Vol. 60, Issue 4, 865-872, October 2001

Novel Site on Sodium Channel -Subunit Responsible for the Differential Sensitivity of Grayanotoxin in Skeletal and Cardiac Muscle

Takahiro Kimura, Kaoru Yamaoka, Eiji Kinoshita, Hiroshi Maejima, Tsunetsugu Yuki, Masuhide Yakehiro, and Issei Seyama

Department of Physiology (T.K., K.Y., E.K., T.Y., I.S.) and Institute of Health Sciences (H.M.), School of Medicine, Hiroshima University, Kasumi, Hiroshima, Japan; and Division of Physiology, Department of Clinical Engineering, Faculty of Health Sciences, Hiroshima International University, Gakuendai, Hiroshima Prefecture, Japan (M.Y.)

We searched for sites on the -subunit of the fast Na⁺ channel responsible for the difference in GTX (grayanotoxin) sensitivity of the skeletal- and cardiac-muscle Na⁺ current. cDNA clones, encoding the skeletal or cardiac isoforms of the -subunit, were inserted into a mammalian expression vector and transiently transfected into human embryonic kidney cells. The expressed channels were measured using whole-cell patch-clamp techniques and examined for GTX sensitivity. As a measure of GTX sensitivity, we used relative chord conductance (ratio of maximum chord conductance of noninactivating GTX-modified Na⁺ currents to that of unmodified peak currents). Wild-type channels from skeletal muscle (µ1) were more sensitive to GTX modification than wild-type cardiac channels (rH1) by a factor of 1.6. To facilitate exploration of -subunit sites determining GTX sensitivity, we used SHHH, a chimera of skeletal muscle (S) domain D1 and heart muscle (H) domains D2D3D4 with supernormal sensitivity to GTX I (1.5-fold of wild-type µ1). Successive replacement of Ser-251 (D1S4-S5 intracellular loop) and Ile-433 (D1S6 transmembrane segment), with corresponding rH1 residues Ala and Val, reduced, in a stepwise manner, the GTX sensitivity of the chimera and related mutants to that of wild-type rHl. We concluded that, in addition to Ile-433, known as the GTX-binding site, Ser-251 represents a novel site for GTX modification.