Different functional states of tetrodotoxin sensitive and tetrodotoxin resistant Na+ channels occur during the in vitro development of rat skeletal muscle.

There are three stages of differentiation of voltage dependent Na+ channels during the in vitro development of rat skeletal muscle. Myoblasts which are less than 60 h old in culture have Na+ channels which normally do not give rise to action potentials but do so after treatment of the cells with very low concentrations of sea anemone toxin. These Na+ channels revealed by sea anemone toxin are resistant to TTX. Myoblasts prior to fusion are electrically excitable (Vmax = 10 V/s). Electrically activated Na+ channels are only blocked by high concentrations of TTX. Titration of TTX resistant Na+ channels with a tritiated derivative of TTX indicates a dissociation constant of the TTX-Na+ channel complex of 50 nM. Myotubes have both high and low affinity binding sites for TTX (Frelin et al. 1983). Action potentials (Vmax = 100-200 V/s) are only inhibited at high concentrations of TTX. Experiments with rat myoballs indicate that only Na+ channels with a low affinity binding site for TTX are functional in voltage-clamp studies. The K0.5 value for TTX inhibition of the peak Na+ current is observed at 70 nM. Spontaneous contractions of myotubes are blocked by TTX with a K0.5 value of 100 nM, suggesting that TTX resistant Na+ channels are also the ones responsible for the spontaneous contractions in rat myotubes in culture. 22Na+ flux studies after activation of the Na+ channel with neurotoxins have been carried out at the different stages of differentiation. Toxin activated Na+ channels have the same high affinity for sea anemone toxins at all stages of development; likewise, the sensitivity for TTX is the same.(ABSTRACT TRUNCATED AT 250 WORDS)