Lainé Muriel, Lin Meng-chin A, Bannister John P A, Silverman William R, Mock Allan F, Roux Benoit, Papazian Diane M
Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California 90095, USA.
Neuron. 2003 Jul 31;39(3):467-81. doi: 10.1016/s0896-6273(03)00468-9.
A recently proposed model for voltage-dependent activation in K+ channels, largely influenced by the KvAP X-ray structure, suggests that S4 is located at the periphery of the channel and moves through the lipid bilayer upon depolarization. To investigate the physical distance between S4 and the pore domain in functional channels in a native membrane environment, we engineered pairs of cysteines, one each in S4 and the pore of Shaker channels, and identified two instances of spontaneous intersubunit disulfide bond formation, between R362C/A419C and R362C/F416C. After reduction, these cysteine pairs bound Cd2+ with high affinity, verifying that the residues are in atomic proximity. Molecular modeling based on the MthK structure revealed a single position for S4 that was consistent with our results and many other experimental constraints. The model predicts that S4 is located in the groove between pore domains from different subunits, rather than at the periphery of the protein.
最近提出的一种受KvAP X射线结构很大影响的钾离子通道电压依赖性激活模型表明，S4位于通道外围，并在去极化时穿过脂质双层。为了研究在天然膜环境中功能通道中S4与孔结构域之间的物理距离，我们设计了成对的半胱氨酸，分别位于Shaker通道的S4和孔中，并确定了R362C/A419C和R362C/F416C之间两个亚基间自发形成二硫键的实例。还原后，这些半胱氨酸对以高亲和力结合Cd2+，证实这些残基处于原子接近距离。基于MthK结构的分子建模揭示了一个与我们的结果以及许多其他实验限制相一致的S4单一位置。该模型预测S4位于来自不同亚基的孔结构域之间的凹槽中，而不是在蛋白质的外围。
