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Simulations of Promising Indolizidine-α6-β2 Nicotinic Acetylcholine Receptor Complexes.

ABSTRACT: Smoking-cessation drugs bind many off-target nicotinic acetylcholine receptors (nAChRs) and cause severe side effects if they are based on nicotine. New drugs that bind only those receptors, such as α6β2* nAChR, implicated in nicotine addiction would avoid the off-target binding. Indolizidine (-)-237D (IND (-)-237D), a bicyclic alkaloid, has been shown to block α6β2* containing nAChRs and functionally inhibit the nicotine-evoked dopamine release. To improve the affinity of indolizidine (-)-237D for α6β2*, we built a library of 2226 analogs. We screened virtually the library against a homology model of α6β2 nAChR that we derived from the recent crystal structure of α4β2 nAChR. We also screened the crystal structure of α4β2 nAChR as a control on specificity. We ranked the compounds based on their predicted free energy of binding. We selected the top eight compounds bound in their best pose and subjected the complexes to 100 ns molecular dynamics simulations to assess the stability of the complexes. All eight analogs formed stable complexes for the duration of the simulations. The results from this work highlight nine distinct analogs of IND (-)-237D with high affinity towards α6β2* nAChR. These leads can be synthesized and tested in in vitro and in vivo studies as lead candidates for drugs to treat nicotine addiction.

SUBMITTER: Acquah FA

PROVIDER: S-EPMC8347036 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Modulation of gain-of-function α6*-nicotinic acetylcholine receptor by β3 subunits.

Project description:We previously have shown that β3 subunits either eliminate (e.g. for all-human (h) or all-mouse (m) α6β4β3-nAChR) or potentiate (e.g. for hybrid mα6hβ4hβ3- or mα6mβ4hβ3-nAChR containing subunits from different species) function of α6*-nAChR expressed in Xenopus oocytes, and that nAChR hα6 subunit residues Asn-143 and Met-145 in N-terminal domain loop E are important for dominant-negative effects of nAChR hβ3 subunits on hα6*-nAChR function. Here, we tested the hypothesis that these effects of β3 subunits would be preserved even if nAChR α6 subunits harbored gain-of-function, leucine- or valine-to-serine mutations at 9' or 13' positions (L9'S or V13'S) in their second transmembrane domains, yielding receptors with heightened functional activity and more amenable to assessment of effects of β3 subunit incorporation. However, coexpression with β3 subunits potentiates rather than suppresses function of all-human, all-mouse, or hybrid α6((L9'S or V13'S))β4*- or α6(N143D+M145V)(L9'S)β2*-nAChR. This contrasts with the lack of consistent function when α6((L9'S or V13'S)) and β2 subunits are expressed alone or in the presence of wild-type β3 subunits. These results provide evidence that gain-of-function hα6hβ2*-nAChR (i.e. hα6(N143D+M145V)(L9'S)hβ2hβ3 nAChR) could be produced in vitro. These studies also indicate that nAChR β3 subunits can be assembly partners in functional α6*-nAChR and that 9' or 13' mutations in the nAChR α6 subunit second transmembrane domain can act as gain-of-function and/or reporter mutations. Moreover, our findings suggest that β3 subunit coexpression promotes function of α6*-nAChR.

| S-EPMC3340145 | biostudies-literature

High-Throughput Patch Clamp Screening in Human α6-Containing Nicotinic Acetylcholine Receptors.

Project description:Nicotine, the addictive component of tobacco products, is an agonist at nicotinic acetylcholine receptors (nAChRs) in the brain. The subtypes of nAChR are defined by their α- and β-subunit composition. The α6β2β3 nAChR subtype is expressed in terminals of dopaminergic neurons that project to the nucleus accumbens and striatum and modulate dopamine release in brain regions involved in nicotine addiction. Although subtype-dependent selectivity of nicotine is well documented, subtype-selective profiles of other tobacco product constituents are largely unknown and could be essential for understanding the addiction-related neurological effects of tobacco products. We describe the development and validation of a recombinant cell line expressing human α6/3β2β3V273S nAChR for screening and profiling assays in an automated patch clamp platform (IonWorks Barracuda). The cell line was pharmacologically characterized by subtype-selective and nonselective reference agonists, pore blockers, and competitive antagonists. Agonist and antagonist effects detected by the automated patch clamp approach were comparable to those obtained by conventional electrophysiological assays. A pilot screen of a library of Food and Drug Administration-approved drugs identified compounds, previously not known to modulate nAChRs, which selectively inhibited the α6/3β2β3V273S subtype. These assays provide new tools for screening and subtype-selective profiling of compounds that act at α6β2β3 nicotinic receptors.

