Bestrophin Channel Function in Heart

描述（由申请人提供）：钙激活的氯离子电流（ICl.Ca）已在心肌细胞中被记录，并在心脏动作电位的复极化中发挥作用。到目前为止，还没有关于基因编码这个传导通道的信息。Bestrophins是一种新的蛋白质家族，最近被描述为钙激活的氯离子通道。我们研究的总体目标是了解Bestrophin离子通道介导心脏细胞生理的机制。作为第一步，我们已经鉴定了小鼠心脏中表达的Bestrophin通道亚型（RT-PCR, Northern blot，免疫荧光和免疫印迹）。我们从小鼠心脏中分离并克隆了Bestrophin转录本，并对一种Bestrophin异构体（mBest-3）产生的电流进行了初步表征。为了实现总体目标，提出了以下具体目标：specific Aim 1检查Bestrophin蛋白是否作为钙活化的氯离子通道起作用。我们将确定Bestrophin通道的电生理特性（在体外表达，膜片钳），并研究在小鼠心脏细胞中哪些特定的同种异构体参与钙激活的氯化物电流。特异性目的2检测心脏中Bestrophin蛋白的组织和细胞定位。我们将检查Bestrophin异构体的膜定位（共聚焦显微镜/免疫荧光）和与辅助蛋白的可能关联（免疫沉淀，下拉试验，质谱分析），包括与其他Bestrophin的相互作用。特异性目的3将评估Bestrophin通道在体内调节心脏细胞生理中的作用，特别是在整个生物体的背景下。我们将描述特定的Bestrophin通道在突变小鼠心脏兴奋性中的相对贡献（靶向基因破坏）。为了完成这些目标，PI将采用现有的以及新的模型和方法，包括：细胞生物学、细胞成像、拉下试验、电生理学、转基因小鼠基因敲除和心血管表型技术。从拟议的研究中获得的信息将极大地增强我们对钙活化的氯离子通道在心脏兴奋性中的作用的理解，并将为治疗靶点提供潜在的领域。公共卫生相关性：本项目的主要目的是阐明Bestrophin通道蛋白在调节心脏兴奋性，特别是在心脏动作电位复极化期间的功能。这些研究可能为我们对Bestrophin通道在心脏生理和疾病状态中的作用的理解提供新的见解，从而为治疗靶点提供潜在的领域。

DESCRIPTION (provided by applicant): Calcium-activated chloride currents (ICl.Ca) have been recorded in cardiac muscle cells and play a role in the repolarization of the cardiac action potential. To date, there is no information as to the gene encoding the channel underlying this conductance. Bestrophins are a novel family of proteins that have recently been described to function as calcium-activated chloride channels. The overall goal of our research is to understand the mechanisms by which Bestrophin ion channels mediate cardiac cell physiology. As a first step, we have identified the Bestrophin channel isoforms expressed in mouse heart (RT-PCR, Northern blot, immunofluorescence and immunoblot). We have isolated and cloned the Bestrophin transcripts from mouse heart and performed a preliminary characterization of the current generated from one Bestrophin isoform (mBest-3). The following specific aims are proposed to achieve the overall goal: Specific Aim 1 examines whether Bestrophin proteins function as calcium-activated chloride channels. We will determine the electrophysiological properties of Bestrophin channels (in vitro expression, patch clamp) and investigate which specific isoforms participate in the calcium activated chloride current in mouse cardiac cells. Specific Aim 2 examines the tissue and cellular localization of Bestrophin proteins in heart. We will examine membrane localization of Bestrophin isoforms (confocal microscopy/immunofluorescence) and possible association with accessory proteins (immunoprecipitation, pull-down assays, mass spectrometry), including interactions with other Bestrophins. Specific Aim 3 will evaluate the roles of Bestrophin channels in regulating cardiac cell physiology in vivo, particularly in the context of whole organisms. We will delineate the relative contribution of specific Bestrophin channels in cardiac excitability in mutant mice (targeted gene disruption). To complete these aims, the PI will employ established as well as new models and methods including: cell biology, cellular imaging, pull down assays, electrophysiology, gene knock-out in transgenic mice and cardiovascular phenotyping techniques. The information gained from the proposed study will greatly enhance our understanding the role of calcium-activated chloride channels in cardiac excitability and will provide potential areas for therapeutic targets. PUBLIC HEALTH RELEVANCE: The main trust of this project is to elucidate the function of Bestrophin channel proteins in regulating cardiac excitability especially during repolarization of the cardiac action potential. The proposed studies may provide novel insights into our understanding of the role of Bestrophin channels in cardiac physiology and disease states and thus provide potential areas for therapeutic targets.