Fibroblast Growth Factor Homologous Factors: Modulation of L-type Calcium Channel

成纤维细胞生长因子同源因子：L 型钙通道的调节

• 批准号: 8432546
• 负责人: Jessica Amenta Hennessey
• 金额: $ 4.18万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8432546
• 关键词: Action Potentials; Affect; Anabolism; Anti-Arrhythmia Agents; Arrhythmia; Binding; Biochemical; Biological Models; Biotinylation; Calcium; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cardiovascular Diseases; Cell surface; Cells; Cessation of life; Co-Immunoprecipitations; Coupling; Data; Disease; Etiology; Family; Fibroblast Growth Factor; Fibroblast Growth Factor Receptors; Functional disorder; Genetic; Genetic Markers; Grant; Growth Factor; Heart; Heart failure; In Vitro; Individual; Inherited; Ion Channel; L-Type Calcium Channels; L-type calcium channel alpha(1C); Lead; Life; Link; Measures; Mediator of activation protein; Methods; Molecular; Mus; Muscle Cells; Mutation; Neurons; Patients; Pharmaceutical Preparations; Physiological; Play; Protein Isoforms; Proteins; Regulation; Resistance; Risk; Role; Sodium Channel; Spinocerebellar Ataxias; Surface; System; Testing; United States; Ventricular; Work; fibroblast growth factor 13; knock-down; mutant; neuronal excitability; novel; overexpression; patch clamp; protein phosphatase inhibitor-2; research study; small hairpin RNA; sudden cardiac death; therapeutic target; trafficking; voltage

DESCRIPTION (provided by applicant): Background: A growing number of cardiovascular disorders such as arrhythmias and certain cardiomyopathies are attributable to ion channel dysfunction that results from mutations within channels. These "channelopathies" have been instrumental in providing an understanding about how perturbation of ion channels can lead to disease. Recent work has shown that ion channel auxiliary and modulatory subunits are potent regulators of channels and often impart effects upon multiple ionic currents. Fibroblast growth factor homologous factors (FHFs, FGF11-FGF14) are modulators of voltage-gated sodium channels and the Pitt lab has recently characterized the role of FGF13 in modulating voltage-gated sodium channels in the heart. Preliminary data show that FHFs, particularly FGF13 can affect Cav1.2, the L-type Ca2+ channel (LTCC) in the heart, as well, which suggests an important role for FHFs in excitation-contraction coupling and arrhythmogenesis. This study will build upon these observations with the following two aims: Aim 1: Define the mechanisms by which FGF13 regulates CaV1.2. The hypothesis to be tested is: FGF13 regulates the cardiac calcium channel, CaV1.2, through a direct interaction. Aim 2: Determine the role of endogenous FGF13 in regulating the CaV1.2 in ventricular myocytes. The hypothesis to be tested is: FGF13 modulates CaV1.2 currents and localization in cardiac myocytes, thereby affecting the cardiac action potential and excitation-contraction coupling. Methods: Aim 1 will employ a heterologous expression system to define the roles of various FGF13 isoforms in modulating Cav1.2 curent via whole-cell patch clamp. These studies will be followed with biochemical analyses to define how FGF13 affects the CaV1.2 current, utilizing co-immunoprecipitation, surface biotinylation, and in vitro binding asays. Aim 2 wil utilize whole-cell patch clamp of mouse ventricular myocytes to define the effects on the Ca2+ current with FGF13 knocked down. The mechanism of action will be defined with immunocytochemical analysis for changes in Cav1.2 distribution after knockdown. The roles of individual FGF13 isoforms in affecting current and Cav1.2 targeting wil be defined using a knockdown of endogenous FGF13 combined with overexpression of a single tagged isoform. Objectives: The results of Aim 1 will define how specific isoforms of the prominent heart FHF, FGF13, affect LTCC function. The interactions between FGF13 and CaV1.2 subunits will be further determined and it will be shown whether FGF13 plays a role in targeting Cav1.2 to the cell surface. The results will inform Aim 2, in which the effects of endogenous FGF13 on the LTCC will be elucidated through knockdown studies and concomitant knockdown and overexpression of specific isoforms. This will demonstrate that FGF13 is a prominent player in the cardiac action potential through its modulation of the LTCC and define FGF13 dysfunction as a mediator of acquired and inherited arrhythmias.

描述（由申请人提供）： 背景：越来越多的心血管疾病（例如心律失常和某些心肌病）可归因于通道内突变导致的离子通道功能障碍。这些“通道病”有助于了解离子通道的扰动如何导致疾病。最近的工作表明，离子通道辅助和调节亚基是通道的有效调节剂，并且经常对多个离子电流产生影响。成纤维细胞生长因子同源因子（FHF、FGF11-FGF14）是电压门控钠通道的调节剂，皮特实验室最近描述了 FGF13 在调节心脏电压门控钠通道中的作用。初步数据表明，FHF，特别是 FGF13 也可以影响 Cav1.2，即心脏中的 L 型 Ca2+ 通道 (LTCC)，这表明 FHF 在兴奋-收缩耦合和心律失常发生中发挥重要作用。本研究将基于这些观察结果，实现以下两个目标： 目标 1：明确 FGF13 调节 CaV1.2 的机制。待检验的假设是：FGF13 通过直接相互作用调节心脏钙通道 CaV1.2。目标 2：确定内源性 FGF13 在调节心室肌细胞 CaV1.2 中的作用。待检验的假设是：FGF13调节CaV1.2电流和在心肌细胞中的定位，从而影响心脏动作电位和兴奋-收缩耦合。方法：目标 1 将采用异源表达系统来定义各种 FGF13 同工型在通过全细胞膜片钳调节 Cav1.2 电流中的作用。这些研究之后将进行生化分析，利用免疫共沉淀、表面生物素化和体外结合测定来确定 FGF13 如何影响 CaV1.2 电流。目标 2 将利用小鼠心室肌细胞的全细胞膜片钳来确定 FGF13 敲低对 Ca2+ 电流的影响。作用机制将通过免疫细胞化学分析敲除后 Cav1.2 分布的变化来确定。单个 FGF13 同工型在影响当前和 Cav1.2 靶向方面的作用将通过内源 FGF13 的敲低结合单个标记同工型的过表达来定义。目标：目标 1 的结果将确定重要心脏 FHF、FGF13 的特定亚型如何影响 LTCC 功能。 FGF13 和 CaV1.2 亚基之间的相互作用将进一步确定，并将显示 FGF13 是否在将 Cav1.2 靶向细胞表面方面发挥作用。结果将告知目标 2，其中 内源性 FGF13 对 LTCC 的影响将通过敲低研究以及伴随的敲低和特定亚型的过度表达来阐明。这将证明 FGF13 通过调节 LTCC 在心脏动作电位中发挥重要作用，并将 FGF13 功能障碍定义为获得性和遗传性心律失常的介质。