辅助亚基KChIP调节Kv4通道的分子机制及功能意义研究

Cytosolic Kv channel-interacting proteins (KChIPs) co-assemble with pore-forming Kv4 α-subunits to form a native complex that encodes the somatodendritic A-type K+ current (IA) in neurons and the transient outward current (Ito) in cardiac myocytes. Neuronal IA plays a critical role in regulating dendritic excitability， somatodendritic signal integration and synaptic plasticity. Cardiac Ito is important for repolarization of cardiac action potential. Auxiliary KChIP1-4 subunits which belong to the neuronal calcium sensor superfamily consist of a conserved C-terminal core region containing four EF-hand-like calcium binding motifs,and a variable N-terminal region that has been proposed to mediate diverse modulation on Kv4 function. For instance,most KChIP1-3 dramatically increase Kv4 cell surface expression and peak current, slow fast inactivation moderately and accelerate recovery from inactivation. Previous studies show that both IA and Ito current amplitudes are determined by the expression levels of positive-modulatory KChIPs, but the functional role of negative modulator is still largely unknown. Different from the aforementioned KChIPs,the KChIP4 splice variant KChIP4a that shares a conserved C-terminal core with other KChIPs functions as an inhibitory subunit and exhibits a distinct modulation on Kv4 channel gating and surface expression via its unique N terminus. The first N-terminal 34 residues of KChIP4a act as a modular domain that abolishes the core-mediated enhancement of Kv4 current and eliminates fast inactivation (therefore also termed K+ channel inactivation suppressor (KIS) domain). Our and other labs have previously shown that core region of KChIPs promotes Kv4 function by stabilizing the tetrameric assembly of Kv4 through a clamping action and trafficking enhancement. The N-terminus of KChIP4a, however, functions as a specific Kv4 inhibitory domain (KID) to suppress A-type potassium current by a mechanism that remains unknown. In this proposal, we plan to use some new technologies such as genetically-encoded photocrosslinking amino acids and single-molecule subunit counting, to explore the molecular mechanism and physiological implication of Kv4 inhibition by inhibitory KChIP subunits.

胞浆蛋白辅助亚基KChIP与电压门控Kv4钾通道共同组装构成神经元中的瞬时低阈值A型钾电流（IA）和心肌细胞中的瞬时外向钾电流（Ito），其中IA调节神经元树突的兴奋性、信号整合及突触可塑性，而Ito对心脏动作电位的复极化发挥重要的作用。四种KChIP1-4均由保守的C-端核心区域（core region）和多变的N-末端组成。研究表明，KChIP1-3增加Kv4通道的膜表达和电流密度、加快失活后恢复及减慢失活，这类辅助蛋白定义为增强型KChIP亚基。此外，还有抑制型KChIP4亚基，其独特的N-末端对Kv4的调节作用与其他KChIP1-3截然相反，其作用机制及生理学意义尚不清楚。本课题计划以此为主要研究目标，在我们的前期结构生物学工作基础上，利用非天然氨基酸定点标记、单分子TIRF计数、电生理记录和整体动物行为等手段，深入研究KChIP调节Kv4通道的分子机制和功能意义。

胞浆蛋白辅助亚基KChIPs与电压门控Kv4钾通道-亚基共同组装形成天然的通道复合体，在神经元中介导胞体-树突间的低阈值瞬时A型钾电流(ISA)，调节神经元树突兴奋性、胞体树突信号整合及突触可塑性。不同种类的KChIPs对Kv4通道的功能调节不同，但其作用机制及生理学意义尚不十分清楚。为此，本项目深入研究KChIPs调节Kv4通道的分子机制和功能意义，获得结果如下：1.应用共聚焦成像、表面生物素标记和电生理记录等技术，发现辅助亚基KChIP4a通过内质网滞留及促进关闭态失活抑制ISA。2.揭示了Kv4通道关闭态失活的结构基础,发现四聚化T1序列在通道构象变化中通过结合KChIP4a的KID序列，抑制通道关闭态失活，进而加强了Kv4通道的功能。3.应用单分子亚基计数、免疫共沉淀和电生理记录等方法证明，KChIP4a和KChIP4bl可以竞争性与Kv4通道结合，并依赖于其表达水平，从而在病生理状态下调节神经元兴奋性；不同种类的KChIPs可以同时结合并对Kv4通道进行增强或抑制的调节。4.在HEK293细胞、原代培养的海马神经元，以及藻人酸引起的大鼠癫痫模型实验中，发现热休克蛋白Hsp70可以通过降解Kv4-KChIP4a通道复合体调节ISA，进而影响神经元的兴奋性。总之，本项目深入研究KChIPs调节Kv4通道的分子机制和功能意义，为研究相关神经精神疾病发生发展提供了理论依据和实验基础。