Isoform‐specific mechanisms of a3b4*‐nicotinic acetylcholine receptor modulation by the prototoxin lynx1

Abstract

This study investigates—for the first time to our knowledge—the existence and mechanisms of functional interactions between the endogenous mammalian prototoxin, lynx1, and α3‐ and β4‐subunit–containing human nicotinic acetylcholine receptors (α3β4*‐nAChRs). Concatenated gene constructs were used to express precisely defined α3β4*‐nAChR isoforms (α3β4) 2 β4‐, (α3β4) 2 α3‐, (α3β4) 2 α5(398D)‐, and (α3β4) 2 α5(398N)‐nAChR in Xenopus oocytes. In the presence or absence of lynx1, α3β4*‐nAChR agonist responses were recorded by using 2‐electrode voltage clamp and single‐channel electrophysiology, whereas radioimmunolabeling measured cell‐surface expression. Lynx1 reduced (α3β4) 2 β4‐nAChR function principally by lowering cell‐surface expression, whereas single‐channel effects were primarily responsible for reducing (α3β4) 2 α3‐nAChR function [decreased unitary conductance (≥50%), altered burst proportions (3‐fold reduction in the proportion of long bursts), and enhanced closed dwell times (3‐ to 6‐fold increase)]. Alterations in both cell‐surface expression and single‐channel properties accounted for the reduction in (α3β4) 2 α5‐nAChR function that was mediated by lynx1. No effects were observed when α3β4*‐nAChRs were coexpressed with mutated lynx1 (control). Lynx1 is expressed in the habenulopeduncular tract, where α3β4*‐α5*‐nAChR subtypes are critical contributors to the balance between nicotine aversion and reward. This gives our findings a high likelihood of physiologic significance. The exquisite isoform selectivity of lynx1 interactions provides new insights into the mechanisms and allosteric sites [α(−)‐interface containing] by which prototoxins can modulate nAChR function. —George, A. A., Bloy, A., Miwa, J. M., Lindstrom, J. M., Lukas, R. J., Whiteaker, P. Isoform‐specific mechanisms of α3β4*‐nicotinic acetylcholine receptor modulation by the prototoxin lynx1. FASEB J . 31, 1398–1420 (2017) www.fasebj.org