R bitter taste receptors, neuronal nicotinic acetylcholine receptors (nAChRs) have recently been shown to be involved in the bitter taste transduction of nicotine, acetylcholine and ethanol. However, at present it is not clear if nAChRs are expressed in enteroendocrine cells other than beta cells of the pancreas and enterochromaffin cells, and if they play a role in the synthesis and release of neurohumoral peptides. Accordingly, we investigated the expression and functional role of nAChRs in enteroendocrine STC-1 cells. Our studies using RT-PCR, qRT-PCR, immunohistochemical and Western blotting techniques demonstrate that STC-1 cells express several α and β nAChR subunits. Exposing STC-1 cells to nicotine acutely (24h) or chronically (4 days) induced a differential increase in the expression of nAChR subunit mRNA and protein in a dose- and time-dependent fashion. Mecamylamine, a non-selective antagonist of nAChRs, inhibited the nicotine-induced increase in mRNA expression of nAChRs. Exposing STC-1 cells to nicotine increased intracellular Ca2+ in a dose-dependent manner that was inhibited in the presence of mecamylamine or dihydro-β-erythroidine, a α4β2 nAChR antagonist. Brain-derived neurotrophic factor (BDNF) mRNA and protein were detected in STC-1 cells using RT-PCR, specific BDNF antibody, and enzyme-linked immunosorbent assay. Acute nicotine exposure (30 min) decreased the cellular content of BDNF in STC-1 cells. The nicotine-induced decrease in BDNF was inhibited in the presence of mecamylamine. We also detected α3 and β4 mRNA in intestinal mucosal cells and α3 protein expression in intestinal enteroendocrine cells. We conclude that STC-1 cells and intestinal enteroendocrine cells express nAChRs. In STC-1 cells nAChR expression is modulated by exposure to nicotine in a dose- and time-dependent manner. Nicotine interacts with nAChRs and inhibits BDNF expression in STC-1 cells.

β-Cryptoxanthin Reduced Lung Tumor Multiplicity and Inhibited Lung Cancer Cell Motility by Downregulating Nicotinic Acetylcholine Receptor α7 Signaling.

Despite the consistent association between a higher intake of the provitamin A carotenoid β-cryptoxanthin (BCX) and a lower risk of lung cancer among smokers, potential mechanisms supporting BCX as a chemopreventive agent are needed. We first examined the effects of BCX on 4-[methyl nitrosamino]-1-[3-pyridyl]-1-butanone (NNK)-induced lung tumorigenesis in A/J mice. BCX supplementation was given daily to the mice starting 2 weeks prior to the injection of NNK and continued 16 weeks after NNK injection. BCX supplementation resulted in a dose-dependent increase of BCX concentration in both serum and lungs of the mice without a significant alteration of vitamin A (retinol and retinyl palmitate) concentration. BCX significantly reduced the multiplicity of the NNK-induced lung tumor by 52% to 63% compared with the NNK-treated mice without BCX supplementation. The protective effect of BCX in the lungs was associated with reductions of both mRNA and protein of the homopentameric neuronal nicotinic acetylcholine receptor α7 (α7-nAChR), which has been implicated in lung tumorigenesis. We then conducted an in vitro cell culture study and found that BCX treatment suppressed α7-nAChR expression and inhibited the migration and invasion of α7-nAChR-positive lung cancer cells but not in cells lacking α7-nAChR. The activities of BCX were significantly attenuated by activators of α7-nAChR/PI3K signaling or by overexpression of constitutively active PI3K. Collectively, the results suggest that BCX inhibits lung tumorigenesis and cancer cell motility through the downregulation of α7-nAChR/PI3K signaling, independent of its provitamin A activity. Therefore, BCX can be used as a chemopreventive agent or a chemotherapeutic compound against lung cancer. Cancer Prev Res; 9(11); 875-86. ©2016 AACR.

Antiepileptic Drugs Based on the α-Substituted Amide Group Pharmacophore: From Chemical Crystallography to Molecular Pharmaceutics.

