Curran Jerry, Musa Hassan, Kline Crystal F, Makara Michael A, Little Sean C, Higgins John D, Hund Thomas J, Band Hamid, Mohler Peter J
From the Dorothy M. Davis Heart and Lung Research Institute, the Departments of Physiology and Cell Biology,
From the Dorothy M. Davis Heart and Lung Research Institute, the Departments of Physiology and Cell Biology.
J Biol Chem. 2015 May 8;290(19):12210-21. doi: 10.1074/jbc.M115.646893. Epub 2015 Mar 30.
Proper trafficking of membrane-bound ion channels and transporters is requisite for normal cardiac function. Endosome-based protein trafficking of membrane-bound ion channels and transporters in the heart is poorly understood, particularly in vivo. In fact, for select cardiac cell types such as atrial myocytes, virtually nothing is known regarding endosomal transport. We previously linked the C-terminal Eps15 homology domain-containing protein 3 (EHD3) with endosome-based protein trafficking in ventricular cardiomyocytes. Here we sought to define the roles and membrane protein targets for EHD3 in atria. We identify the voltage-gated T-type Ca(2+) channels (CaV3.1, CaV3.2) as substrates for EHD3-dependent trafficking in atria. Mice selectively lacking EHD3 in heart display reduced expression and targeting of both Cav3.1 and CaV3.2 in the atria. Furthermore, functional experiments identify a significant loss of T-type-mediated Ca(2+) current in EHD3-deficient atrial myocytes. Moreover, EHD3 associates with both CaV3.1 and CaV3.2 in co-immunoprecipitation experiments. T-type Ca(2+) channel function is critical for proper electrical conduction through the atria. Consistent with these roles, EHD3-deficient mice demonstrate heart rate variability, sinus pause, and atrioventricular conduction block. In summary, our findings identify CaV3.1 and CaV3.2 as substrates for EHD3-dependent protein trafficking in heart, provide in vivo data on endosome-based trafficking pathways in atria, and implicate EHD3 as a key player in the regulation of atrial myocyte excitability and cardiac conduction.
膜结合离子通道和转运蛋白的正确运输对于正常心脏功能是必需的。基于内体的心脏中膜结合离子通道和转运蛋白的蛋白质运输了解甚少，尤其是在体内。事实上，对于某些心脏细胞类型，如心房肌细胞，关于内体运输几乎一无所知。我们之前将含C末端Eps15同源结构域蛋白3（EHD3）与心室心肌细胞中基于内体的蛋白质运输联系起来。在这里，我们试图确定EHD3在心房中的作用和膜蛋白靶点。我们确定电压门控T型钙通道（CaV3.1、CaV3.2）是心房中EHD3依赖性运输的底物。心脏中选择性缺乏EHD3的小鼠心房中Cav3.1和CaV3.2的表达和靶向均降低。此外，功能实验表明EHD3缺陷型心房肌细胞中T型介导的钙电流显著丧失。此外，在共免疫沉淀实验中，EHD3与CaV3.1和CaV3.2均相关。T型钙通道功能对于心房的正常电传导至关重要。与这些作用一致，EHD3缺陷型小鼠表现出心率变异性、窦性停搏和房室传导阻滞。总之，我们的研究结果确定CaV3.1和CaV3.2是心脏中EHD3依赖性蛋白质运输的底物，提供了关于心房中基于内体的运输途径的体内数据，并表明EHD3是调节心房肌细胞兴奋性和心脏传导的关键因素。
