Regulation of ␣4␤2 Nicotinic Receptor Desensitization by Calcium and Protein Kinase C

ABSTRACT Neuronal nicotinic acetylcholine receptor (nAChR) desensitization is hypothesized to be a trigger for long-term changes in receptor number and function observed after chronic administration of nicotine at levels similar to those found in persons who use tobacco. Factors that regulate desensitization could potentially influence the outcome of long-lasting exposure to nicotine. The roles of Ca 2ϩ and protein kinase C (PKC) on desensitization of ␣4␤2 nAChRs expressed in Xenopus laevis oocytes were investigated. Nicotine-induced (300 nM; 30 min) desensitization of ␣4␤2 receptors in the presence of Ca 2ϩ developed in a biphasic manner with fast and slow exponential time constants of f ϭ 1.4 min (65% relative amplitude) and s ϭ 17 min, respectively. Recovery from desensitization was reasonably well described by a single exponential with rec ϭ 43 min. Recovery was largely eliminated after replacement of external Ca 2ϩ with Ba 2ϩ and slowed by calphostin C ( rec ϭ 48 min), an inhibitor of PKC. Conversely, the rate of recovery was enhanced by phorbol-12-myristate-13-acetate ( rec ϭ 14 min), a PKC activator, or by cyclosporin A (with rec ϭ 8 min), a phosphatase inhibitor. ␣4␤2 receptors containing a mutant ␣4 subunit that lacks a consensus PKC phosphorylation site exhibited little recovery from desensitization. Based on a twodesensitized-state cyclical model, it is proposed that after prolonged nicotine treatment, ␣4␤2 nAChRs accumulate in a "deep" desensitized state, from which recovery is very slow. We suggest that PKC-dependent phosphorylation of ␣4 subunits changes the rates governing the transitions from "deep" to "shallow" desensitized conformations and effectively increases the overall rate of recovery from desensitization. Longlasting dephosphorylation may underlie the "permanent" inactivation of ␣4␤2 receptors observed after chronic nicotine treatment