The antiepileptic activity of α-substituted acetamides, lactams, and cyclic imides has been known for over six decades. We recently proposed an α-substituted amide group as the minimum pharmacophore responsible for inhibition of neuronal nicotinic acetylcholine receptors by these compounds, with the implication that inhibition of these receptors in the brain might be the unifying mechanism of action for these classes of antiepileptic drugs. In order to realize the pharmacological potential of these orally administered drugs, the relevant aspects of solid-state chemistry and pharmaceutics (including solubility and stability) need to be addressed. A better - more cohesive and generalized - understanding of the solid-state properties of these drugs would pave the road for a rational approach to their development, formulation, and manufacturing. In this paper, Pharmaceutically relevant aspects of the crystal structure and solid-state chemistry of antiepileptic drugs containing the α-substituted amide bond pharmacophore - α-substituted acetamides, lactams, and cyclic imides and the structurally related barbiturates, hydantoins, and acetylureas are reviewed. The applicable experimental and computational approaches are also briefly mentioned.

Neuronal Nicotinic Receptors Are Crucial for Tuning of E/I Balance in Prelimbic Cortex and for Decision-Making Processes.

Decision-making is an essential component of our everyday life commonly disabled in a myriad of psychiatric conditions, such as bipolar and impulsive control disorders, addiction and pathological gambling, or schizophrenia. A large cerebral network encompassing the prefrontal cortex, the amygdala, and the nucleus accumbens is activated for efficient decision-making. We developed a mouse gambling task well suited to investigate the influence of uncertainty and risk in decision-making and the role of neurobiological circuits and their monoaminergic inputs. Neuronal nicotinic acetylcholine receptors (nAChRs) of the PFC are important for decision-making processes but their presumed roles in risk-taking and uncertainty management, as well as in cellular balance of excitation and inhibition (E/I) need to be investigated. Using mice lacking nAChRs - β2-/- mice, we evidence for the first time the crucial role of nAChRs in the fine tuning of prefrontal E/I balance together with the PFC, insular, and hippocampal alterations in gambling behavior likely due to sensitivity to penalties and flexibility alterations. Risky behaviors and perseveration in extinction task were largely increased in β2-/- mice as compared to control mice, suggesting the important role of nAChRs in the ability to make appropriate choices adapted to the outcome.

Molecular simulation study of the unbinding of α-conotoxin [ϒ4E]GID at the α7 and α4β2 neuronal nicotinic acetylcholine receptors

The α7 and α4β2 neuronal nicotinic receptors belonging to the family of ligand-gated ion channels are most prevalent in the brain, and are implicated in various neurodegenerative disorders. α-conotoxin GID (and its analogue [ϒ4E]GID) specifically inhibits these subtypes, with more affinity towards the human α7 (hα7) subtype, and is valuable in understanding the physiological roles of these receptors. In this study, we use umbrella-sampling molecular dynamics simulations to understand the mechanism of interaction between [ϒ4E]GID and the agonist binding pockets of the α4β2 and the hα7 receptors, and to estimate their relative binding affinities (ΔGbind). The obtained ΔGbind values indicate stronger interaction with the hα7 receptor, in agreement with previous experimental studies. Simulations also revealed different unbinding pathways between the two receptor subtypes, enabling identification of a number of interactions at locations far from the orthosteric binding site which may explain the difference in [ϒ4E]GID potency. The pathways identified will help in the design of novel conotoxins with increased potency at α4β2, for which there is currently no known highly potent conotoxin inhibitor. Computational mutational free energy analyses also revealed a number of possible single-site mutations to GID which might enhance its selective binding to α4β2 over α7.

Crystal structure of a human neuronal nAChR extracellular domain in pentameric assembly: Ligand-bound α2 homopentamer

In this study we report the X-ray crystal structure of the extracellular domain (ECD) of the human neuronal α2 nicotinic acetylcholine receptor (nAChR) subunit in complex with the agonist epibatidine at 3.2 Å. Interestingly, α2 was crystallized as a pentamer, revealing the intersubunit interactions in a wild type neuronal nAChR ECD and the full ligand binding pocket conferred by two adjacent α subunits. The pentameric assembly presents the conserved structural scaffold observed in homologous proteins, as well as distinctive features, providing unique structural information of the binding site between principal and complementary faces. Structure-guided mutagenesis and electrophysiological data confirmed the presence of the α2(+)/α2(-) binding site on the heteromeric low sensitivity α2β2 nAChR and validated the functional importance of specific residues in α2 and β2 nAChR subunits. Given the pathological importance of the α2 nAChR subunit and the high sequence identity with α4 (78%) and other neuronal nAChR subunits, our findings offer valuable information for modeling several nAChRs and ultimately for structure-based design of subtype specific drugs against the nAChR associated diseases.