Bestrophin Channel Function in Heart

描述（由申请人提供）：已在心肌细胞中记录了钙激活氯电流（ICl.Ca），并在心脏动作电位的复极化中发挥作用。到目前为止，还没有关于编码这种传导的通道的基因的信息。Bestrophins是一个新的蛋白质家族，最近被描述为具有钙激活氯离子通道的功能。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学的机制。作为第一步，我们已经鉴定了在小鼠心脏中表达的Bestrophin通道同种型（RT-PCR、北方印迹、免疫荧光和免疫印迹）。我们已经从小鼠心脏中分离并克隆了Bestrophin转录本，并对从一种Bestrophin亚型（mBest-3）产生的电流进行了初步表征。提出了以下具体目标以实现总体目标：具体目标1检查Bestrophin蛋白是否作为钙激活氯离子通道发挥功能。我们将确定Bestrophin通道的电生理特性（体外表达，膜片钳），并研究哪些特定亚型参与小鼠心肌细胞中的钙激活氯电流。特异性目的2检查Bestrophin蛋白在心脏中的组织和细胞定位。我们将研究Bestrophin亚型的膜定位（共聚焦显微镜/免疫荧光）和可能与辅助蛋白（免疫沉淀，下拉测定，质谱），包括与其他Bestrophins的相互作用。具体目标3将评估Bestrophin通道在体内调节心脏细胞生理学中的作用，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）。为了实现这些目标，PI将采用已建立的以及新的模型和方法，包括：细胞生物学、细胞成像、下拉试验、电生理学、转基因小鼠的基因敲除和心血管表型分析技术。从拟议的研究中获得的信息将大大提高我们对钙激活氯离子通道在心脏兴奋性中的作用的理解，并将提供潜在的治疗靶点。公共卫生相关性：本课题的主要目的是阐明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过程中的作用。拟议的研究可能为我们理解Bestrophin通道在心脏生理学和疾病状态中的作用提供新的见解，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.