MOLECULAR MECHANISMS OF PACEMAKER CHANNEL FUNCTION

描述(逐字摘自申请者摘要)：该项目的总体目标 项目是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和 神经元，并且与K+通道的ERG家族关系最密切(例如， Herg)。总而言之，HCN和HERG通道是自动化的重要调节器 在心房起搏细胞中。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 超极化。造成这种差异的分子机制尚不清楚。我们 假设特定的静电和氢键之间的相互作用 HcN的S4和其他跨膜结构域促进通道开放 响应超极化，但防止响应超极化的通道开放 去极化。我们进一步假设S4-S5连接子将 将S4结构域移动到激活门。这一假设是基于 HERG基因S4-S5连接子D540K点突变的发现 使关闭状态不稳定，并且与HCN类似，允许通道在 对超极化的反应。 野生型和突变型HCN和HERG通道将在 非洲爪哇卵母细胞与电压钳替代半胱氨酸的研究 可获得性、诱变和生化技术。具体目标是 确定HCN超极化依赖开放的结构基础 和突变的Herg通道，以表征HCN2的S4结构域在 对膜去极化和超极化的响应，并确定 S4与其他跨膜结构域的静电相互作用 在HCN通道的折叠和选通中。这些研究将阐明 HCN通道门控的分子机制及进一步认识 心脏起搏器。

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.