Staurosporine inhibits the extent of acetylcholine receptor recovery from carbachol‐induced desensitization in snake twitch fibres

Abstract

1 The effect of the protein kinase inhibitor, staurosporine, on the extent and time course of recovery following carbachol-induced desensitization was studied in snake twitch-muscle fibres maintained in an isotonic potassium propionate solution and voltage-clamped to + 30 mV. 3 Staurosporine also produced a concentration-dependent (10nM to 0.5 pM) decrease in the amplitude of a second carbachol-induced current, following a wash period, as compared to the amplitude of the current produced by the initial carbachol application. Pretreatment with 0.5,UM K252a, another wide spectrum protein kinase inhibitor, also decreased the extent of recovery of the response to a second carbachol application following desensitization.4 Staurosporine pretreatment (0.5jUM) had no effect on either the kinetics of receptor-channel gating or the initial endplate sensitivity to agonist. This was determined by comparing the amplitude of the carbachol (540Mm)-induced currents and the amplitude and decay rate of m.e.p.cs in control and staurosporine-treated fibres. 5 Staurosporine had no effect on the time course of desensitization onset produced during the initial application of 540piM carbachol or the depth of desensitization produced by the end of a 2-3 min exposure to 540pM carbachol.6 Elevation of the external calcium concentration from 1 to 10mM during the 540pM carbachol application completely antagonized the decreased extent of recovery of m.e.p.c. amplitude produced by pretreatment with 0.5JM staurosporine. 7 We suggest that phosphorylation of a population of acetylcholine receptors is required for complete recovery from desensitization, and that staurosporine inhibits the protein kinases responsible for this phosphorylation. 8 We further propose that a transient increase in intracellular calcium, produced by an increase in calcium influx through agonist-activated endplate channels, stimulates additional protein kinase activity, which in turn, antagonizes the effect of staurosporine-treatment on recovery.

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

“…Snakes were killed by rapid decapitation and the costocutaneous muscles, with portions of the rib and skin, were pinned out on sylgard-coated petri dishes in a control physiological solution containing in mM: NaCl 159, KCl 2.5, MgCl2 4.2, CaCl2 1.0, HEPES 1.0, pH 7.3 (Dionne & Parsons, 1981 (Dionne & Parsons, 1981;Connor et al, 1984;Hardwick et al, 1991;Hardwick & Parsons, 1993a,b (Connor et al, 1984;Hardwick et al, 1991 Single channel recordings were made on enzymatically cleaned endplates using the cell attached patch technique as previously described (Hamill et al, 1981;Hardwick & Parsons, 1993a Figure 1). However, as the duration of carbachol exposure or concentration was increased, the extent of recovery was progressively decreased.…”

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

“…However, the following scheme is presented as one possible explanation. First, exposure to high concentration of agonist for a prolonged time activates and desensitizes virtually all functional AChRs (Hardwick et al, 1991). We propose that a subpopulation of these receptors become destabilized in the membrane, perhaps due to an activationdependent dephosphorylation of either an anchoring protein or the AChR itself.…”

“…Values represent the mean ± s.e.mean from n endplates. significantly decreased (Hardwick et al, 1991;Hardwick & Parsons, 1993a (Table 2). Treatment with colchicine alone produced a maximal inhibition to -70% of control amplitudes, whereas the combination of colchicine and staurosporine produced a maximal inhibition to -60%, a value similar to that produced by staurosporine alone.…”

“…Thereafter, whenever possible, recordings were also made at several other voltages to estimate the slope conductance (Figure 3). Because the rate of desensitization is accelerated following carbachol exposure (Hardwick et al, 1991;Hardwick & Parsons, 1993a), it was often impossible to obtain recordings at several voltages from a single patch.…”

“…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.…”