The Unique Î±4(1)/(2)Î±4 Agonist Binding Site In (Î±4)3(Î²2)2 Subtype Nicotinic Acetylcholine Receptors Permits Differential Agonist Desensitization Pharmacology Versus The (Î±4)2(Î²2)3 Subtypes
Selected nicotinic agonists were used to activate and desensitize high-sensitivity (HS) (Î±4)2(Î²2)3) or low-sensitivity (LS) (Î±4)3(Î²2)2) isoforms of human Î±4Î²2-nicotinic acetylcholine receptors (nAChRs). Function was assessed using 86Rb1 efflux in a stably transfected SH-EP1-hÎ±4Î²2 human epithelial cell line, and twoelectrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing concatenated pentameric HS or LS Î±4Î²2- 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 Î±4Î²2-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 Î±4(1)/(2)Î±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 Î²2-subunit-specific [125I]Î¼Ab295 labeling. However, maximum function is approximately five times greater on a \"per-receptor\" basis for unmodified LSP versus HSP Î±4Î²2-nAChRs. Thus, recruitment of the Î±4(1)/(2)Î±4 site at higher agonist concentrations appears to augment otherwisesimilar function mediated by the pair of Î±4(1)/(2)Î²2 sites shared by both isoforms. These studies elucidate the receptor-level differences underlying the differential pharmacology of the two Î±4Î²2-nAChR isoforms, and demonstrate that HS versus LS Î±4Î²2-nAChR activity can be selectively manipulated using pharmacological approaches. Since Î±4Î²2 nAChRs are the predominant neuronal subtype, these discoveries likely have significant functional implications, and may provide important insights for drug discovery and development. Copyright Â© 2013 by The American Society for Pharmacology and Experimental Therapeutics.
Journal of Pharmacology and Experimental Therapeutics
Digital Object Identifier (DOI)
Eaton, J. Brek; Lucero, Linda M.; Stratton, Harrison; Chang, Yongchang; Cooper, John F.; Lindstrom, Jon M.; Lukas, Ronald J.; and Whiteaker, Paul, "The Unique Î±4(1)/(2)Î±4 Agonist Binding Site In (Î±4)3(Î²2)2 Subtype Nicotinic Acetylcholine Receptors Permits Differential Agonist Desensitization Pharmacology Versus The (Î±4)2(Î²2)3 Subtypes" (2014). Neurobiology. 285.