Localization of voltage-sensitive sodium channels on the extrasynaptic membrane surface of mouse skeletal muscle by autoradiography of scorpion toxin binding sites.

Voltage-dependent sodium channels (Na+ channels) were localized by autoradiography on mouse skeletal muscle using both light and electron microscopy. 125I-scorpion toxins (ScTx) of both the alpha and beta type were used as probes. The specificity of labelling was verified by competitive inhibition with unlabelled toxin and by inhibition of alpha ScTx labelling in depolarizing conditions. Under light microscopy, the labelling of the myocyte surface appeared randomly distributed with both the alpha and beta toxins. No difference in the labelling density obtained with beta ScTx was observed between a 2 mm central segment of the fibre containing the endplate and an adjacent segment not containing the endplate. At the endplate, however, the beta ScTx binding site density was about seven fold higher at the edge of the synaptic primary clefts. This density decreased with distance from the synaptic cleft reaching the extrasynaptic value at 30-40 microns. An analysis of myocyte labelling using electron microscopy provided evidence for a specific, but very low labelling of the myocyte interior which can be attributed to the T-tubules. These results confirm a relatively high density of Na+ channels in a perijunctional zone about 50 microns in width, which could ensure the initial spread of the surface depolarization with a high safety factor, and a homogeneous distribution over the remaining surface with a low density evaluated at 5-10 per microns2. However, the very low labelling of T-tubules could be attributed mainly to a low density of tubular Na+ channels.