Grayanotoxin opens Na channels from inside the squid axonal membrane.

External application of alpha-dihydro-grayanotoxin II (alpha-H2-GTX II) to squid giant axon under nonperfused condition caused substantial membrane depolarization. Intracellular perfusion of the fibers retarded this depolarization appreciably. Tritium-labeled alpha-dihydro-grayanotoxin II ([3H]alpha-H2-GTX II) in the external medium can permeate through the cell membrane, but permeation of alpha-H2-GTX II does not occur either with the carrier-mediated system or through the pores of the Na channel. The finding that the most hydrophilic grayanotoxin analogue, desacyl asebotoxin VII, is effective only when applied internally, strongly suggests that the receptor for grayanotoxin does not exist on the external surface of the membrane. The linear relationship between the concentration of [3H]alpha-H2-GTX II in the external medium and the count in the effluent from the perfused axon indicates that GTX II diffuses through the cell membrane's lipid phase and reaches the site of action only approached from the internal medium.