Voltage clamp analysis of tetrodotoxin-sensitive and -insensitive sodium channels in rat muscle cells developing in vitro

Abstract

Sodium currents in cultured rat muscle cells converted to myoballs by treatment with colchicine were recorded using a giga-ohm seal voltage clamp procedure in the whole cell configuration. The mean peak Na+ conductance of the myoballs was 90 pS/microns2 of surface membrane. Half-maximal activation of Na+ currents was observed for test pulses to -31 mV and half-maximal inactivation was observed for prepulses to -74 mV. Titration of the inhibition of Na+ currents by tetrodotoxin (TTX) yielded a biphasic inhibition curve consistent with the presence of two classes of Na+ channels differing in affinity for TTX. The TTX-sensitive channels carried 28% of the Na+ current and had an apparent KD for TTX of 13 nM at 20 degrees C. The TTX-insensitive Na+ channels had an apparent KD for TTX of 3.2 microns. Inhibition of TTX-insensitive Na+ channels by TTX was enhanced by repetitive stimulation of the myoballs at 2 Hz, whereas the inhibition of TTX-sensitive Na+ channels by TTX was not frequency dependent. We conclude that rat muscle cells developing in vitro synthesize physiologically functional, TTX-sensitive Na+ channels in the absence of innervation. These channels, which are characteristic of adult skeletal muscle, function in parallel with TTX-insensitive Na+ channels that are present in embryonic muscle.