Is the site of action of grayanotoxin the sodium channel gating of squid axon?

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Abstract
The site of action of grayanotoxin (GTX) in the nerve membrane was investigated by using various endopeptidases. The experiment was conducted on squid axons isolated from Doryteuthis bleekeli with both voltage clamp and internal perfusion methods. Intracellular application of various endopeptidases for more than 30 min eliminated the gating action from both Na current and K current systems. When GTX (100 .mu.M) was subsequently applied to the internal medium, the membranes could depolarize to various extents. The site of action of GTX is not confined to the channel gating but is present in a part of the Na channel having both voltage sensor and ion filter functions. With the application of trypsin Streptomyces fradiae trypsin, pronase, BPN'' and S. fradiae protease (group B), GTX-induced depolarization was much smaller than that with the application of .alpha.-chymotrypsin, N-protease and thermolysin (group A). The difference in the sensitivity to GTX between group A and group B became remarkable as the time for application of the enzymes was prolonged. Since all enzymes belonging to group B retain trypsin-like activity and are more effective in removing the sensitivity to GTX the molecular moiety around the binding site of GTX is rich in basic amino acids or the essential part for opening the Na channel should be protected by basic amino acids.