KCNH channel regulation by intracellular ligands

KCNH家族中的离子通道具有多种重要的生理功能， 包括对癌症进展、神经元兴奋性和心脏的调节 收缩。KCNH通道的N端包含Per-Arnt-Sim(PAS)结构域 和C-末端的环核苷酸结合同源(CNBH)结构域。 这些细胞内域是主要负责的关键功能域 由于KCNH通道的不同属性决定了它们的生理 申请。这项提议的目标是识别直接 结合PAS和CNBH结构域并揭示其作用机制 KCNH频道。我们将确定KCNH通道配体并对其进行功能表征 使用一种基于表面等离子体共振(SPR)组合的新策略 电生理学为主要方法。我们将首先确定小分子 通过筛选针对PAS和CNBH结构域的小分子文库来筛选配体 使用高通量SPR方法的KCNH信道。然后我们将确定 已鉴定的小分子配体与中通量的功能效应 电极电压钳位法。最后要揭示其作用的分子机制 识别的配体我们将使用膜片钳电流记录的组合， 诱变、X射线结晶学、表面等离子体共振和结构模拟。这些研究将 细胞内配体和Pas对KCNH通道调控的研究进展 和CNBH结构域，为研究生理作用提供了药理学工具 并将极大地促进新型药物的开发 治疗心律失常、癌症和癫痫的药物。

Ion channels in the KCNH family perform diverse and important physiological functions, including the regulation of cancer progression, neuronal excitability and cardiac contraction. KCNH channels contain a Per-Arnt-Sim (PAS) domain in their N-terminal and cyclic nucleotide-binding homology (CNBH) domain in their C-terminal regions. These intracellular domains are the key functional domains that are largely responsible for the distinct properties of KCNH channels that determine their physiological applications. The goal of this proposal is to identify small molecule ligands that directly bind to the PAS and CNBH domains and uncover the mechanisms of their action on KCNH channels. We will identify and functionally characterize KCNH channel ligands using a novel strategy based on the combination of surface plasmon resonance (SPR) and electrophysiology as the principal methods. We will first identify small molecule ligands by screening libraries of small molecules against the PAS and CNBH domains of KCNH channels with the high-throughput SPR method. We will then determine the functional effects of the identified small molecule ligands with medium-throughput two- electrode voltage-clamp method. Finally to uncover molecular mechanisms of action of the identified ligands we will use a combination of patch-clamp current recordings, mutagenesis, X-ray crystallography, SPR and structural modeling. These studies will advance our knowledge of KCNH channel regulation by intracellular ligands and PAS and CNBH domains, provide pharmacological tools to study physiological contributions of KCNH channels and will greatly facilitate the development of novel pharmaceutical agents for treatment of cardiac arrhythmias, cancer and epilepsy.