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， 包括调节癌症进展、神经元兴奋性和心脏 收缩。KCNH通道在其N-末端含有Per-Arnt-Sim（PAS）结构域 和环核苷酸结合同源（CNBH）结构域。 这些胞内结构域是主要负责 KCNH通道的独特特性决定了它们的生理特性， 应用.该提案的目标是鉴定直接 结合PAS和CNBH结构域，并揭示其作用机制， KCNH频道。我们将确定和功能特性KCNH通道配体 使用基于表面等离子体共振（SPR） 和电生理学作为主要方法。我们将首先识别小分子 通过筛选针对PAS和CNBH结构域的小分子文库 的KCNH通道的高通量SPR方法。然后我们将确定 鉴定的小分子配体的功能效应与中等通量双- 电极电压箝位法最后，为了揭示分子作用机制， 我们将使用膜片钳电流记录的组合来鉴定配体， 诱变、X射线晶体学、SPR和结构建模。这些研究将 进一步了解细胞内配体和PAS对KCNH通道的调节作用 和CNBH结构域，提供药理学工具来研究生理贡献 KCNH通道，将大大促进新药物的开发 用于治疗心律失常、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.