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

背景：越来越多的心血管疾病，如心律失常和某些心肌病可归因于离子通道突变导致的离子通道功能障碍。这些“通道病变”有助于理解离子通道的扰动如何导致疾病。最近的研究表明，离子通道辅助和调节亚基是通道的有效调节剂，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（FHFs, FGF11-FGF14）是电压门控钠通道的调节剂，皮特实验室最近表征了FGF13在调节心脏电压门控钠通道中的作用。初步数据显示，FHFs，特别是FGF13可以影响心脏中l型Ca2+通道（LTCC） Cav1.2，这表明FHFs在兴奋-收缩耦合和心律失常发生中起重要作用。本研究将以这些观察结果为基础，有以下两个目标：目标1：确定FGF13调节CaV1.2的机制。待检验的假设为：FGF13通过直接相互作用调节心脏钙通道CaV1.2。目的2：确定内源性FGF13在调节心室肌细胞CaV1.2中的作用。待验证的假设为：FGF13调节心肌细胞内CaV1.2电流和定位，从而影响心脏动作电位和兴奋-收缩耦合。方法：目的1将采用异种表达系统，通过全细胞膜片钳确定各种FGF13亚型在调节Cav1.2电流中的作用。这些研究之后将进行生化分析，以确定FGF13如何影响CaV1.2电流，利用共免疫沉淀，表面生物素化和体外结合试验。目的2将利用小鼠心室肌细胞的全细胞膜片钳来确定FGF13敲除对Ca2+电流的影响。作用机制将通过免疫细胞化学分析确定敲除后Cav1.2分布的变化。单个FGF13异构体在影响电流和Cav1.2靶向中的作用将通过敲低内源性FGF13结合过表达单个标记异构体来确定。目的：Aim 1的结果将确定突出的心脏FHF， FGF13的特定亚型如何影响LTCC功能。FGF13与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靶向到细胞表面中发挥作用。结果将通知Aim 2，其中

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.