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 Ca²⁺ and protein kinase C (PKC) on desensitization of α4β2 nAChRs expressed in Xenopus laevis oocytes were investigated. Nicotine-induced (300 nM; 30 min) desensitization of α4β2 receptors in the presence of Ca²⁺ developed in a biphasic manner with fast and slow exponential time constants of τ_f = 1.4 min (65% relative amplitude) and τ_s = 17 min, respectively. Recovery from desensitization was reasonably well described by a single exponential with τ_rec = 43 min. Recovery was largely eliminated after replacement of external Ca²⁺ with Ba²⁺ and slowed by calphostin C (τ_rec = 48 min), an inhibitor of PKC. Conversely, the rate of recovery was enhanced by phorbol-12-myristate-13-acetate (τ_rec = 14 min), a PKC activator, or by cyclosporin A (with τ_rec = 8 min), a phosphatase inhibitor. α4β2 receptors containing a mutant α4 subunit that lacks a consensus PKC phosphorylation site exhibited little recovery from desensitization. Based on a two-desensitized-state cyclical model, it is proposed that after prolonged nicotine treatment, α4β2 nAChRs accumulate in a “deep” desensitized state, from which recovery is very slow. We suggest that PKC-dependent phosphorylation of α4 subunits changes the rates governing the transitions from “deep” to “shallow” desensitized conformations and effectively increases the overall rate of recovery from desensitization. Long-lasting dephosphorylation may underlie the “permanent” inactivation of α4β2 receptors observed after chronic nicotine treatment.