Slow-inactivation induced conformational change in domain 2-segment 6 of cardiac Na+ channel.
Slow-inactivation induced conformational change in domain 2-segment 6 of cardiac Na+ channel.
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DOI：
10.1016/j.bbrc.2006.04.049
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发表时间：
2006-06
影响因子：
3.1
通讯作者：
J. O'Reilly;P. Shockett
中科院分区：
文献类型：
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作者：
J. O'Reilly;P. Shockett
为了研究人心肌钠离子通道(hNav1.5)结构域2-S6(D2-S6)在缓慢失活过程中的构象变化，我们应用了甲硫磺酸乙铵(MTSEA)取代半胱氨酸可及性方法(SCAM)。我们用半胱氨酸(C)取代了抗MTSEA的hNav1.5突变体C373Y中D2-S6的930位天然缬氨酸(V)，产生了双突变体C373Y-V930C。用膜片钳技术记录瞬时转基因HEK细胞的全细胞Na+电流。在C373Y-V930C中，我们发现在闭合状态(−160 mV)施加1.5 mM的MTSEA对全细胞Na+电流没有显著影响，而在缓慢失活状态(0 mV延长去极化)施加MTSEA会降低电流。我们认为hNav1.5中的D2-S6在缓慢失活过程中经历了分子重排，暴露了残基930的侧链，从而使其易于被MTSEA修饰。
To examine conformational changes during slow inactivation involving domain 2-segment 6 (D2-S6) of human cardiac Na+channel (hNav1.5), we applied the substituted-cysteine accessibility method (SCAM) using methanethiosulfonate ethylammonium (MTSEA). We substituted cysteine (C) for native valine (V) at position 930 of D2-S6 in the MTSEA-resistant hNav1.5 mutant C373Y to produce the double mutant C373Y-V930C. Whole-cell Na+currents were recorded using patch-clamp techniques in transiently transfected HEK cells. In C373Y-V930C, we find that MTSEA (1.5mM) applied in the closed state (−160mV) has no significant effect on whole-cell Na+current, while MTSEA applied in the slow-inactivated state (prolonged depolarization at 0mV) decreases current. We propose that D2-S6 in hNav1.5 undergoes molecular rearrangement during slow inactivation exposing the side chain of residue 930 such that it becomes accessible to modification by MTSEA.