Structural determinants for the action of grayanotoxin in D1 S4.S5 and D4 S4.S5 intracellular linkers of sodium channel α-subunits

Maejima Hiroshi

Kinoshita Eiji

Yuki Tsunetsugu

Yakehiro Masuhide

Seyama Issei

Yamaoka Kaoru

Biochemical and Biophysical Research Communications

We located a novel binding site for grayanotoxin on the cytoplasmic linkers of voltage-dependent cardiac (rH1) or skeletalmuscle (l1) Nat channel isoforms (segments S4.S5 in domains D1 and D4), using the alanine scanning substitution method. GTXmodification of Nat channels, transiently expressed in HEK 293 cells, was evaluated under whole-cell voltage clamp, from the ratio of maximum chord conductance for modified and unmodified Nat channels. In l1, mutations K237A, L243A, S246A, K248A, K249A, L250A, S251A, or T1463A, caused a moderate, but statistically significant decrease in this ratio. On making corresponding mutations in rH1, only L244A dramatically reduced the ratio. Because in l1, the serine at position 251 is the only heterologous residue with respect to rH1 (Ala-252), we made a double mutant L243A&S251A to match the sequence of l1 and rH1 in S4.S5 linkers of both domains. This double mutation resulted in a significant decrease in the ratio, to the same extent as L244A substitution in rH1 did, indicating that the site at Leu-244 in rH1 or at Leu-243 in l1 is a novel one, exhibiting a synergistic effect of grayanotoxin.

Grayanotoxin

Sodium channel

S4.S5 linker

Whole-cell patch clamp

HEK 293

Biology [ 460 ]

eng

Elsevier

Copyright (c) 2002 Elsevier Ltd.

[ISSN] 0006-291X

[DOI] 10.1016/S0006-291X(02)00702-7

[NCID] AA00564395

[PMID] 12150970

[DOI] http://dx.doi.org/10.1016/S0006-291X(02)00702-7 isVersionOf