Zhuang Wenhui, Yan Zhiqiang
State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Department of Physiology and Biophysics, School of Life Sciences, Human Phenome Institute, Fudan University, Shanghai, China.
Shenzhen Bay Laboratory, Institute of Molecular Physiology, Shenzhen, China.
Front Physiol. 2021 Jan 20;11:604134. doi: 10.3389/fphys.2020.604134. eCollection 2020.
Kv7.4 (KCNQ4) voltage-gated potassium channels control excitability in the inner ear and the central auditory pathway. Mutations in Kv7.4 channels result in inherited progressive deafness in humans. Calmodulin (CaM) is crucial for regulating Kv7 channels, but how CaM affects Kv7 activity has remained unclear. Here, based on electrophysiological recordings, we report that the third EF hand (EF3) of CaM controls the calcium-dependent regulation of Kv7.4 activation and that the S2-S3 loop of Kv7.4 is essential for the regulation mediated by CaM. Overexpression of the mutant CaM, which loses the calcium binding ability of all four EF hands, facilitates Kv7.4 activation by accelerating activation kinetics and shifting the voltage dependence of activation leftwards. The single mutant CaM, which loses the calcium binding ability of the EF3, phenocopies facilitating effects of CaM on Kv7.4 activation. Kv7.4 channels co-expressed with wild-type (WT) CaM show inhibited activation when intracellular calcium levels increase, while Kv7.4 channels co-expressed with CaM or CaM are insensitive to calcium. Mutations C156A, C157A, C158V, R159, and R161A, which are located within the Kv7.4 S2-S3 loop, dramatically facilitate activation of Kv7.4 channels co-expressed with WT CaM but have no effect on activation of Kv7.4 channels co-expressed with CaM, indicating that these five mutations decrease the inhibitory effect of Ca/CaM. The double mutation C156A/R159A decreases Ca/CaM binding and completely abolishes CaM-mediated calcium-dependent regulation of Kv7.4 activation. Taken together, our results provide mechanistic insights into CaM regulation of Kv7.4 activation and highlight the crucial role of the Kv7.4 S2-S3 loop in CaM regulation.
Kv7.4（KCNQ4）电压门控钾通道控制内耳和中枢听觉通路的兴奋性。Kv7.4通道的突变会导致人类遗传性进行性耳聋。钙调蛋白（CaM）对调节Kv7通道至关重要，但CaM如何影响Kv7活性仍不清楚。在此，基于电生理记录，我们报告CaM的第三个EF手（EF3）控制Kv7.4激活的钙依赖性调节，并且Kv7.4的S2-S3环对于CaM介导的调节至关重要。失去所有四个EF手的钙结合能力的突变型CaM的过表达通过加速激活动力学并将激活的电压依赖性向左移动来促进Kv7.4激活。失去EF3的钙结合能力的单突变CaM模拟了CaM对Kv7.4激活的促进作用。与野生型（WT）CaM共表达的Kv7.4通道在细胞内钙水平升高时显示激活受到抑制，而与CaM或CaM共表达的Kv7.4通道对钙不敏感。位于Kv7.4 S2-S3环内的突变C156A、C157A、C158V、R159和R161A显著促进与WT CaM共表达的Kv7.4通道的激活，但对与CaM共表达的Kv7.4通道的激活没有影响，表明这五个突变降低了Ca/CaM的抑制作用。双突变C156A/R159A降低了Ca/CaM结合并完全消除了CaM介导的Kv7.4激活的钙依赖性调节。综上所述，我们的结果为CaM对Kv7.4激活的调节提供了机制见解，并突出了Kv7.4 S2-S3环在CaM调节中的关键作用。
