Abstract
Grayanotoxin (GTX) exerts selective effects on voltage dependent sodium channels by eliminating fast inactivation and causing a hyperpolarizing shift in voltage dependence of channel activation. We have detected several sites that affect GTX action in all S6 transmembrane segments of four domains, and succeeded in distinguishing specific roles in the two sites (Nav1.4-Tyr-1586 and Nav1.4-Phe-1579) in D4S6 by measuring binding and unbinding constants of GTX (kon and koff) in mutant sodium channels. The former regulates the accessibility of the toxin to the receptor and the latter behaves as a receptor. Systematic substitutions of Nav1.4 at Phe-1579 increased both kon and koff resulting in no prominent effect on dissociation constant (Kd). Such substitutions of Phe-1579 with several amino acids indicated that the size of molecule was the most critical factor to affect GTX binding. Systematic substitutions of Tyr-1586 with amino acids having hydrophobic or aromatic side chains decreased koff causing drastic increase in the affinity of the toxin. In this study we applied similar analysis on the sites in D1. Nav1.4-Leu-437 in D1S6 had a similar property for Tyr-1586, indicating this site also behaves as a receptor site. These results indicate that a receptor for GTX may be composed of multiple sites broadly distributed in different domains. [Jpn J Physiol 54 Suppl:S135 (2004)]
