Voltage Sensor Movement in the HERG Potassium Channel

描述(由申请人提供)：本提案的总体目标是确定电压传感器移动的结构基础，以及将传感器移动与HERG延迟整流K通道的打开相耦合的机制。HERG是介导心脏动作电位复极的几个电压门控IC通道之一。HERG通道功能障碍是由突变或常用处方药引起的，与危及生命的心律失常有关。HERG在维持正常心脏电活动中的关键作用来自其不同寻常的门控特性：缓慢的通道激活和快速的失活。激活和失活门控是电压依赖的，这意味着这些过程与带电残基在膜内的运动耦合。电压传感器运动的结构基础和传感器运动与HERG通道开口的耦合机制尚不清楚。在本方案中，我们首次描述了HERG电压传感器通过门控电流测量的运动。我们假设(1)S4结构域(可能的电压传感器)周围的结构域影响S4运动的速度，并解释HERG离子电流的缓慢激活，(2)失活和激活门控耦合到相同的基本电压传感机制，(3)电压传感器运动通过细胞内S4-S5连接子和S6结构域之间的直接相互作用耦合到通道开放。该建议的目的是确定HERG通道中缓慢激活的结构基础，定义与快速HERG失活相关的电压传感器运动，并确定电压传感器运动与通道开放相耦合的机制。这些目标将通过定点突变、HERG与结构上相关的EAG通道之间的嵌合构建、生化分析、双微电极电压钳和切开卵母细胞电压钳技术来实现。深入了解HERG通道电压感知的分子基础将促进我们理解该通道如何有助于心脏的正常电稳定，并可能促进我们对心律失常易感性的理解。

DESCRIPTION (provided by applicant): The overall goals of this proposal are to define the structural basis of voltage sensor movement and the mechanism of coupling sensor movement to opening of HERG delayed rectifier K+ channels. HERG is one of several voltage-gated IC channels that mediate repolarization of the cardiac action potential. HERG channel dysfunction, caused by mutations or commonly prescribed medications, is associated with life-threatening arrhythmias. The critical role of HERG in the maintenance of normal cardiac electrical activity derives from its unusual gating properties: slow channel activation and fast inactivation. Activation and inactivation gating are voltage dependent, implying that these processes are coupled to movement of charged residues within the membrane. The structural basis of voltage sensor movement and mechanism of coupling sensor movement to HERG channel opening are not known. In this proposal, we provide the first description of HERG voltage sensor movement as measured by gating current. We hypothesize (1) the domains surrounding the S4 domain (the putative voltage sensor) influence the rate of S4 movement and account for the slow activation of HERG ionic current, (2) inactivation and activation gating are coupled to the same fundamental voltage sensing mechanism and (3) voltage sensor movement is coupled to channel opening via a direct interaction between the intracellular S4-S5 linker and the S6 domain. The aims of this proposal are to determine the structural basis of slow activation in HERG channels, to define the voltage sensor movement associated with fast HERG inactivation and to determine the mechanism whereby movement of the voltage sensor is coupled to channel opening. These aims will be accomplished using site-directed mutagenesis, chimeric constructs between HERG and the structurally related EAG channel, biochemical assays, two microelectrode voltage clamp and cut-open oocyte voltage clamp techniques. Insight into the molecular basis of voltage sensing in the HERG channel will advance our understanding of how this channel contributes to normal electrical stability in the heart and may advance our understanding of arrhythmia susceptibility.