Molecular Physiology of HERG (KCNH2) Pottasium Channels

描述（由申请人提供）：人类以太相关基因1a (HERG1a, Kv11.1) K+通道在维持基本心律中起关键作用。HERG1a通道的意义在于它们是心脏快速延迟整流K+通道（IKr）的中心组成部分。HERG和IKr专门传导向外的K+电流，驱动心脏动作电位后期的复极化。编码HERG通道的基因的遗传突变强调了HERG1a在健康和疾病中的关键作用。HERG突变与长QT综合征（LQTS）有关，LQTS是一种引起心律失常、晕厥和猝死的心脏疾病。HERG通道具有额外的意义，因为越来越多的药物的副作用是通过抑制HERG通道的功能产生获得性LQTS （aLQTS）。HERG和IKr通道的开启和关闭（门控）对正常的心脏电生理和心跳至关重要。特别是，原生IKr通道的关闭速率对于再极化期间外向IKr电流的完美定时至关重要。一些进展，包括我们之前的工作，已经描绘了通道关闭（失活）机制的关键分子成分，包括HERG1a n端区域内的两个关键结构域。这些是‘ PAS’结构域和上游的一个短区域称为PAS- cap。失活机制的多样性来自HERG1a变体，HERG1b缺乏关键的PAS和PAS- cap结构域，因此比HERG1a关闭得快得多。HERG1b在心脏中的存在可能解释了IKr更快的失活动力学。尽管取得了这些进展，但通道失活的机制仍然难以捉摸。实验的目的是确定HERG和IKr关闭的综合分子机制。具体目的是：1)测试PAS- cap区域通过与通道的静电相互作用决定失活门控的假设；2)测试PAS结构域的疏水表面与通道中的疏水“PAS受体位点”相互作用以介导失活的假设；3)测试HERG1b亚基是天然IKr的关键功能成分的假设，以及ERG1b解释了天然IKr的更快动力学。为了实现具体目标，我们将使用多学科方法，包括异种表达系统和天然细胞中的膜片钳和电压钳电生理学，荧光光谱，基因转移到肌细胞和天然细胞培养技术。我们的长期目标是确定心脏IKr通道门控和调节的基本分子基础，以更好地治疗遗传性LQTS和预防获得性LQTS。

DESCRIPTION (provided by applicant): Human ether a go-go-related gene 1a (HERG1a, Kv11.1) K+ channels play a critical role in maintaining the fundamental cardiac rhythm. The significance of HERG1a channels is that they are the central component of the rapid delayed-rectifier K+ channel (IKr) in heart. HERG and IKr are specialized to conduct an outward K+ current that drives repolarization of the late phase of the cardiac action potential. The critical role of HERG1a in health and disease is emphasized by inherited mutations in the gene encoding HERG channels. Mutations in HERG are associated with the long QT syndrome (LQTS) a cardiac disorder that causes arrhythmia, syncope and sudden death. HERG channels are of additional significance as a side-effect of an increasing number of pharmaceuticals is to produce an acquired form of LQTS (aLQTS) by inhibiting the function of HERG channels. The opening and closing (gating) of HERG and IKr channels are critical for normal cardiac electrophysiology and the normal heartbeat. In particular, the closing rate of native IKr channels is vital for the perfect timing of the outward IKr current during repolarization. Some advances, including our previous work, have delineated key molecular components of the channel closing (deactivation) mechanism, including two critical domains within the HERG1a N-terminal region. These are the `PAS' domain and a short region upstream here termed the PAS-CAP. Diversity in the mechanism of deactivation comes from a HERG1a variant, HERG1b that lacks the key PAS and PAS-CAP domains and consequently closes much faster than HERG1a. The presence of HERG1b in heart may explain the faster kinetics of deactivation measured for IKr. Despite these advances, a mechanism for channel deactivation has remained elusive. The goals of the proposed experiments are to determine a comprehensive molecular mechanism for closing in HERG and IKr. The Specific Aims are to 1) test the hypothesis that the PAS-CAP region determines deactivation gating via an electrostatic interaction with the channel 2) to test the hypothesis that the hydrophobic surface of the PAS domain interacts with a hydrophobic `PAS receptor site' in the channel to mediate deactivation and 3) to test the hypothesis that the HERG1b subunit is a key functional component of native IKr and that ERG1b accounts for the faster kinetics described for native IKr. To carry out the specific aims we will use a multidisciplinary approach that includes patch-clamp and voltage-clamp electrophysiology in heterologous expression systems and native cells, fluorescence spectroscopy, gene transfer to myocytes and native cell culture techniques. Our long-term objectives are to determine the fundamental molecular basis of gating and modulation in cardiac IKr channels, in an effort to better treat inherited LQTS and prevent acquired LQTS.