Destruction of the sodium conductance inactivation by a specific protease in perfused nerve fibres from Loligo.

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Abstract
Intracellular perfusion of giant axons from L. forbesi with a crude protein extract of pronase dissolved in a KF solution suppressed the process of fast inactivation of the Na conductance (the h-process in the Hodgkin-Huxley terminology). The results with protease inhibitors indicated that the most substrate specific endopeptidase present in pronase, alkaline proteinase b, destroyed the h-process. After destruction of the inactivation the conductance rise upon depolarization followed cube law kinetics. Values of the time constant .tau.m before and after destruction of the h-process were very similar. After destruction of the inactivation process the properties tested were cation selectivity, instantaneous conductance and internal receptor sites for tetrodotoxin (TTX) and tetraethylammonium (TEA). No detectable changes in selectivity or instantaneous conductance were observed. No internal receptors for TTX affecting the Na conductance were found but a TEA receptor was exposed by the protein hydrolysis. TEA derivatives (triethylammonium, TEA-, with an aliphatic chain, Cn) induced a partial block of the steady-state Na current and induced a time-dependent blockage of the conductance. The 1st effect of TEA-Cn could be described in terms of a unimolecular reaction with the following equilibrium constants: 50, 2.5, 1.0, 0.4 and 0.025 mM for TEA-C2, TEA-C4, TEA-C5, TEA-C7 and TEA-C9, respectively. From the dependence of the equilibrium dissociation constant on the length of the alkyl chain the free-energy change was estimated in 560 cal/mol of CH2. The gain in free energy per CH2 group transferred from aqueous medium to the interior of a non-polar medium was 1000 cal. While it was not possible to propose the amino-acid sequence of the site cleaved by alkaline proteinase b, an important functional component seems to be arginine (or lysine).