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

Abstract

1The 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.) 6 Only large amplitude ACh-activated channels (-50 pS) were recorded from fibres either in the presence of 50 liM sphingosine or from fibres chronically exposed to PMA. However, following recovery from a 10 min exposure to 540 tiM 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 staurosporinetreated 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.

Requirement of a colchicine‐sensitive component of the cytoskeleton for acetylcholine receptor recovery

“…The amplitude, open times and slope conductances of the large channels were similar to those seen in the colchicine-treated fibres not exposed to carbachol (Table 3). In addition, the small conductance channels had a similar amplitude, open time and conductance to those seen in staurosporine-treated fibres following carbachol exposure (Table 3, Hardwick & Parsons, 1995).…”

“…amplitudes and ACh-activated single channel currents were obtained by the same methods as described in the preceding manuscript (Hardwick & Parsons, 1995).…”

“…In (Table 3, Hardwick & Parsons, 1995). In 1 patch from a colchicine-treated fibre, two channel populations were observed, a typical large conductance channel and a second small conductance channel.…”

“…reduced by the addition of agents which inhibit protein kinase C (Hardwick & Parsons, 1995). Since both the AChR itself and several of the subsynaptic proteins associated with the receptor can be substrates for PKC (Huganir 1988;Hall & Sanes, 1993;Wagner et al, 1993), we wanted to examine the effects of an agent known to alter the subsynaptic cytoskeleton on the recovery of AChR function following agonist-induced activation and desensitization.…”

“…Pharmacology (1995) 114. 442-446 tocutaneous muscles isolated from garter snakes (Thamnophis) as described previously (Conor et al, 1984;Hardwick et al, 1991;Hardwick & Parsons, 1993a,b) and in the preceding manuscript (Hardwick & Parsons, 1995). Colchicine (Sigma) was added to the control physiological solution (10 tiM to 1 mM) and the muscle preparations were incubated at room temperature for 16-18 h. Control preparations were maintained in the physiological sodium solution without colchicine for identical time periods.…”