The appearance of voltage-sensitive Na+ channels during the in vitro differentiation of embryonic chick skeletal muscle cells.

The properties of Na+ channels at different stages of the in vitro development of embyronic chick skeletal muscle were analyzed using three different classes of toxins that act specifically on Na+ channels: alkaloids like veratridine and batrachotoxin, which stabilize an open form of the Na+ channel; polypeptide neurotoxins like sea anemone and scorpion neurotoxins that prolong the life time of the conducting form of the channel by slowing down the inactivation process; and tetrodotoxin, which inhibits the entry of Na+ through the channel. The rate of 22Na+ uptake, which is activated by a mixture of 10 p~ sea anemone toxin II and 100 pM veratridine and which is inhibited by 0.1 p~ tetrodotoxin, was used as an estimate of the amount of Na+ channels. Toxin-activated rates of “Na+ uptake were very low in unfused cultures; they increased in parallel with the extent of fusion to reach a maximum at 100 h of culture, and then stabilized. Dose-response curves for the action of the different neurotoxins on the initial rate of ‘*Na+ uptake were obtained at different ages of culture between 2 and 7 days. The apparent affinities of the different toxins for their receptors on the Na’ channel remained invariant, indicating that the same type of functional, tetrodotoxin-sensitive Na+ channel is present at all stages of myotube development. Only the number of Na+ channels is increasing during myogenesis. Direct biochemical titration of Na+ channels was carried out with a highly radiolabeled derivative of tetrodotoxin. Specific tetrodotoxin binding could not be de