Intermolecular Interactions of NaV1.5 and Kir2.1 In Ion Channel Diseases

描述（由申请人提供）：该提案重点关注内向整流钾通道蛋白Kir2.1与主要心脏钠通道NaV1.5亚基之间分子相互作用的机制和电生理学后果。我们的初步结果强烈表明，NaV1.5和Kir2.1通过大分子复合物内各自的PDZ结合结构域来调节彼此的表面表达和功能，以控制心脏兴奋性。这种动态互惠是翻译后的，至少部分涉及两种通道蛋白在共同膜隔室的运输和靶向的相互调节以及内化。我们专注于已知破坏NaV1.5（Brugada综合征，BS）或Kir2.1（Andersen-Tawil综合征，ATS）的遗传突变。我们推测，破坏一种通道蛋白类型（例如，NaV1.5）也会影响其他类型（例如，Kir2.1通过干扰它们相互作用的共同大分子复合物，从而有助于电生理表型和致突变潜力。我们将测试以下三个主要假设：1）NaV1.5和Kir2.1蛋白通道经历PDZ结构域介导的与控制其膜稳定性的大分子复合物中的共同伴侣的相互作用; 2）大分子复合物的形成影响Kir2.1和NaV1.5的顺行和/或逆行运输;和3）人诱导多能干细胞衍生的心肌细胞表达影响蛋白运输的ATS或BS突变的hiPSC-CM将显示降低的兴奋性，反映两种通道蛋白的表达改变，这应该对遗传性心律失常表型有很大贡献。我们建议将联合收割机蛋白质组学（例如，蛋白质纯化，酵母双杂交测定和相互作用结构域作图）和遗传（例如，诱变和沉默）工具、共聚焦显微镜、活细胞成像、光漂白后的荧光恢复、膜片钳、光学作图、异源系统中的基因转移和沉默以及单个高度成熟的心室样hiPSC-CM和hiPSC-CM单层。我们还将进行计算机模拟，以使虚拟解剖和解释的电生理学和mammogenic的变化所造成的NaV1.5-Kir2.1相互作用及其突变体的大分子复合物。

DESCRIPTION (provided by applicant): This proposal focuses on the mechanisms and electrophysiological consequences of the molecular interactions between the inward rectifier potassium channel protein Kir2.1 and the �ubunit of the major cardiac sodium channel NaV1.5. Our preliminary results strongly suggest that NaV1.5 and Kir2.1 modulate each other's surface expression and function through their respective PDZ binding domains within a macromolecular complex to control cardiac excitability. Such a dynamic reciprocity is post-translational, involving, at least in part, mutual regulation of trafficking and targeting of both channel proteins at common membrane compartments, as well as internalization. We focus on inheritable mutations that are known to disrupt trafficking of NaV1.5 (Brugada Syndrome, BS) or Kir2.1 (Andersen-Tawil Syndrome, ATS). We surmise that a mutation that disrupts the expression of one channel protein type (e.g., NaV1.5) will also affect the other type (e.g., Kir2.1 by disturbing the common macromolecular complex through which they interact, thus contributing to both the electrophysiological phenotype and arrhythmogenic potential. We will test the following three major hypotheses: 1) NaV1.5 and Kir2.1 protein channels undergo PDZ-domain mediated interactions with common partners in a macromolecular complex that controls their membrane stability; 2) macromolecular complex formation affects anterograde and/or retrograde trafficking of Kir2.1 and NaV1.5; and 3) human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CMs) expressing either ATS or BS mutations that affect protein trafficking will show reduced excitability reflecting altered expression of both channel proteins, which should contribute strongly to the inherited arrhythmia phenotype. We propose to combine proteomics (e.g., protein purification, yeast two-hybrid assay and interaction domain mapping) and genetic (e.g., mutagenesis and silencing) tools, confocal microscopy, live cell imaging, fluorescence recovery after photobleaching, patch clamping, optical mapping, gene transfer and silencing in heterologous systems, and in single, highly mature ventricular-like hiPSC-CMs and hiPSC-CM monolayers. We will also conduct computer simulations to enable the virtual dissection and interpretation of the electrophysiological and arrhythmogenic changes resulting from NaV1.5-Kir2.1 interactions and their mutants in a macromolecular complex.