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

Abstract

There are three stages of differentiation of voltage dependent Na⁺ channels during the in vitro development of rat skeletal muscle.

• (i) 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.

• (ii) Myoblasts prior to fusion are electrically excitable ($\dot V$ _max=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.

• (iii) Myotubes have both high and low affinity binding sites for TTX (Frelin et al. 1983). Action potentials ($\dot V$ _max=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 K_0.5 value for TTX inhibition of the peak Na⁺ current is observed at 70 nM. Spontaneous contractions of myotubes are blocked by TTX with a K_0.5 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.

The in vitro differentiation of Na⁺ channels in rat and chick skeletal muscle cells is compared and discussed in relation to the action of neurotrophic influences.