Mechanism of staurosporine-induced decrease in acetylcholine receptor recovery from desensitization.

Abstract

1. Previously, we showed in voltage-clamped snake twitch muscle fibres that the extent of recovery of the nicotinic acetylcholine (ACh) receptor from carbachol-induced desensitization is reduced by pretreatment with the protein kinase inhibitor staurosporine. The present studies were undertaken to determine the mechanism underlying the staurosporine-induced inhibition of recovery. 2. Pretreatment with 0.5 microM staurosporine significantly decreased the extent of recovery of spontaneous miniature endplate current (m.e.p.c.) amplitudes in preparations exposed to 540 microM carbachol. The decrease in recovery of m.e.p.c. amplitude by staurosporine was dependent on the duration of carbachol exposure. No significant decrease in m.e.p.c. amplitude was observed with a 1 min exposure to agonist, whereas a significant decrease in recovery was seen with agonist exposures between 5-10 min. Further, the effect of staurosporine pretreatment on ACh receptor recovery was long-lasting such that m.e.p.c. amplitude remained decreased for at least 60 min. 3. Estimation of mean channel conductance by noise analysis during local perfusion of 20 microM carbachol demonstrated a decrease in conductance from 52 pS to 23 pS in staurosporine-treated preparations following recovery from desensitization. Staurosporine treatment in the absence of desensitization did not alter the mean channel conductance. 4. A single population of ACh-activated single channel currents with a conductance of 45-49 pS was recorded in cell-attached patches from enzymatically cleaned endplates in control and staurosporine-treated preparations not exposed to carbachol. 5. At staurosporine-treated endplates exposed to carbachol and then allowed to recover, a population of small conductance (23 pS) channels was observed. These channels were not normally seen in control preparations which had undergone carbachol-induced desensitization and recovery.6. We suggest that the decrease in m.e.p.c. amplitude observed following recovery from desensitization in staurosporine-treated endplates results from the activation of a mixture of small and large conductance ACh receptor channels.