Interactions of Globular and Ribbon [γ4E]GID with α4β2 Neuronal Nicotinic Acetylcholine Receptor.
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ABSTRACT:
SUBMITTER: Wu X
PROVIDER: S-EPMC8469569 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Alterations in expression patterns of α4β2 nicotinic acetylcholine receptors have been demonstrated to alter cholinergic neurotransmission and are implicated in neurologic disorders, including autism, nicotine addiction, Alzheimer's disease, and Parkinson's disease. Positive allosteric modulators (PAMs) represent promising new leads in the development of therapeutic agents for the treatment of these disorders. This study investigates the involvement of the β2-containing subunit interfaces of α4β2 receptors in the modulation of acetylcholine (ACh)-induced responses by the PAM desformylflustrabromine (dFBr). Eight amino acids on the principal face of the β2 subunit were mutated to alanine to explore the involvement of this region in the potentiation of ACh-induced currents by dFBr. ACh-induced responses obtained from wild-type and mutant α4β2 receptors expressed in Xenopus laevis oocytes were recorded in the presence and absence of dFBr using two-electrode voltage clamp electrophysiology. Wild-type and mutant receptors were expressed in both high and low ACh sensitivity isoforms by using biased injection ratios of 1:5 or 5:1 α4 to β2 complementary RNA. Mutations were made in the B, C, and A loops of the principal face of the β2 subunit, which are regions not involved in the binding of ACh. Mutant β2(Y120A) significantly eliminated dFBr potency in both isoform preparations. Several other mutations altered dFBr potentiation levels in both preparations. Our findings support the involvement of the principal face of the β2 subunit in dFBr modulation of ACh-induced responses. Findings from this study will aid in the improved design of dFBr-like PAMs for potential therapeutic use.
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Project description:Background and purposeThe heteromeric α4β2 nicotinic acetylcholine receptor (nAChR) is abundant in the human brain and is associated with a range of CNS disorders. This nAChR subtype has been recently crystallised in a conformation that was proposed to represent a desensitised state. Here, we investigated the conformational transition mechanism of this nAChR from a desensitised to a closed/resting state.Experimental approachThe competitive antagonist dihydro-β-erythroidine (DHβE) was modelled by replacement of the agonist nicotine in the α4β2 nAChR experimental structure. DHβE is used both in vitro and in vivo for its ability to block α4β2 nAChRs. This system was studied by three molecular dynamics simulations with a combined simulation time of 2.6 μs. Electrophysiological studies of mutated receptors were performed to validate the simulation results.Key resultsThe relative positions of the extracellular and transmembrane domains in the models are distinct from those of the desensitised state structure and are compatible with experimental structures of Cys-loop receptors captured in a closed/resting state.Conclusions and implicationsOur model suggests that the side chains of α4 L257 (9') and α4 L264 (16') are the main constrictions in the transmembrane pore. The involvement of position 9' in channel gating is well established, but position 16' was only previously identified as a gate for the bacterial channels, ELIC and GLIC. L257 but not L264 was found to influence the slow component of desensitisation. The structure of the antagonist-bound state proposed here should be valuable for the development of therapeutic or insecticide compounds.
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Project description:Marine cone snails are a large family of gastropods that have evolved highly potent venoms for predation and defense. The cone snail venom has exceptional molecular diversity in neuropharmacologically active compounds, targeting a range of receptors, ion channels, and transporters. These conotoxins have helped to dissect the structure and function of many of these therapeutically significant targets in the central and peripheral nervous systems, as well as unravelling the complex cellular mechanisms modulated by these receptors and ion channels. This review provides an overview of α-conotoxins targeting neuronal nicotinic acetylcholine receptors. The structure and activity of both classical and non-classical α-conotoxins are discussed, along with their contributions towards understanding nicotinic acetylcholine receptor (nAChR) structure and function.
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Project description:Excess sugar consumption has been shown to contribute directly to weight gain, thus contributing to the growing worldwide obesity epidemic. Interestingly, increased sugar consumption has been shown to repeatedly elevate dopamine levels in the nucleus accumbens (NAc), in the mesolimbic reward pathway of the brain similar to many drugs of abuse. We report that varenicline, an FDA-approved nicotinic acetylcholine receptor (nAChR) partial agonist that modulates dopamine in the mesolimbic reward pathway of the brain, significantly reduces sucrose consumption, especially in a long-term consumption paradigm. Similar results were observed with other nAChR drugs, namely mecamylamine and cytisine. Furthermore, we show that long-term sucrose consumption increases ?4?2 * and decreases ?6?2* nAChRs in the nucleus accumbens, a key brain region associated with reward. Taken together, our results suggest that nAChR drugs such as varenicline may represent a novel treatment strategy for reducing sugar consumption.