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Project description:α-Conotoxin LvIA (α-CTx LvIA) is a small peptide from the venom of the carnivorous marine gastropod Conus lividus and is the most selective inhibitor of α3β2 nicotinic acetylcholine receptors (nAChRs) known to date. It can distinguish the α3β2 nAChR subtype from the α6β2* (* indicates the other subunit) and α3β4 nAChR subtypes. In this study, we performed mutational studies to assess the influence of residues of the β2 subunit versus those of the β4 subunit on the binding of α-CTx LvIA. Although two β2 mutations, α3β2[F119Q] and α3β2[T59K], strongly enhanced the affinity of LvIA, the β2 mutation α3β2[V111I] substantially reduced the binding of LvIA. Increased activity of LvIA was also observed when the β2-T59L mutant was combined with the α3 subunit. There were no significant difference in inhibition of α3β2[T59I], α3β2[Q34A], and α3β2[K79A] nAChRs when compared with wild-type α3β2 nAChR. α-CTx LvIA displayed slower off-rate kinetics at α3β2[F119Q] and α3β2[T59K] than at the wild-type receptor, with the latter mutant having the most pronounced effect. Taken together, these data provide evidence that the β2 subunit contributes to α-CTx LvIA binding and selectivity. The results demonstrate that Val(111) is critical and facilitates LvIA binding; this position has not previously been identified as important to binding of other 4/7 framework α-conotoxins. Thr(59) and Phe(119) of the β2 subunit appear to interfere with LvIA binding, and their replacement by the corresponding residues of the β4 subunit leads to increased affinity.

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Project description:α6β4 nAChR is expressed in the peripheral and central nervous systems and is associated with pain, addiction, and movement disorders. Natural α-conotoxins (α-CTxs) can effectively block different nAChR subtypes with higher efficacy and selectivity. However, the research on α6β4 nAChR is relatively poor, partly because of the lack of available target-specific α-CTxs. In this study, we synthesized a novel α-4/7 conotoxin QuIA that was found from Conus quercinus. We investigated the efficacy of this peptide to different nAChR subtypes using a two-electrode voltage-clamp technique. Remarkably, we found α-QuIA inhibited the neuronal α3β2 and α6/α3β4 nAChR subtypes with significantly high affinity (IC50 was 55.7 nM and 90.68 nM, respectively), and did not block other nAChR subtypes even at a high concentration of 10 μM. In contrast, most α-CTxs have been determined so far to effectively block the α6/α3β4 nAChR subtype while also maintaining a similar higher efficacy against the closely related α6β2β3 and/or α3β4 subtypes, which are different from QuIA. In conclusion, α-QuIA is a novel α4/7-CTx, which has the potential to develop as an effective neuropharmacology tool to detect the function of α6β4 nAChR.

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Assessing potentiation of the (α4)3(β2)2 nicotinic acetylcholine receptor by the allosteric agonist CMPI.

Project description:The extracellular domain of the nicotinic acetylcholine receptor isoforms formed by three α4 and two β2 subunits ((α4)3(β2)2 nAChR) harbors two high-affinity "canonical" acetylcholine (ACh)-binding sites located in the two α4:β2 intersubunit interfaces and a low-affinity "noncanonical" ACh-binding site located in the α4:α4 intersubunit interface. In this study, we used ACh, cytisine, and nicotine (which bind at both the α4:α4 and α4:β2 interfaces), TC-2559 (which binds at the α4:β2 but not at the α4:α4 interface), and 3-(2-chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)-1H-pyrrazol-4-yl)isoxazole (CMPI, which binds at the α4:α4 but not at the α4:β2 interface), to investigate the binding and gating properties of CMPI at the α4:α4 interface. We recorded whole-cell currents from Xenopus laevis oocytes expressing (α4)3(β2)2 nAChR in response to applications of these ligands, alone or in combination. The electrophysiological data were analyzed in the framework of a modified Monod-Wyman-Changeux allosteric activation model. We show that CMPI is a high-affinity, high-efficacy agonist at the α4:α4 binding site and that its weak direct activating effect is accounted for by its inability to productively interact with the α4:β2 sites. The data presented here enhance our understanding of the functional contributions of ligand binding at the α4:α4 subunit interface to (α4)3(β2)2 nAChR-channel gating. These findings support the potential use of α4:α4 specific ligands to increase the efficacy of the neurotransmitter ACh in conditions associated with decline in nAChRs activity in the brain.

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LY2087101 and dFBr share transmembrane binding sites in the (α4)3(β2)2 Nicotinic Acetylcholine Receptor.

