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Atypical nicotinic agonist bound conformations conferring subtype selectivity.

ABSTRACT: The nicotinic acetylcholine (ACh) receptor (nAChR) plays a crucial role in excitatory neurotransmission and is an important target for drugs and insecticides. Diverse nAChR subtypes with various subunit combinations confer differential selectivity for nicotinic drugs. We investigated the subtype selectivity of nAChR agonists by comparing two ACh-binding proteins (AChBPs) as structural surrogates with distinct pharmacological profiles [i.e., Lymnaea stagnalis (Ls) AChBP of low neonicotinoid and high nicotinoid sensitivities and Aplysia californica (Ac) AChBP of high neonicotinoid sensitivity] mimicking vertebrate and insect nAChR subtypes, respectively. The structural basis of subtype selectivity was examined here by photoaffinity labeling. Two azidoneonicotinoid probes in the Ls-AChBP surprisingly modified two distinct and distant subunit interface sites: loop F Y164 of the complementary or (-)-face subunit and loop C Y192 of the principal or (+)-face subunit, whereas three azidonicotinoid probes derivatized only Y192. Both the neonicotinoid and nicotinoid probes labeled Ac-AChBP at only one position at the interface between loop C Y195 and loop E M116. These findings were used to establish structural models of the two AChBP subtypes. In the Ac-AChBP, the neonicotinoids and nicotinoids are nestled in similar bound conformations. Intriguingly, for the Ls-AChBP, the neonicotinoids have two bound conformations that are inverted relative to each other, whereas nicotinoids appear buried in only one conserved conformation as seen for the Ac-AChBP subtype. Accordingly, the subtype selectivity is based on two disparate bound conformations of nicotinic agonists, thereby establishing an atypical concept for neonicotinoid versus nicotinoid selectivity between insect and vertebrate nAChRs.

SUBMITTER: Tomizawa M

PROVIDER: S-EPMC2234212 | biostudies-literature | 2008 Feb

REPOSITORIES: biostudies-literature

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Atypical nicotinic agonist bound conformations conferring subtype selectivity.

Tomizawa Motohiro M Maltby David D Talley Todd T TT Durkin Kathleen A KA Medzihradszky Katalin F KF Burlingame Alma L AL Taylor Palmer P Casida John E JE

Proceedings of the National Academy of Sciences of the United States of America 20080129 5

The nicotinic acetylcholine (ACh) receptor (nAChR) plays a crucial role in excitatory neurotransmission and is an important target for drugs and insecticides. Diverse nAChR subtypes with various subunit combinations confer differential selectivity for nicotinic drugs. We investigated the subtype selectivity of nAChR agonists by comparing two ACh-binding proteins (AChBPs) as structural surrogates with distinct pharmacological profiles [i.e., Lymnaea stagnalis (Ls) AChBP of low neonicotinoid and h ...[more]

PMID: 18230720

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Project description:Selected nicotinic agonists were used to activate and desensitize high-sensitivity (HS) (?4)2(?2)3) or low-sensitivity (LS) (?4)3(?2)2) isoforms of human ?4?2-nicotinic acetylcholine receptors (nAChRs). Function was assessed using (86)Rb(+) efflux in a stably transfected SH-EP1-h?4?2 human epithelial cell line, and two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing concatenated pentameric HS or LS ?4?2-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 ?4?2-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 ?4(+)/(-)?4 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 ?2-subunit-specific [(125)I]mAb295 labeling. However, maximum function is approximately five times greater on a "per-receptor" basis for unmodified LSP versus HSP ?4?2-nAChRs. Thus, recruitment of the ?4(+)/(-)?4 site at higher agonist concentrations appears to augment otherwise-similar function mediated by the pair of ?4(+)/(-)?2 sites shared by both isoforms. These studies elucidate the receptor-level differences underlying the differential pharmacology of the two ?4?2-nAChR isoforms, and demonstrate that HS versus LS ?4?2-nAChR activity can be selectively manipulated using pharmacological approaches. Since ?4?2 nAChRs are the predominant neuronal subtype, these discoveries likely have significant functional implications, and may provide important insights for drug discovery and development.

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Atypical nicotinic agonist bound conformations conferring subtype selectivity.

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Atypical nicotinic agonist bound conformations conferring subtype selectivity.

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