Residue-specific effects on slow inactivation at V787 in D2-S6 of Nav1.4 sodium channels
Residue-specific effects on slow inactivation at V787 in D2-S6 of Nav1.4 sodium channels
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DOI：
10.1016/s0006-3495(01)75858-4
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发表时间：
2001-10-01
影响因子：
3.4
通讯作者：
Wang, GK
中科院分区：
文献类型：
--
作者：
O'Reilly, JP;Wang, SY;Wang, GK
电压门控钠通道（NaChs）的缓慢失活响应于数秒至数分钟的去极化而发生，并且被认为在调节膜兴奋性和动作电位放电模式中起重要作用。然而，缓慢失活的分子机制还没有很好地理解。为了验证跨膜片段6结构域2（D2-S6）在NaCh缓慢失活中起作用的假设，我们在大鼠骨骼肌NaCh mu 1（Na（v）1.4）的D2-S6中的位置V787（缬氨酸）处替换不同的氨基酸。使用来自HEK细胞中瞬时表达的NaChs的全细胞记录来研究和比较突变体和野生型之间的慢失活表型。V787 K（赖氨酸取代）显示出缓慢失活的显着增强。V787 K进入缓慢失活状态的速度比野生型快约100倍（tau（1）约为30 ms vs.约为3 s），并且发生的超极化电位比野生型高得多（V-1/2 s（无穷大）曲线约为-130 mV vs.约为-75 mV）。V787 C（半胱氨酸取代）显示出对缓慢失活的抗性，即，与V787 K相反。与野生型相比，V787 C进入缓慢失活状态更慢（tau（1）约为5 s），更不完全，电压依赖性更低（s（无穷大）曲线的V-1/2约为-50 mV）。将半胱氨酸修饰剂甲硫基磺酸乙铵（MTSEA）应用于V787 C表明，787位经历了与缓慢失活状态相关的分子构象的相对变化。我们的研究结果表明，在Na（v）1.4中的V737位置在缓慢失活门控中起着重要作用，并且在NaCh缓慢失活期间，分子重排发生在D2-S6中的残基V787处或附近。
Slow inactivation in voltage-gated sodium channels (NaChs) occurs in response to depolarizations of seconds to minutes and is thought to play an important role in regulating membrane excitability and action potential firing patterns. However, the molecular mechanisms of slow inactivation are not well understood. To test the hypothesis that transmembrane segment 6 of domain 2 (D2-S6) plays a role in NaCh slow inactivation, we substituted different amino acids at position V787 (valine) in D2-S6 of rat skeletal muscle NaCh mu1 (Na(v)1.4). Whole-cell recordings from transiently expressed NaChs in HEK cells were used to study and compare slow inactivation phenotypes between mutants and wild type. V787K (lysine substitution) showed a marked enhancement of slow inactivation. V787K enters the slow-inactivated state approximate to 100X faster than wild type (tau (1) approximate to 30 ms vs. approximate to3 s), and occurs at much more hyperpolarized potentials than wild type (V-1/2 of s(infinity) curve approximate to -130 mV vs. approximate to -75 mV). V787C (cysteine substitution) showed a resistance to slow inactivation, i.e., opposite to that of V787K. Entry into the slow inactivation state in V787C was slower (tau (1) approximate to 5 s), less complete, and less voltage-dependent (V-1/2 of s(infinity) curve approximate to -50 mV) than in wild type. Application of the cysteine modification agent methanethiosulfonate ethylammonium (MTSEA) to V787C demonstrated that the 787 position undergoes a relative change in molecular conformation that is associated with the slow inactivation state. Our results suggest that the V737 position in Na(v)1.4 plays an important role in slow inactivation gating and that molecular rearrangement occurs at or near residue V787 in D2-S6 during NaCh slow inactivation.