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J. Biol. Chem., Vol. 278, Issue 11, 9464-9471, March 14, 2003

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Distinct Sites Regulating Grayanotoxin Binding and Unbinding to D4S6 of Na_v1.4 Sodium Channel as Revealed by Improved Estimation of Toxin Sensitivity^*

Hiroshi Maejima, Eiji Kinoshita^§, Issei Seyama^¶, and Kaoru Yamaoka^§

From the  Institute of Health Sciences and the ^§ Department of Physiology, School of Medicine, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551 and the ^¶ Faculty of Human Life Science, Hiroshima Jogakuin University, Ushita Higashi 4-13-1, Higashi-ku, Hiroshima 732-0063, Japan

Grayanotoxin (GTX) exerts selective effects on voltage-dependent sodium channels by eliminating fast sodium inactivation and causing a hyperpolarizing shift in voltage dependence of channel activation. In this study, we adopted a newly developed protocol that provides independent estimates of the binding and unbinding rate constants of GTX (k_on and k_off) to GTX sites on the sodium channel protein, important in the molecular analysis of channel modification. Novel GTX sites were determined in D2S6 (Asn-784) and D3S6 (Ser-1276) by means of site-directed mutagenesis; the results suggested that the GTX receptor consists of the S6 transmembrane segments of four homologous domains facing the ion-conducting pore. We systematically introduced at two sites in D4S6 (Na_v1.4-Phe-1579 and Na_v1.4-Tyr-1586) amino acid substituents with residues containing hydrophobic, aromatic, charged, or polar groups. Generally, substitutions at Phe-1579 increased both k_on and k_off, resulting in no prominent change in dissociation constant (K_d). It seems that the smaller the molecular size of the residue at Na_v1.4-Phe-1579, the larger the rates of k_on and k_off, indicating that this site acts as a gate regulating access of toxin molecules to a receptor site. Substitutions at Tyr-1586 selectively increased k_off but had virtually no effect on k_on, thus causing a drastic increase in K_d. At position Tyr-1586, a hydrophobic or aromatic amino acid side chain was required to maintain normal sensitivity to GTX. These results suggest that the residue at position Tyr-1586 has a more critical role in mediating GTX binding than the one at position Phe-1579. Here, we propose that the affinity of GTX to Na_v1.4 sodium channels might be regulated by two residues (Phe and Tyr) at positions Phe-1579 and Tyr-1586, which, respectively, control access and binding of GTX to its receptor.

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