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Project description:Nicotine, the principal reinforcing compound in tobacco, acts in the brain by activating neuronal nicotinic acetylcholine receptors (nAChRs). This review summarizes our current knowledge regarding how the α5 accessory nAChR subunit, encoded by the CHRNA5 gene, differentially modulates α4β2* and α3β4* receptors at the cellular level. Genome-wide association studies have linked a gene cluster in chromosomal region 15q25 to increased susceptibility to nicotine addiction, lung cancer, chronic obstructive pulmonary disease, and peripheral arterial disease. Interestingly, this gene cluster contains a non-synonymous single-nucleotide polymorphism (SNP) in the human CHRNA5 gene, causing an aspartic acid (D) to asparagine (N) substitution at amino acid position 398 in the α5 nAChR subunit. Although other SNPs have been associated with tobacco smoking behavior, efforts have focused predominantly on the D398 and N398 variants in the α5 subunit. In recent years, significant progress has been made toward understanding the role that the α5 nAChR subunit-and the role of the D398 and N398 variants-plays on nAChR function at the cellular level. These insights stem primarily from a wide range of experimental models, including receptors expressed heterologously in Xenopus oocytes, various cell lines, and neurons derived from human induced pluripotent stem cells (iPSCs), as well as endogenous receptors in genetically engineered mice and-more recently-rats. Despite providing a wealth of available data, however, these studies have yielded conflicting results, and our understanding of the modulatory role that the α5 subunit plays remains incomplete. Here, we review these reports and the various techniques used for expression and analysis in order to examine how the α5 subunit modulates key functions in α4β2* and α3β4* receptors, including receptor trafficking, sensitivity, efficacy, and desensitization. In addition, we highlight the strikingly different role that the α5 subunit plays in Ca2+ signaling between α4β2* and α3β4* receptors, and we discuss whether the N398 α5 subunit variant can partially replace the D398 variant.
| S-EPMC7744819 | biostudies-literature
Project description:Two α4β2 nicotinic acetylcholine receptor (α4β2-nAChR) isoforms exist with (α4)2(β2)3 and (α4)3(β2)2 subunit stoichiometries and high versus low agonist sensitivities (HS and LS), respectively. Both isoforms contain a pair of α4(+)/(-)β2 agonist-binding sites. The LS isoform also contains a unique α4(+)/(-)α4 site with lower agonist affinity than the α4(+)/(-)β2 sites. However, the relative roles of the conserved α4(+)/(-)β2 agonist-binding sites in and between the isoforms have not been studied. We used a fully linked subunit concatemeric nAChR approach to express pure populations of HS or LS isoform α4β2*-nAChR. This approach also allowed us to mutate individual subunit interfaces, or combinations thereof, on each isoform background. We used this approach to systematically mutate a triplet of β2 subunit (-)-face E-loop residues to their non-conserved α4 subunit counterparts or vice versa (β2HQT and α4VFL, respectively). Mutant-nAChR constructs (and unmodified controls) were expressed in Xenopus oocytes. Acetylcholine concentration-response curves and maximum function were measured using two-electrode voltage clamp electrophysiology. Surface expression was measured with (125)I-mAb 295 binding and was used to define function/nAChR. If the α4(+)/(-)β2 sites contribute equally to function, making identical β2HQT substitutions at either site should produce similar functional outcomes. Instead, highly differential outcomes within the HS isoform, and between the two isoforms, were observed. In contrast, α4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise equivalent α4(+)/(-)β2 agonist sites modifies their contributions to nAChR activation and that E-loop residues are an important contributor to this neighbor effect.
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Project description:KTM is a 16 amino acid peptide with the sequence WCCSYPGCYWSSSKWC. Here, we present the nuclear magnetic resonance (NMR) structure and bioactivity of this rationally designed ?-conotoxin (?-CTx) that demonstrates potent inhibition of rat ?3?2-nicotinic acetylcholine receptors (r?3?2-nAChRs). Two bioassays were used to test the efficacy of KTM. First, a qualitative PC12 cell-based assay confirmed that KTM acts as a nAChR antagonist. Second, bioactivity evaluation by two-electrode voltage clamp electrophysiology was used to measure the inhibition of r?3?2-nAChRs by KTM (IC50 = 0.19 ± 0.02 nM), and inhibition of the same nAChR isoform by ?-CTx MII (IC50 = 0.35 ± 0.8 nM). The three-dimensional structure of KTM was determined by NMR spectroscopy, and the final set of 20 structures derived from 32 distance restraints, four dihedral angle constraints, and two disulfide bond constraints overlapped with a mean global backbone root-mean-square deviation (RMSD) of 1.7 ± 0.5 Å. The structure of KTM did not adopt the disulfide fold of ?-CTx MII for which it was designed, but instead adopted a flexible ribbon backbone and disulfide connectivity of C2-C16 and C3-C8 with an estimated 12.5% ?-helical content. In contrast, ?-CTx MII, which has a native fold of C2-C8 and C3-C16, has an estimated 38.1% ?-helical secondary structure. KTM is the first reported instance of a Framework I (CC-C-C) ?-CTx with ribbon connectivity to display sub-nanomolar inhibitory potency of r?3?2-nAChR subtypes.
