The Unique α4(+)/(-) a4 Agonist Binding Site in (α4)3(β2)2 Subtype Nicotinic Acetylcholine Receptors Permits Differential Agonist Desensitization Pharmacology versus the α4)2(β2)73 Subtypes

Selected nicotinic agonists were used to activate and desensitize high-sensitivity (HS) (alpha 4)(3)(beta 2)(2) or low-sensitivity (LS) (alpha 4)(3)(beta 2)(2)) isoforms of human alpha 4 beta 2-nicotinic acetylcholine receptors (nAChRs). Function was assessed using Rb-86(+) efflux in a stably transfected SH-EP1-h alpha 4 beta 2 human epithelial cell line, and twoelectrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing concatenated pentameric HS or LS alpha 4 beta 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 alpha 4 beta 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 alpha 4(+)/(-) a4 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 b2-subunit-specific [125I] mAb295 labeling. However, maximum function is approximately five times greater on a per-receptor basis for unmodified LSP versus HSP alpha 4 beta 2-nAChR. Thus, recruitment of the alpha 4(+)/(-) a4 site at higher agonist concentrations appears to augment otherwisesimilar function mediated by the pair of alpha 4(+)/(-) b2 sites shared by both isoforms. These studies elucidate the receptor-level differences underlying the differential pharmacology of the two alpha 4 beta 2-nAChR isoforms, and demonstrate that HS versus LS alpha 4 beta 2-nAChR activity can be selectively manipulated using pharmacological approaches. Since alpha 4 beta 2-nAChR are the predominant neuronal subtype, these discoveries likely have significant functional implications, and may provide important insights for drug discovery and development.