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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Location Of Kcne1 Relative To Kcnq1 In The I_(ks) Potassium Channel By Disulfide Cross-linking Of Substituted Cysteines
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Location Of Kcne1 Relative To Kcnq1 In The I_(ks) Potassium Channel By Disulfide Cross-linking Of Substituted Cysteines

机译：取代半胱氨酸二硫键交联在I_（ks）钾通道中Kcne1相对于Kcnq1的位置

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摘要

The cardiac-delayed rectifier K~+ current (I_(KS)) is carried by a complex of KCNQ1 (Q1) subunits, containing the voltage-sensor domains and the pore, and auxiliary KCNE1 (E1) subunits, required for the characteristic I_(KS) voltage dependence and kinetics. To locate the transmembrane helix of E1 (E1-TM) relative to the Q1 TM helices (S1-S6), we mutated, one at a time, the first four residues flanking the extracellular ends of S1-S6 and E1-TM to Cys, coexpressed all combinations of Q1 and E1 Cys-substituted mutants in CHO cells, and determined the extents of spontaneous disulfide-bond formation. Cys-flanking E1-TM readily formed disulfides with Cys-flanking S1 and S6, much less so with the S3-S4 linker, and not at all with S2 or S5. These results imply that the extracellular flank of the E1-TM is located between S1 and S6 on different subunits of Q1. The salient functional effects of selected cross-links were as follows. A disulfide from E1 K41C to S1 1145C strongly slowed deactivation, and one from E1 L42C to S6 V324C eliminated deac-tivation. Given that E1-TM is between S1 and S6 and that K41C and L42C are likely to point approximately oppositely, these two cross-links are likely to favor similar axial rotations of E1-TM. In the opposite orientation, a disulfide from E1 K41C to S6 V324C slightly slowed activation, and one from E1 L42C to S1 I145C slightly speeded deactivation. Thus, the first E1 orientation strongly favors the open state, while the approximately opposite orientation favors the closed state.
机译：心脏延迟整流器K〜+电流（I_（KS））由KCNQ1（Q1）子单元的复合体承载，其中包含电压传感器域和孔以及特性I_所需的辅助KCNE1（E1）子单元。 （KS）电压依赖性和动力学。为了找到相对于Q1 TM螺旋（S1-S6）的E1（E1-TM）跨膜螺旋，我们一次将一个位于S1-S6和E1-TM胞外末端侧翼的前四个残基突变为Cys ，在CHO细胞中共表达了Q1和E1 Cys取代突变体的所有组合，并确定了自发二硫键形成的程度。半胱氨酸侧翼的E1-TM容易与半胱氨酸侧翼的S1和S6形成二硫键，而与S3-S4连接子形成的二硫化物则少得多，而与S2或S5根本不形成。这些结果表明，E1-TM的细胞外侧面位于Q1不同亚基上的S1和S6之间。所选交联的显着功能作用如下。从E1 K41C到S1 1145C的二硫化物极大地减缓了失活，从E1 L42C到S6 V324C的二硫化物消除了失活。假设E1-TM在S1和S6之间，并且K41C和L42C可能指向相反的方向，则这两个交联点可能会促进E1-TM的类似轴向旋转。在相反的方向上，从E1 K41C到S6 V324C的二硫化物稍微减慢了活化，而从E1 L42C到S1 I145C的二硫化物则稍微减慢了失活速度。因此，第一E1方向强烈有利于打开状态，而大致相反的方向有利于关闭状态。

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