Necessity of divalent cations for recovery from carbachol‐induced nicotinic acetylcholine receptor inactivation at snake twitch fibre endplates
Authors: Hardwick Jean C. , Parsons Rodney L. 
Published: July 2012

Previous studies demonstrated that elevation of the extracellular calcium concentration during a prolonged exposure to a high concentration of carbachol reverses the staurosporine‐induced decrease in the extent of endplate resensitization in voltage‐clamped snake twitch fibres. The present studies were designed to establish the site, specificity and potential mechanisms by which calcium could reverse the effects of staurosporine on acetylcholine receptor recovery.
Pretreatment of potassium‐depolarized muscle preparations with 0.5 μm staurosporine, followed by a 10 min incubation with 540 μm carbachol, produced a significant decrease in the recovery of miniature endplate current (m.e.p.c.) amplitudes. Raising the extracellular calcium concentration from 1 mm to 10 mm during the agonist application reversed this inhibition. In addition, a brief (3 min) incubation in an elevated calcium (10 mm) solution in staurosporine‐treated preparations previously exposed to 540 μm carbachol also reversed the reduction in m.e.p.c. amplitude.
Substitution of calcium with 10 mm strontium had no effect on the staurosporine‐induced decrease in m.e.p.c. amplitude, whereas 10 mm magnesium partially substituted for calcium. Inclusion of (+)‐tubocurarine (13 μm) in the 10 mm calcium solution to prevent the influx of calcium through agonist‐activated channels did not prevent the reversal of the staurosporine‐induced decrease in m.e.p.c. amplitudes. This suggested that the site of action of calcium on endplate resensitization was extracellular.
Analysis of acetylcholine (ACh)‐induced single channel currents demonstrated that a population of small conductance channels seen only in the staurosporine‐treated preparations following carbachol exposure, was still present in staurosporine‐treated preparations incubated in 540 μm carbachol solution containing 10 mm calcium. Thus the effect of calcium on ACh receptor recovery was not due to the conversion of the small conductance channels to the normal, large conductance channels.
Removal of calcium from the extracellular solution with the magnesium concentration unchanged, resulted in no change in the extent of m.e.p.c. amplitude recovery, nor did it alter the ability of staurosporine to inhibit recovery. Removal of both calcium and magnesium resulted in a significant decrease in the extent of recovery and staurosporine produced no additional decrease. This decrease in m.e.p.c. recovery in the divalent cation‐free solution was not associated with a change in mean channel conductance as determined by noise analysis.
Based on the results from these experiments, we suggest that, with prolonged exposure to agonist, some ACh receptors at the endplate become irreversibly inactivated. For full recovery of endplate sensitivity to occur, inactivated ACh receptors must be replaced. Part of the replacement ACh receptors appear to be recruited from a readily available but not previously activated pool and this process is dependent on extracellular calcium and/or magnesium. Thus, elevation of extracellular calcium may be able to reverse the apparent decrease in endplate sensitivity in staurosporine‐treated fibres following carbachol exposure by increasing the number of functional receptors at the endplate.