Bestrophin Channel Function in Heart

心脏中的卵黄蛋白通道功能

基本信息

批准号：
8101990
负责人：
FIONA Catherine BRITTON
金额：
$ 35.13万
依托单位：
UNIVERSITY OF NEVADA RENO
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-08-15 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8101990
关键词：
4-Aminopyridine Action Potentials Address Animals Antibodies Area Arrhythmia Biological Biological Assay Calcium Cardiac Cardiac Myocytes Cardiovascular system Cell physiology Cells Cellular biology Chloride Channels Chloride Ion Chlorides Co-Immunoprecipitations Confocal Microscopy Cyclic AMP Development Disease Echocardiography Electrocardiogram Electrophysiology (science)Engineered Gene Gel Gene Family Gene Targeting Genes Genetically Engineered Mouse Goals Health Heart Immunoblotting Immunofluorescence Immunologic Immunohistochemistry Immunoprecipitation In Vitro Ion Channel Knock-out Label Mammalian Cell Mass Spectrum Analysis Mediating Membrane Methods Modeling Molecular Mus Mutant Strains Mice Northern Blotting Phase Phenotype Physiological Physiology Play Property Protein Family Protein Isoforms Protein Kinase C Proteins Relative (related person)Research Research Proposals Reverse Transcriptase Polymerase Chain Reaction Role Swelling System Techniques Telemetry Testing Tetracyclines Tissues Transcript Transgenic Mice Trust Whole Organism cellular imaging clinically significant expression cloning in vivo insight member novel patch clamp protein function protein protein interaction therapeutic target

项目摘要

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.

描述（由申请人提供）：钙激活氯电流（ICl.Ca）已在心肌细胞中被记录，并且在心脏动作电位的复极化中发挥作用。迄今为止，还没有关于编码该电导通道的基因的信息。卵黄蛋白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卵黄蛋白离子通道介导心脏细胞生理学的机制。第一步，我们鉴定了在小鼠心脏中表达的黄蜂蛋白通道亚型（RT-PCR、Northern 印迹、免疫荧光和免疫印迹）。我们从小鼠心脏中分离并克隆了 Bestropin 转录本，并对一种 Bestropin 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：具体目标 1 检查 Bestropin 蛋白是否充当钙激活氯离子通道。我们将确定 Bestropin 通道的电生理特性（体外表达、膜片钳），并研究哪些特定异构体参与小鼠心肌细胞中的钙激活氯电流。具体目标 2 检查 Bestropin 蛋白在心脏中的组织和细胞定位。我们将检查 Bestropin 异构体的膜定位（共焦显微镜/免疫荧光）以及与辅助蛋白的可能关联（免疫沉淀、下拉分析、质谱法），包括与其他 Bestropin 的相互作用。具体目标 3 将评估 Bestropin 通道在体内调节心肌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 Bestropin 通道对突变小鼠心脏兴奋性的相对贡献（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、下拉分析、电生理学、转基因小鼠中的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激活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。公共健康相关性：该项目的主要目的是阐明黄斑黄蛋白通道蛋白在调节心脏兴奋性中的功能，特别是在心脏动作电位复极化过程中。拟议的研究可能为我们理解 Bestropin 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。