Chen Xin, Ruan Mei-Yu, Cai Shi-Qing
Institute of Neuroscience and State Key Laboratory of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China, and University of Chinese Academy of Sciences, Shanghai 200031, P. R. China.
Institute of Neuroscience and State Key Laboratory of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China, and.
J Neurosci. 2015 Feb 4;35(5):1880-91. doi: 10.1523/JNEUROSCI.3429-14.2015.
Voltage-gated Kv4 channels control the excitability of neurons and cardiac myocytes by conducting rapidly activating-inactivating currents. The function of Kv4 channels is profoundly modulated by K(+) channel interacting protein (KChIP) soluble auxiliary subunits. However, the in vivo mechanism of the modulation is not fully understood. Here, we identified three C. elegans KChIP-like (ceKChIP) proteins, NCS-4, NCS-5, and NCS-7. All three ceKChIPs alter electrical characteristics of SHL-1, a C. elegans Kv4 channel ortholog, currents by slowing down inactivation kinetics and shifting voltage dependence of activation to more hyperpolarizing potentials. Native SHL-1 current is completely abolished in cultured myocytes of Triple KO worms in which all three ceKChIP genes are deleted. Reexpression of NCS-4 partially restored expression of functional SHL-1 channels, whereas NCS-4(efm), a NCS-4 mutant with impaired Ca(2+)-binding ability, only enhanced expression of SHL-1 proteins, but failed to transport them from the Golgi apparatus to the cell membrane in body wall muscles of Triple KO worms. Moreover, translational reporter revealed that NCS-4 assembles with SHL-1 K(+) channels in male diagonal muscles. Deletion of either ncs-4 or shl-1 significantly impairs male turning, a behavior controlled by diagonal muscles during mating. The phenotype of the ncs-4 null mutant could be rescued by reexpression of NCS-4, but not NCS-4(efm), further emphasizing the importance of Ca(2+) binding to ceKChIPs in regulating native SHL-1 channel function. Together, these data reveal an evolutionarily conserved mechanism underlying the regulation of Kv4 channels by KChIPs and unravel critical roles of ceKChIPs in regulating muscle cell excitability and animal behavior in C. elegans.
电压门控Kv4通道通过传导快速激活-失活电流来控制神经元和心肌细胞的兴奋性。Kv4通道的功能受到K⁺通道相互作用蛋白(KChIP)可溶性辅助亚基的深刻调节。然而,这种调节的体内机制尚未完全了解。在这里,我们鉴定出三种秀丽隐杆线虫KChIP样(ceKChIP)蛋白,即NCS-4、NCS-5和NCS-7。所有这三种ceKChIPs都会改变秀丽隐杆线虫Kv4通道直系同源物SHL-1的电流的电学特性,通过减慢失活动力学并将激活的电压依赖性转移到更超极化的电位。在所有三个ceKChIP基因均被缺失的三重敲除蠕虫的培养肌细胞中,天然SHL-1电流完全消失。NCS-4的重新表达部分恢复了功能性SHL-1通道的表达,而NCS-4(efm)是一种Ca²⁺结合能力受损的NCS-4突变体,仅增强了SHL-1蛋白的表达,但未能将它们从高尔基体转运到三重敲除蠕虫体壁肌肉的细胞膜上。此外,翻译报告基因显示NCS-4与雄性对角肌中的SHL-1 K⁺通道组装在一起。ncs-4或shl-1的缺失会显著损害雄性转向,这是交配过程中由对角肌控制的一种行为。ncs-4缺失突变体的表型可以通过NCS-4的重新表达来挽救,但不能通过NCS-4(efm)来挽救,这进一步强调了Ca²⁺与ceKChIPs结合在调节天然SHL-1通道功能中的重要性。总之,这些数据揭示了KChIPs调节Kv4通道的进化保守机制,并揭示了ceKChIPs在调节秀丽隐杆线虫肌肉细胞兴奋性和动物行为中的关键作用。