Disulfide locking a sodium channel voltage sensor reveals ion pair formation during activation
Disulfide locking a sodium channel voltage sensor reveals ion pair formation during activation
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DOI：
10.1073/pnas.0806486105
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发表时间：
2008-09-30
影响因子：
11.1
通讯作者：
Catterall, William A.
中科院分区：
文献类型：
--
作者：
DeCaen, Paul G.;Yarov-Yarovoy, Vladimir;Catterall, William A.
电压门控离子通道的S4跨膜段在去极化时向外移动，引发构象变化，打开孔洞，但S4移动的机制尚未解决。一种结构模型预测在门控过程中S4门控电荷与相邻的S2和S3跨膜片段中的负电荷之间的离子对的顺序形成。在这里，我们证明了细菌钠通道NaChBac的S4和S2中的第三个门控电荷(R3)和D60的半胱氨酸对在激活过程中形成了一个二硫键，从而“锁定”了S4片段，并导致该通道的缓慢失活。二硫键锁定与激活的动力学和电压依赖性密切相关，并被超极化逆转。与单个半胱氨酸突变体相比，D60C：R3C通道的激活更有利，突变循环分析表明这些残基之间存在强烈的自由能耦合，进一步支持了R3和D60在门控过程中的相互作用。我们的结果表明，在电压传感器激活期间，离子对的形成依赖于电压，并表明这种相互作用催化了S4运动和通道激活。
The S4 transmembrane segments of voltage-gated ion channels move outward on depolarization, initiating a conformational change that opens the pore, but the mechanism of S4 movement is unresolved. One structural model predicts sequential formation of ion pairs between the S4 gating charges and negative charges in neighboring S2 and S3 transmembrane segments during gating. Here, we show that paired cysteine substitutions for the third gating charge (R3) in S4 and D60 in S2 of the bacterial sodium channel NaChBac form a disulfide bond during activation, thus "locking'' the S4 segment and inducing slow inactivation of the channel. Disulfide locking closely followed the kinetics and voltage dependence of activation and was reversed by hyperpolarization. Activation of D60C:R3C channels is favored compared with single cysteine mutants, and mutant cycle analysis revealed strong free-energy coupling between these residues, further supporting interaction of R3 and D60 during gating. Our results demonstrate voltage-dependent formation of an ion pair during activation of the voltage sensor in real time and suggest that this interaction catalyzes S4 movement and channel activation.