The unique ?4+/-?4 agonist binding site in (?4)3(?2)2 subtype nicotinic acetylcholine receptors permits differential agonist desensitization pharmacology versus the (?4)2(?2)3 subtype.

ABSTRACT: 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.

SUBMITTER: Eaton JB 

PROVIDER: S-EPMC3868879 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature