Maejima Hiroshi, Kinoshita Eiji, Yuki Tsunetsugu, Yakehiro Masuhide, Seyama Issei, Yamaoka Kaoru
Institute of Health Sciences, School of Medicine, Hiroshima University, Hiroshima, Kasumi 1-2-3, Hiroshima 734-8551, Japan.
Biochem Biophys Res Commun. 2002 Jul 12;295(2):452-7. doi: 10.1016/s0006-291x(02)00702-7.
We located a novel binding site for grayanotoxin on the cytoplasmic linkers of voltage-dependent cardiac (rH1) or skeletal-muscle (mu 1) Na(+) channel isoforms (segments S4-S5 in domains D1 and D4), using the alanine scanning substitution method. GTX-modification of Na(+) 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 Na(+) channels. In mu 1, 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 mu 1, 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 mu 1 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 mu 1 is a novel one, exhibiting a synergistic effect of grayanotoxin.
我们采用丙氨酸扫描替换法，在电压依赖性心脏（rH1）或骨骼肌（mu 1）钠通道亚型的细胞质连接子（结构域D1和D4中的S4-S5片段）上定位了一个新的灰藜毒素结合位点。通过全细胞膜片钳记录，从修饰和未修饰钠通道的最大弦电导比值，评估了在HEK 293细胞中瞬时表达的钠通道的GTX修饰情况。在mu 1中，K237A、L243A、S246A、K248A、K249A、L250A、S251A或T1463A突变导致该比值出现中度但具有统计学意义的下降。在rH1中进行相应突变时，只有L244A显著降低了该比值。由于在mu 1中，第251位的丝氨酸是相对于rH1（丙氨酸-252）的唯一异源残基，我们构建了双突变体L243A&S251A，以匹配两个结构域S4-S5连接子中mu 1和rH1的序列。这种双突变导致该比值显著下降，下降程度与rH1中的L244A替换相同，表明rH1中Leu-244或mu 1中Leu-243处的位点是一个新位点，具有灰藜毒素的协同效应。
