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Unraveling amino acid residues critical for allosteric potentiation of (?4)3(?2)2-type nicotinic acetylcholine receptor responses.

ABSTRACT: Neuronal nicotinic acetylcholine receptors (nAChRs) are promising drug targets to manage several neurological disorders and nicotine addiction. Growing evidence indicates that positive allosteric modulators of nAChRs improve pharmacological specificity by binding to unique sites present only in a subpopulation of nAChRs. Furthermore, nAChR positive allosteric modulators such as NS9283 and CMPI have been shown to potentiate responses of (?4)3(?2)2 but not (?4)2(?2)3 nAChR isoforms. This selective potentiation underlines that the ?4:?4 interface, which is present only in the (?4)3(?2)2 nAChR, is an important and promising drug target. In this report we used site-directed mutagenesis to substitute specific amino acid residues and computational analyses to elucidate CMPI's binding mode at the ?4:?4 subunit extracellular interface and identified a unique set of amino acid residues that determined its affinity. We found that amino acid residues ?4Gly-41, ?4Lys-64, and ?4Thr-66 were critical for (?4)3(?2)2 nAChR potentiation by CMPI, but not by NS9283, whereas amino acid substitution at ?4His-116, a known determinant of NS9283 and of agonist binding at the ?4:?4 subunit interface, did not reduce CMPI potentiation. In contrast, substitutions at ?4Gln-124 and ?4Thr-126 reduced potentiation by CMPI and NS9283, indicating that their binding sites partially overlap. These results delineate the role of amino acid residues contributing to the ?4:?4 subunit extracellular interface in nAChR potentiation. These findings also provide structural information that will facilitate the structure-based design of novel therapeutics that target selectively the (?4)3(?2)2 nAChR.

SUBMITTER: Wang ZJ

PROVIDER: S-EPMC5473250 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Unraveling amino acid residues critical for allosteric potentiation of (α4)3(β2)2-type nicotinic acetylcholine receptor responses.

Wang Ze-Jun ZJ Deba Farah F Mohamed Tasnim S TS Chiara David C DC Ramos Kara K Hamouda Ayman K AK

The Journal of biological chemistry 20170426 24

Neuronal nicotinic acetylcholine receptors (nAChRs) are promising drug targets to manage several neurological disorders and nicotine addiction. Growing evidence indicates that positive allosteric modulators of nAChRs improve pharmacological specificity by binding to unique sites present only in a subpopulation of nAChRs. Furthermore, nAChR positive allosteric modulators such as NS9283 and CMPI have been shown to potentiate responses of (α4)3(β2)2 but not (α4)2(β2)3 nAChR isoforms. This selective ...[more]

PMID: 28446611

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Project description:Selected nicotinic agonists were used to activate and desensitize high-sensitivity (HS) (α4)2(β2)3) or low-sensitivity (LS) (α4)3(β2)2) isoforms of human α4β2-nicotinic acetylcholine receptors (nAChRs). Function was assessed using (86)Rb(+) efflux in a stably transfected SH-EP1-hα4β2 human epithelial cell line, and two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing concatenated pentameric HS or LS α4β2-nAChR constructs (HSP and LSP). Unlike previously studied agonists, desensitization by the highly selective agonists A-85380 [3-(2(S)-azetidinylmethoxy)pyridine] and sazetidine-A (Saz-A) preferentially reduced α4β2-nAChR HS-phase versus LS-phase responses. The concatenated-nAChR experiments confirmed that approximately 20% of LS-isoform acetylcholine-induced function occurs in an HS-like phase, which is abolished by Saz-A preincubation. Six mutant LSPs were generated, each targeting a conserved agonist binding residue within the LS-isoform-only α4(+)/(-)α4 interface agonist binding site. Every mutation reduced the percentage of LS-phase function, demonstrating that this site underpins LS-phase function. Oocyte-surface expression of the HSP and each of the LSP constructs was statistically indistinguishable, as measured using β2-subunit-specific [(125)I]mAb295 labeling. However, maximum function is approximately five times greater on a "per-receptor" basis for unmodified LSP versus HSP α4β2-nAChRs. Thus, recruitment of the α4(+)/(-)α4 site at higher agonist concentrations appears to augment otherwise-similar function mediated by the pair of α4(+)/(-)β2 sites shared by both isoforms. These studies elucidate the receptor-level differences underlying the differential pharmacology of the two α4β2-nAChR isoforms, and demonstrate that HS versus LS α4β2-nAChR activity can be selectively manipulated using pharmacological approaches. Since α4β2 nAChRs are the predominant neuronal subtype, these discoveries likely have significant functional implications, and may provide important insights for drug discovery and development.

Dataset Information

Unraveling amino acid residues critical for allosteric potentiation of (?4)3(?2)2-type nicotinic acetylcholine receptor responses.

Publications

Unraveling amino acid residues critical for allosteric potentiation of (α4)3(β2)2-type nicotinic acetylcholine receptor responses.

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