Nicotine-Modulated Subunit Stoichiometry Affects Stability and Trafficking of α3β4 Nicotinic Receptor

Heteromeric nAChRs are pentameric cation channels, composed of combinations of two or three alpha and three or two beta subunits, which play key physiological roles in the central and peripheral nervous systems. The prototypical agonist nicotine acts intracellularly to upregulate many nAChR subtypes, a phenomenon that is thought to contribute to the nicotine dependence of cigarette smokers. The alpha 3 beta 4 subtype has recently been genetically linked to nicotine dependence and lung cancer; however, the mode of action of nicotine on this receptor subtype has been incompletely investigated. Here, using transfected mammalian cells as model system, we characterized the response of the human alpha 3 beta 4 receptor subtype to nicotine and the mechanism of action of the drug. Nicotine, when present at 1 mM concentration, elicited a similar to 5-fold increase of cell surface alpha 3 beta 4 and showed a more modest upregulatory effect also at concentrations as low as 10 mu M. Upregulation was obtained if nicotine was present during, but not after, pentamer assembly and was caused by increased stability and trafficking of receptors assembled in the presence of the drug. Experimental determinations as well as computational studies of subunit stoichiometry showed that nicotine favors assembly of pentamers with (alpha 3)(2)(beta 4)(3) stoichiometry; these are less prone than (alpha 3)(3)(beta 4)(2) receptors to proteasomal degradation and, because of the presence in the beta subunit of an endoplasmic reticulum export motif, more efficiently transported to the plasma membrane. Our findings uncover a novel mechanism of nicotine-induced alpha 3 beta 4 nAChR upregulation that may be relevant also for other nAChR subtypes.