Movement of the Na+ channel inactivation gate during inactivation
Movement of the Na+ channel inactivation gate during inactivation
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DOI：
10.1074/jbc.271.48.30971
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发表时间：
1996-11-29
影响因子：
4.8
通讯作者：
Catterall, WA
中科院分区：
文献类型：
--
作者：
Kellenberger, S;Scheuer, T;Catterall, WA
大鼠脑IIA钠通道cu亚基失活门中的苯丙氨酸1489是稳定失活所必需的。建议在灭活过程中移动到孔的细胞内口并将其闭塞，但尚未提供该残留物在灭活过程中移动的直接证据。我们使用取代的半胱氨酸可及性方法来测试在位置1489处取代的半胱氨酸残基对从细胞质侧施加的甲硫基磺酸盐试剂修饰的可用性。Phe-1489突变为Cys导致一小部分（8%）非失活电流，Ag+和甲硫基磺酸盐试剂不可逆地减慢失活速率并增加F1489 C的非失活电流，但不增加野生型通道的非失活电流。单通道分析表明，修饰减缓了封闭和开放状态的失活，并使失活状态不稳定。去极化阻止了这些试剂对Cys-1489的快速修饰，并且它们的反应速率的电压依赖性与稳态失活密切相关。在比通道门控更负的电压下，修饰没有可检测的电压依赖性。我们的研究结果表明，失活后，Phe-1489在失活门移动从一个暴露的和可修改的位置外的膜电场到一个埋藏的和不可访问的位置，也许在或附近的通道孔的细胞内口。
Phenylalanine 1489 in the inactivation gate of the rat brain IIA sodium channel cu subunit is required for stable inactivation. It is proposed to move into the intracellular mouth of the pore and occlude it during inactivation, but direct evidence for movement of this residue during inactivation has not been presented. We used the substituted cysteine accessibility method to test the availability of a cysteine residue substituted at position 1489 to modification by methanethiosulfonate reagents applied from the cytoplasmic side. Mutation of Phe-1489 to Cys results in a small (8%) fraction of noninactivating current, Ag+ and methanethiosulfonate reagents irreversibly slowed the inactivation rate and increased the fraction of noninactivating current of F1489C but not wild-type channels. Single channel analysis showed that modification slowed inactivation from both closed and open states and destabilized the inactivated state. Depolarization prevented rapid modification of Cys-1489 by these reagents, and the voltage dependence of their reaction rate correlated closely with steady-state inactivation, Modification was not detectably voltage-dependent at voltages more negative than channel gating. Our results show that, upon inactivation, Phe-1489 in the inactivation gate moves from an exposed and modifiable position outside the membrane electric field to a buried and inaccessible position, perhaps in or near the intracellular mouth of the channel pore.