Bestrophin Channel Function in Heart

心脏中的卵黄蛋白通道功能

• 批准号: 7886561
• 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
• 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/7886561
• 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; 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; public health relevance; therapeutic target

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）：钙活化的氯化物电流（ICL.CA）已记录在心脏肌肉细胞中，并在心脏作用电位的复极化中发挥作用。迄今为止，尚无有关编码该电导基础通道的基因的信息。 Bestrophins是一种新型的蛋白质家族，最近被描述为充当钙活化的氯化物通道。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，我们已经确定了在小鼠心脏（RT-PCR，Northern印迹，免疫荧光和免疫印迹）中表达的BESTROPHIN通道同工型。我们已经从小鼠心脏分离并克隆了BestRophin的转录本，并对一种从一种Bestrophin同工型（MBEST-3）产生的电流进行了初步表征。提出了以下特定目的来实现总体目标：特定目标1检查BESTRophin蛋白是否充当钙活化的氯化物通道。我们将确定Bestrophin通道（体外表达，斑块夹）的电生理特性，并研究哪些特异性同工型参与小鼠心脏细胞中钙激活的氯化物电流。特定目标2检查了心脏中蛋白质蛋白的组织和细胞定位。我们将检查BestRophin同工型（共聚焦显微镜/免疫荧光）的膜定位，并可能与辅助蛋白（免疫沉淀，下拉测定，质谱法）相关联，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，尤其是在整个生物体的背景下。我们将描述特异性BESTROPHIN通道在突变小鼠中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将采用已建立的新模型和方法，包括：细胞生物学，细胞成像，下拉测定，电生理学，转基因小鼠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大大增强我们的理解，理解钙激活的氯化物通道在心脏兴奋性中的作用，并将为治疗靶标提供潜在的领域。公共卫生相关性：该项目的主要信任是阐明Bestrophin通道蛋白在调节心脏兴奋性方面的功能，尤其是在心脏动作潜力的复极化过程中。拟议的研究可能会提供新的见解，以了解我们对Bestrophin通道在心脏生理和疾病状态的作用的理解，从而为治疗靶标提供潜在的领域。