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Project description:Neuronal nicotinic AChRs (nAChRs) are implicated in the pathogenesis of diverse neurological disorders and in the regulation of small-cell lung carcinoma growth. Twelve subunits have been identified in vertebrates, and mutations of one are recognized in a rare form of human epilepsy. Mice with genetically manipulated neuronal nAChR subunits exhibit behavioral or autonomic phenotypes. Here, we report the first model of an acquired neuronal nAChR disorder and evidence for its pertinence to paraneoplastic neurological autoimmunity. Rabbits immunized once with recombinant alpha3 subunit (residues 1-205) develop profound gastrointestinal hypomotility, dilated pupils with impaired light response, and grossly distended bladders. As in patients with idiopathic and paraneoplastic autoimmune autonomic neuropathy, the severity parallels serum levels of ganglionic nAChR autoantibody. Failure of neurotransmission through abdominal sympathetic ganglia, with retention of neuronal viability, confirms that the disorder is a postsynaptic channelopathy. In addition, we found ganglionic nAChR protein in small-cell carcinoma lines, identifying this cancer as a potential initiator of ganglionic nAChR autoimmunity. The data support our hypothesis that immune responses driven by distinct neuronal nAChR subtypes expressed in small-cell carcinomas account for several lung cancer-related paraneoplastic disorders affecting cholinergic systems, including autoimmune autonomic neuropathy, seizures, dementia, and movement disorders.
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Project description:To characterize the binding sites of mecamylamine enantiomers on the transmembrane domain (TMD) of human (h) (α4)3(β2)2 and (α4)2(β2)3 nicotinic acetylcholine receptors (AChRs), we used nuclear magnetic resonance (NMR), molecular docking, and radioligand binding approaches. The interactions of (S)-(+)- and (R)-(-)-mecamylamine with several residues, determined by high-resolution NMR, within the hα4β2-TMD indicate different modes of binding at several luminal (L) and nonluminal (NL) sites. In general, the residues sensitive to each mecamylamine enantiomer are similar at both receptor stoichiometries. However, some differences were observed. The molecular docking experiments were crucial for delineating the location and orientation of each enantiomer in its binding site. In the (α4)2(β2)3-TMD, (S)-(+)-mecamylamine interacts with the L1 (i.e., between positions -3' and -5') and L2 (i.e., between positions 16' and 20') sites, whereas the β2-intersubunit (i.e., cytoplasmic end of two β2-TMDs) and α4/β2-intersubunit (i.e., cytoplasmic end of α4-TM1 and β2-TM3) sites are shared by both enantiomers. In the (α4)3(β2)2-TMD, both enantiomers bind with different orientations to the L1' (closer to ring 2') and α4-intrasubunit (i.e., at the cytoplasmic ends of α4-TM1 and α4-TM2) sites, but only (R)-(-)-mecamylamine interacts with the L2' (i.e., closer to ring 20') and α4-TM3-intrasubunit sites. Our findings are important because they provide, for the first time, a structural understanding of the allosteric modulation elicited by mecamylamine enantiomers at each hα4β2 stoichiometry. This advancement could be beneficial for the development of novel therapies for the treatment of several neurological disorders.
| S-EPMC3971955 | biostudies-literature
Project description:The ?7-nicotinic acetylcholine receptor (?7-nAChR) is a ligand-gated ion channel that is expressed widely in vertebrates and is the principal high-affinity ?-bungarotoxin (?-bgtx) binding protein in the mammalian CNS. ?7-nAChRs associate with proteins that can modulate its properties. The ?7-nAChR interactome is the summation of proteins interacting or associating with ?7-nAChRs in a protein complex. To identify an ?7-nAChR interactome in neural tissue, we isolated ?-bgtx-affinity protein complexes from wild-type and ?7-nAChR knockout (?7 KO) mouse whole brain tissue homogenates using ?-bgtx-affinity beads. Affinity precipitated proteins were trypsinized and analyzed with an Orbitrap Fusion mass spectrometer. Proteins isolated with the ?7-nAChR specific ligand, ?-bgtx, were determined to be ?7-nAChR associated proteins. The ?7-nAChR subunit and 120 additional proteins were identified. Additionally, 369 proteins were identified as binding to ?-bgtx in the absence of ?7-nAChR expression, thereby identifying nonspecific proteins for ?7-nAChR investigations using ?-bgtx enrichment. These results expand on our previous investigations of ?7-nAChR interacting proteins using ?-bgtx-affinity bead isolation by controlling for differences between ?7-nAChR and ?-bgtx-specific proteins, developing an improved protein isolation methodology, and incorporating the latest technology in mass spectrometry. The ?7-nAChR interactome identified in this study includes proteins associated with the expression, localization, function, or modulation of ?7-nAChRs, and it provides a foundation for future studies to elucidate how these interactions contribute to human disease.
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