RT Journal Article
SR Electronic
T1 The Unique <em>α</em>4(+)/(−)<em>α</em>4 Agonist Binding Site in (<em>α</em>4)<sub>3</sub>(<em>β</em>2)<sub>2</sub> Subtype Nicotinic Acetylcholine Receptors Permits Differential Agonist Desensitization Pharmacology versus the (<em>α</em>4)<sub>2</sub>(<em>β</em>2)<sub>3</sub> Subtype
JF Journal of Pharmacology and Experimental Therapeutics
JO J Pharmacol Exp Ther
FD American Society for Pharmacology and Experimental Therapeutics
SP 46
OP 58
DO 10.1124/jpet.113.208389
VO 348
IS 1
A1 Eaton, J. Brek
A1 Lucero, Linda M.
A1 Stratton, Harrison
A1 Chang, Yongchang
A1 Cooper, John F.
A1 Lindstrom, Jon M.
A1 Lukas, Ronald J.
A1 Whiteaker, Paul
YR 2014
UL http://jpet.aspetjournals.org/content/348/1/46.abstract
AB 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 86Rb+ 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 [125I]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.