Necessity of protein kinase C activity for maintenance of acetylcholine receptor function at snake twitch fibre endplates.

1. The extent of recovery of endplate sensitivity following a 5 or 10 min exposure to carbachol was determined from measurements of miniature endplate current (m.e.p.c.) amplitudes in voltage-clamped snake twitch fibre endplates. M.e.p.c. amplitude recovery was dependent on the carbachol concentration (0.27-5.4 mM) and duration of application. Staurosporine pretreatment (0.5 microM for approximately 15 min) further decreased the extent of m.e.p.c. amplitude recovery. 2. The decrease in m.e.p.c. amplitude at control endplates exposed to high concentrations of agonist (5.4 mM carbachol for 10 min) was due to an apparent decrease in postsynaptic receptor density, not to a change in the conductance of the acetylcholine (ACh)-activated channels. 3. Pretreatment with either 1 microM lavendustin A or 50 microM KN-62 had no effect on m.e.p.c. amplitude recovery, whereas pretreatment with either 0.5 microM staurosporine, 50 microM sphingosine, or 0.5 microM calphostin C significantly reduced m.e.p.c. amplitude recovery following carbachol exposure. 4. Sphingosine and staurosporine produced a concentration-dependent decrease in the extent of m.e.p.c. amplitude recovery, but had no effect on m.e.p.c. characteristics in the absence of carbachol. In addition, this decrease in m.e.p.c. amplitude was not due to the presence of a subpopulation of small amplitude m.e.p.cs. 5. Prolonged treatment (18-20 h) of muscles with 200 nM phorbol 12-myristate 13-acetate (PMA), to down regulate protein kinase C, resulted in a significant reduction in m.e.p.c. amplitudes following exposure to carbachol. Conversely, treatment with 200 nM 4 alpha PMA, an inactive analogue, had no effect on m.e.p.c. amplitude recovery. 6. Only large amplitude ACh-activated channels (~50 pS) were recorded from fibres either in the presence of 50 micro M sphingosine or from fibres chronically exposed to PMA. However, following recovery from a 10 min exposure to 540 micro M carbachol, both small conductance (-25 pS) and large conductance ACh-activated channels were recorded in both sphingosine- and phorbol-treated preparations. The conductance of these two populations of channels was virtually identical to those seen in staurosporine treated fibres following carbachol exposure.7. We conclude that protein kinase C is required for full recovery of AChR sensitivity following carbachol-induced receptor inactivation. Exposure to high concentrations of agonist for prolonged periods appears to result in the inactivation of a subpopulation of receptors. These receptors must be replaced or reactivated by a process involving protein kinase C. When this phosphorylation step is inhibited, the AChRs remain in an activatable form, but with a reduced conductance.