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The Conus toxin geographutoxin IL distinguishes two functional sodium channel subtypes in rat muscle cells developing in vitro

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. Geographutoxin II (GTX II), a novel polypeptide toxin from the piscivorous marine snail Conus geographus, reduces sodium currents in rat myoballs without marked alteration of the time course or voltage dependence of activation of the remaining current. Titration of the inhibition of sodium currents by GTX II showed that, in individual myoballs, a fraction of the sodium current averaging 49 +/- 9% (SEM) was inhibited by saturating (25 microM) concentrations of GTX II. The concentration-effect curve fit a noncooperative, 1:1 binding isotherm with a single KD for GTX II of 19 nM characteristic of inhibition of the TTX-sensitive sodium channels of adult rat muscle. Titration of the sodium current remaining in the presence of 2.5 microM GTX II with TTX gave complete inhibition. The dose-response curve fit a noncooperative, 1:1 binding isotherm with a single KD for TTX of 1.3 microM characteristic of TTX-insensitive sodium channels of embryonic muscle. The action of GTX II was not frequency dependent. The all-or-none inhibition of these 2 sodium channel subtypes by GTX II suggests substantial structural differences in the region of neurotoxin receptor site 1 on TTX-sensitive and - insensitive sodium channels and provides definitive evidence that these 2 sodium channel subtypes function in parallel in muscle cells developing in the absence of innervation.