Molecular Mechanisms of Pacemaker Channel Function

描述(由申请人提供)：本项目的总体目标是描述HCN和HERG通道门控的分子机制。HCN通道传导起搏器电流(IF)，HERG通道传导快速延迟整流电流Ikr。这些电流的电压和时间依赖门控有助于心脏起搏细胞的节律性放电。HERG通道通过膜去极化打开，与S4电压传感器的向外运动相耦合。相反，HCN通道是通过膜超极化与S4结构域的向内运动耦合而开放的。我们假设，S4运动与HCN和HERG激活之间的相反极性耦合是S4-S5连接子中的残基与S6结构域的C末端之间的通道特异性相互作用的结果。对于HERG，我们已经表征了与S4运动相关的门控电流和药物结合的结构基础；我们将把这些研究扩展到HCN通道。此外，还将确定由外部阳离子变化引起的HERG通道门控改变的分子机制。离子电流和门控电流将使用两个微电极和切开的凡士林间隙电压钳技术记录在非洲爪哇卵母细胞中异源表达的通道。这些研究有望进一步加深我们对控制心率的生物振荡器的理解。

DESCRIPTION (provided by applicant): The overall goal of this project is to characterize the molecular mechanisms of HCN and hERG channel gating. HCN channels conduct the pacemaker current (If), and hERG channels conduct the rapid delayed rectifier current IKr. The voltage and time-dependent gating of these currents contribute to the rhythmic firing of cardiac pacemaker cells. HERG channels are opened by membrane depolarization, coupled to outward motion of the S4 voltage sensor. In contrast, HCN channels are opened by membrane hyperpolarization, coupled to inward motion of the S4 domain. We hypothesize that the opposite polarity of coupling between S4 movement and activation of HCN and hERG results from channel-specific interactions between residues in the S4-S5 linker and the C-terminal end of the S6 domain. For hERG, we have characterized the gating currents associated with S4 movement and the structural basis for drug binding; we will extend these studies to HCN channels. In addition, the molecular mechanisms of altered hERG channel gating induced by changes in external cations will be determined. Ionic and gating currents will be recorded using two microelectrode and cut-open Vaseline gap voltage clamp techniques of channels heterologously expressed in Xenopus oocytes. These studies promise to further our understanding of the biological oscillator that controls heart rhythm.