MOLECULAR MECHANISMS OF PACEMAKER CHANNEL FUNCTION

描述（来自申请人摘要的逐字）：本申请的总体目标 项目是为了确定功能的结构基础， 超极化激活的环核苷酸门控（HCN）通道。这些 阳离子选择性通道传导心肌细胞的起搏电流， 神经元，并且与K+通道的erg家族最密切相关（例如， HERG）。HCN和HERG通道是自律性的重要调节器 心房起搏细胞中。然而，虽然HERG（和所有其他电压门控K+ 通道）通过去极化打开，HCN通道通过去极化打开 超极化这种差异的分子机制尚不清楚。我们 假设在这些分子之间特定静电和氢键相互作用 HCN的S4和其他跨膜结构域促进细胞内的通道开放。 响应于超极化，但阻止响应于超极化的通道开放。 去极化我们进一步假设，S4-S5接头偶联 S4结构域向激活门的移动。这个假设是基于 我们发现HERG的S4-S5连接子中的点突变（D540 K） 使闭合状态不稳定，并且类似于HCN，允许通道打开， 对超极化的反应。 野生型和突变型HCN和HERG通道将在细胞中异源表达。 非洲爪蟾卵母细胞和研究使用电压钳，取代半胱氨酸 可及性诱变和生物化学技术。具体目标是 确定HCN超极化依赖性打开的结构基础 和突变的HERG通道，以表征HCN 2的S4结构域在 反应膜去极化和超极化，并确定 S4和其他跨膜结构域之间静电相互作用的作用 在HCN通道的折叠和门控中。这些研究将阐明 门控HCN通道的分子机制，并进一步了解 心脏起搏器的。

DESCRIPTION (Verbatim from Applicant's Abstract): The overall goal of this project is to determine the structural basis for function of Hyperpolarization-activated, Cyclic Nucleotide-gated (HCN) channels. These cation-selective channels conduct the pacemaker current of cardiac myocytes and neurons, and are most closely related to the erg family of K+ channels (e.g., HERG). Together, HCN and HERG channels are important regulators of automaticity in atrial pacemaker cells. However, while HERG (and all other voltage-gated K+ channels) are opened by depolarizataion, HCN channels are opened by hyperpolarization. The molecular mechanism for this difference is unknown. We hypothesize that specific electrostatic and H-bonding interactions between the S4 and other transmembrane domains of HCN facilitate channel opening in response to hyperpolarization, but prevent channel opening in response to depolarization. We further hypothesize that the S4-S5 linker couples the movement of the S4 domains to the activation gate. This hypothesis is based on our discovery that a point mutation (D540K) in the S4-S5 linker of HERG destabilizes the closed state and, similar to HCN, permits channels to open in response to hyperpolarizaiton. Wild-type and mutant HCN and HERG channels will be heterologously expressed in Xenopus oocytes and studied using voltage clamp, substituted cysteine accessibility mutagenesis and biochemical techniques. The specific aims are to determine the structural basis for hyperpolarization-dependent opening of HCN and mutant HERG channels, to characterize movement of the S4 domain of HCN2 in response to membrane depolarization and hyperpolarization, and to determine the role of electrostatic interactions between S4 and other transmembrane domains in the folding and gating of HCN channels. These studies will elucidate the molecular mechanisms for gating of HCN channels and further our understanding of the cardiac pacemaker.