BIN1 Localizes the L-Type Calcium Channel to Cardiac T-Tubules
BIN1 Localizes the L-Type Calcium Channel to Cardiac T-Tubules
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DOI：
10.1371/journal.pbio.1000312
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发表时间：
2010-02-01
期刊：
影响因子：
9.8
通讯作者：
Shaw, Robin M.
中科院分区：
文献类型：
--
作者：
Hong, Ting-Ting;Smyth, James W.;Shaw, Robin M.
BAR结构域蛋白超家族参与膜内陷和内吞作用，但其在组织膜蛋白方面的作用尚未被探究。特别是，膜支架蛋白BIN1在骨骼肌细胞中具有启动T管发生的功能。在小鼠中持续敲低BIN1会导致围产期致死，这与诱发的扩张型肥厚性心肌病有关。然而，BIN1在心肌细胞中的功能作用尚不清楚。心脏T管的一个重要功能是使L型钙通道（Cav1.2）与位于肌浆网的兰尼碱受体紧密相邻，从而启动细胞内钙瞬变。有效的兴奋 - 收缩（EC）偶联和正常的心脏收缩力依赖于Cav1.2定位到T管。我们假设BIN1不仅存在于心脏T管，而且还将Cav1.2定位到这些膜结构。我们报道BIN1定位到心脏T管，并在那里与Cav1.2聚集。研究涉及使用互补免疫细胞化学、双免疫金标记电子显微镜以及免疫共沉淀技术对新鲜获取的人和小鼠成年心肌细胞进行研究。此外，我们在心肌细胞和细胞系中使用表面生物素化以及活细胞共聚焦和全内反射荧光显微镜成像来探究Cav1.2向BIN1结构的运输。我们通过视觉和定量分析发现，动态微管被束缚到由BIN1支撑的膜上，从而使Cav1.2从微管靶向运输到相关膜。由于Cav1.2向BIN1的运输在简化的非肌细胞系中也会发生，我们发现其他肌细胞特异性结构并非必需，并且基于微管的Cav1.2运输与其BIN1支架之间存在内在联系。在分化的小鼠心肌细胞中，敲低BIN1会减少表面Cav1.2并延迟钙瞬变的发展，这表明Cav1.2靶向BIN1对心脏钙信号传导在功能上很重要。我们已经确定，与膜相关的BIN1不仅诱导膜弯曲，还能指导微管运输的膜蛋白的特定顺向运输。此外，这种模式提供了一种依赖微管和BIN1的Cav1.2向T管运输的机制。这种新的Cav1.2运输途径应作为EC偶联的一个重要调节方面，影响哺乳动物心脏的心脏收缩力。
The BAR domain protein superfamily is involved in membrane invagination and endocytosis, but its role in organizing membrane proteins has not been explored. In particular, the membrane scaffolding protein BIN1 functions to initiate T-tubule genesis in skeletal muscle cells. Constitutive knockdown of BIN1 in mice is perinatal lethal, which is associated with an induced dilated hypertrophic cardiomyopathy. However, the functional role of BIN1 in cardiomyocytes is not known. An important function of cardiac T-tubules is to allow L-type calcium channels (Cav1.2) to be in close proximity to sarcoplasmic reticulum-based ryanodine receptors to initiate the intracellular calcium transient. Efficient excitation-contraction (EC) coupling and normal cardiac contractility depend upon Cav1.2 localization to T-tubules. We hypothesized that BIN1 not only exists at cardiac T-tubules, but it also localizes Cav1.2 to these membrane structures. We report that BIN1 localizes to cardiac T-tubules and clusters there with Cav1.2. Studies involve freshly acquired human and mouse adult cardiomyocytes using complementary immunocytochemistry, electron microscopy with dual immunogold labeling,and co-immunoprecipitation. Furthermore, we use surface biotinylation and live cell confocal and total internal fluorescence microscopy imaging in cardiomyocytes and cell lines to explore delivery of Cav1.2 to BIN1 structures. We find visually and quantitatively that dynamic microtubules are tethered to membrane scaffolded by BIN1, allowing targeted delivery of Cav1.2 from the microtubules to the associated membrane. Since Cav1.2 delivery to BIN1 occurs in reductionist non-myocyte cell lines, we find that other myocyte-specific structures are not essential and there is an intrinsic relationship between microtubule-based Cav1.2 delivery and its BIN1 scaffold. In differentiated mouse cardiomyocytes, knockdown of BIN1 reduces surface Cav1.2 and delays development of the calcium transient, indicating that Cav1.2 targeting to BIN1 is functionally important to cardiac calcium signaling. We have identified that membrane-associated BIN1 not only induces membrane curvature but can direct specific antegrade delivery of microtubule-transported membrane proteins. Furthermore, this paradigm provides a microtubule and BIN1-dependent mechanism of Cav1.2 delivery to T-tubules. This novel Cav1.2 trafficking pathway should serve as an important regulatory aspect of EC coupling, affecting cardiac contractility in mammalian hearts.