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

| S-EPMC5775429 | biostudies-literature

Molecular-dynamics simulations of ELIC-a prokaryotic homologue of the nicotinic acetylcholine receptor.

Project description:The ligand-gated ion channel from Erwinia chrysanthemi (ELIC) is a prokaryotic homolog of the eukaryotic nicotinic acetylcholine receptor (nAChR) that responds to the binding of neurotransmitter acetylcholine and mediates fast signal transmission. ELIC is similar to the nAChR in its primary sequence and overall subunit organization, but despite their structural similarity, it is not clear whether these two ligand-gated ion channels operate in a similar manner. Further, it is not known to what extent mechanistic insights gleaned from the ELIC structure translate to eukaryotic counterparts such as the nAChR. Here we use molecular-dynamics simulations to probe the conformational dynamics and hydration of the transmembrane pore of ELIC. The results are compared with those from our previous simulation of the human alpha7 nAChR. Overall, ELIC displays increased stability compared to the nAChR, whereas the two proteins exhibit remarkable similarity in their global motion and flexibility patterns. The majority of the increased stability of ELIC does not stem from the deficiency of the models used in the simulations, and but rather seems to have a structural basis. Slightly altered dynamical correlation features are also observed among several loops within the membrane region. In sharp contrast to the nAChR, ELIC is completely dehydrated from the pore center to the extracellular end throughout the simulation. Finally, the simulation of an ELIC mutant substantiates the important role of F246 on the stability, hydration and possibly function of the ELIC channel.

| S-EPMC2711484 | biostudies-literature

α-Conotoxin [S9A]TxID Potently Discriminates between α3β4 and α6/α3β4 Nicotinic Acetylcholine Receptors.

Project description:α3β4 nAChRs have been implicated in various pathophysiological conditions. However, the expression profile of α3β4 nAChRs and α6/α3β4 nAChRs overlap in a variety of tissues. To distinguish between these two subtypes, we redesigned peptide 1 (α-conotoxin TxID), which inhibits α3β4 and α6/α3β4 nAChR subtypes. We systematically mutated 1 to evaluate analogue selectivity for α3β4 vs α6/α3β4 nAChRs expressed in Xenopus laevis oocytes. One analogue, peptide 7 ([S9A]TxID), had 46-fold greater potency for α3β4 versus α6/α3β4 nAChRs. Peptide 7 had IC50s > 10 μM for other nAChR subtypes. Molecular dynamics simulations suggested that Ser-9 of TxID was involved in a weak hydrogen bond with β4 Lys-81 in the α6β4 binding site but not in the α3β4 binding site. When Ser-9 was substituted by an Ala, this hydrogen bond interaction was disrupted. These results provide further molecular insights into the selectivity of 7 and provide a guide for designing ligands that block α3β4 nAChRs.

| S-EPMC5572761 | biostudies-literature

Nicotinic acetylcholine receptors containing α6 subunits contribute to alcohol reward-related behaviours.

Project description:Evidence is emerging that neuronal nicotinic acetylcholine receptors (nAChRs) in the mesolimbic dopamine (DA) system are involved in mediating the reinforcing effects of alcohol. Midbrain DA neurons express high levels of α6 subunit-containing nAChRs that modulate DA transmission, implicating their involvement in reward-related behaviours. This study assessed the role of α6-containing nAChRs in modulating alcohol reward using transgenic mice expressing mutant, hypersensitive α6 nAChR subunits (α6L9'S mice). α6L9'S mice and littermate controls were tested in three well-established models of alcohol reward: 24-h two-bottle choice drinking, drinking in the dark (DID), and conditioned place preference (CPP). Confocal microscopy and patch-clamp electrophysiology were used to show the localization and function of hypersensitive α6 subunit-containing nAChRs. Results indicate that female α6L9'S mice showed significantly higher alcohol intake at low concentrations of alcohol (3% and 6%) in the two-bottle choice procedure. Both male and female α6L9'S mice drank significantly more in the DID procedure and displayed an alcohol-induced place preference using a low dose of alcohol (0.5 g/kg) that was ineffective in littermate controls. Confocal microscopy showed that α6 subunit-containing nAChRs are selectively expressed on ventral tegmental area (VTA) DAergic, but not GABAergic neurons. Patch-clamp electrophysiology showed that VTA DA neurons of α6L9'S mice are hypersensitive to ACh. Collectively, these results suggest that α6L9'S mice are more sensitive to the rewarding effects of alcohol, and suggest that VTA α6 subunit-containing nAChRs modulate alcohol reward. Thus, α6 subunit-containing nAChRs may be a promising therapeutic target for treatment of alcohol use disorders.

| S-EPMC4426878 | biostudies-other

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