Depolarization induces intersubunit cross-linking in a S4 cysteine mutant of the shaker potassium channel
Depolarization induces intersubunit cross-linking in a S4 cysteine mutant of the shaker potassium channel
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DOI：
10.1074/jbc.m207258200
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发表时间：
2002-11-08
影响因子：
4.8
通讯作者：
Sivaprasadarao, A
中科院分区：
文献类型：
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作者：
Aziz, QH;Partridge, CJ;Sivaprasadarao, A
电压门控钾离子通道（K-v）是一种完整的膜蛋白，由4个亚基组成，每个亚基包含6个跨膜片段（S1-S6）。S1-S4包含电压敏感结构域，S5-S6与接头P-环形成离子传导孔结构域。在活化过程中，S4经历结构重排，导致通道孔的打开和离子传导。为了获得这些结构变化的细节，我们使用了工程二硫键方法。为此，我们在Shaker K通道S4的细胞外末端引入了L361 C突变，并在非洲爪蟾卵母细胞中表达了突变的通道。当暴露于温和的氧化条件下（环境氧气或铜菲咯啉），半胱氨酸-361形成亚基间二硫键，如蛋白质印迹上出现的二聚体条带所示。因此，突变通道遭受了电导（通过双电极电压钳测量）的显着损失。去除天然半胱氨酸未能阻止二硫化物形成，表明Cys-361与相邻亚基中的对应物形成二硫化物。该效应是电压依赖性的，并且在Cys-361暴露于细胞外相后的通道激活期间发生。虽然二硫桥降低了最大电导，但它引起电导-电压关系的超极化转变，并降低了通道的失活动力学。后两种效果表明稳定的开放状态的通道。总之，我们报告说，在激活过程中，L361 C突变体摇床K通道的S4段的N-末端之间的亚基间距离减少。
Voltage-gated potassium (K-v) channels are integral membrane proteins, composed of four subunits, each comprising six (S1-S6) transmembrane segments. S1-S4 comprise the voltage-sensing domain, and S5-S6 with the linker P-loop forms the ion conducting pore domain. During activation, S4 undergoes structural rearrangements that lead to the opening of the channel pore and ion conduction. To obtain details of these structural changes we have used the engineered disulfide bridge approach. For this we have introduced the L361C mutation at the extracellular end of S4 of the Shaker K channel and expressed the mutant channel in Xenopus oocytes. When exposed to mild oxidizing conditions (ambient oxygen or copper phenanthroline), Cys-361 formed an intersubunit disulfide bridge as revealed by the appearance of a dimeric band on Western blotting. As a consequence, the mutant channel suffered a significant loss in conductance (measured by two-electrode voltage clamp). Removal of native cysteines failed to prevent the disulfide formation, indicating that Cys-361 forms a disulfide with its counterpart in the neighboring subunit. The effect was voltage-dependent and occurred during channel activation after Cys-361 has been exposed to the extracellular phase. Although the disulfide bridge reduced the maximal conductance, it caused a hyperpolarizing shift in the conductance-voltage relationship and reduced the deactivation kinetics of the channel. The latter two effects suggest stabilization of the open state of the channel. In conclusion, we report that during activation the intersubunit distance between the N-terminal ends of the S4 segments of the L361C mutant Shaker K channel is reduced.