NECESSITY OF PROTEIN-KINASE-C ACTIVITY FOR MAINTENANCE OF ACETYLCHOLINE-RECEPTOR FUNCTION AT SNAKE TWITCH FIBER END-PLATES

1 The extent of recovery of endplate sensitivity following a 5 or 10 m in exposure to carbachol was determined from measurements of miniature endplate current (m.e.p.c.) amplitudes in voltage-clamped snake twitc h fibre endplates. M.e.p.c. amplitude recovery was dependent on the ca rbachol concentration (0.27-5.4 mM) and duration of application. Staur osporine pretreatment (0.5 mu M for similar to 15 min) further decreas ed 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 ag onist (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 mu M lavendustin A or 50 mu M KN-62 had no effect on m.e.p.c. ampli tude recovery, whereas pretreatment with either 0.5 mu M staurosporine , 50 mu M sphingosine, or 0.5 mu M calphostin C significantly reduced m.e.p.c. amplitude recovery following carbachol exposure. 4 Sphingosin e 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 decreas e 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 muscl es 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. amp litudes following exposure to carbachol. Conversely, treatment with 20 0 nM 4 alpha PMA, an inactive analogue, had no effect on m.e.p.c. ampl itude recovery. 6 Only large amplitude ACh-activated channels (similar to 50 pS) were recorded from fibres either in the presence of 50 mu M sphingosine or from fibres chronically exposed to PMA. However, follo wing recovery from a 10 min exposure to 540 mu M carbachol, both small conductance (similar to 25 pS) and large conductance ACh-activated ch annels were recorded in both sphingosine- and phorbol-treated preparat ions. The conductance of these two populations of channels was virtual ly identical to those seen in staurosporine-treated fibres following c arbachol exposure. 7 We conclude that protein kinase C is required for 'full recovery of AChR sensitivity following carbachol-induced recepto r inactivation. Exposure to high concentrations of agonist for prolong ed periods appears to result in the inactivation of a subpopulation of receptors. These receptors must be replaced or reactivated by a proce ss involving protein kinase C. When this phosphorylation step is inhib ited, the AChRs remain in an activatable form, but with a reduced cond uctance.