Regulation of alpha 4 beta 2 nicotinic receptor desensitization by calciumand protein kinase C

Neuronal nicotinic acetylcholine receptor (nAChR) desensitization is hypoth esized to be a trigger for long-term changes in receptor number and functio n observed after chronic administration of nicotine at levels similar to th ose found in persons who use tobacco. Factors that regulate desensitization could potentially influence the outcome of long-lasting exposure to nicoti ne. The roles of Ca2+ and protein kinase C (PKC) on desensitization of alph a 4 beta 2 nAChRs expressed in Xenopus laevis oocytes were investigated. Ni cotine-induced (300 nM; 30 min) desensitization of alpha 4 beta 2 receptors in the presence of Ca2+ developed in a biphasic manner with fast and slow exponential time constants of tau(f) = 1.4 min (65% relative amplitude) and tau(s) = 17 min, respectively. Recovery from desensitization was reasonabl y well described by a single exponential with tau(rec) = 43 min. Recovery w as largely eliminated after replacement of external Ca2+ with Ba2+ and slow ed by calphostin C (tau(rec) = 48 min), an inhibitor of PKC. Conversely, th e rate of recovery was enhanced by phorbol-12-myristate-13-acetate (tau(rec ) = 14 min), a PKC activator, or by cyclosporin A (with tau(rec) = 8 min), a phosphatase inhibitor. alpha 4 beta 2 receptors containing a mutant alpha 4 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, alpha 4 beta 2 nAChRs accumulate in a "deep" desensitized state, from which recovery is very slow. We suggest that PKC-dependent phosphorylation of alpha 4 subuni ts changes the rates governing the transitions from "deep" to "shallow" des ensitized conformations and effectively increases the overall rate of recov ery from desensitization. Long-lasting dephosphorylation may underlie the " permanent" inactivation of alpha 4 beta 2 receptors observed after chronic nicotine treatment.