Molecular mechanisms for regulation of HCN channels by TRIP8b subunits

描述（由申请人提供）：神经元响应突触输入而激发动作电位的基本能力在很大程度上由其树突的电特性决定。调节树突电特性的一个关键离子通道是超极化激活的环核苷酸门控（HCN）离子通道。HCN通道在兴奋性阈值下工作，因此通道的数量、定位、电压依赖性和环核苷酸依赖性的微小变化可对细胞的兴奋性产生显著影响。我们的长期目标是了解HCN通道如何控制神经元兴奋性的分子机制。最近，在神经元中发现了HCN通道的辅助亚基，称为TRIP 8b。TRIP 8b对HCN通道的运输、电压依赖性和环核苷酸依赖性具有深远的影响。在这项研究中，我们计划研究TRIP 8b如何与HCN通道结合并调节通道功能的分子机制。我们的方法将是结合联合收割机的X射线晶体学，原子分辨率的结构信息，与电生理学和荧光研究功能通道在其天然的膜环境。我们的实验将揭示HCN 2 C-末端区域与TRIP 8b之间在原子分辨率下的相互作用的结构、相互作用的化学计量以及TRIP 8b调节HCN 2的环核苷酸依赖性和电压依赖性门控的机制。这些发现将为我们理解HCN通道的结构和调节提供重大进展，因为它们存在于神经元中，并进一步阐明它们在大脑生理学和病理学中的作用。

DESCRIPTION (provided by applicant): The fundamental ability of a neuron to fire action potentials in response to synaptic input is largely determined by the electrical properties of its dendrites. One key ion channel that regulates the electrical properties of dendrites is the hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channel. HCN channels operate at the threshold of excitability so small changes in the number, localization, voltage dependence, and cyclic nucleotide dependence of the channels can have a dramatic impact on the excitability of the cell. Our long term goal is to understand the molecular mechanisms for how HCN channels control neuronal excitability. Recently an auxiliary subunit of HCN channels in neurons was discovered, called TRIP8b. TRIP8b has a profound effect on the trafficking, voltage dependence, and cyclic nucleotide dependence of the HCN channels. In this grant, we propose to study the molecular mechanism for how TRIP8b binds to the HCN channel and regulates channel function. Our approach will be to combine x-ray crystallography, for atomic resolution structural information, with electrophysiology and fluorescence to study the functional channel in its native membrane environment. Our experiments will reveal the structure of the interaction between the HCN2 C-terminal region and TRIP8b at atomic resolution, the stoichiometry of the interaction, and the mechanism for TRIP8b regulation of the cyclic nucleotide-dependent and voltage- dependent gating of HCN2. These findings will provide a significant advance in our understanding of the structure and regulation of HCN channels as they exist in the neuron and further illuminate their role in the physiology and pathology of the brain.