Cardiovascular Research Institute, University of California San Francisco, San Francisco, California, USA.
PLoS Biol. 2010 Feb 16;8(2):e1000312. doi: 10.1371/journal.pbio.1000312.
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.
BAR 结构域蛋白超家族参与膜内陷和内吞作用，但它在膜蛋白组织中的作用尚未得到探索。特别是，膜支架蛋白 BIN1 可启动骨骼肌细胞中的 T 小管发生。BIN1 在小鼠中的组成性敲低是围产期致死的，这与诱导的扩张性肥厚性心肌病有关。然而，BIN1 在心肌细胞中的功能作用尚不清楚。心脏 T 小管的一个重要功能是使 L 型钙通道（Cav1.2）与基于肌浆网的ryanodine 受体接近，以启动细胞内钙瞬变。有效的兴奋-收缩（EC）偶联和正常的心脏收缩力依赖于 Cav1.2 向 T 小管的定位。我们假设 BIN1 不仅存在于心脏 T 小管上，而且还将 Cav1.2 定位到这些膜结构上。我们报告 BIN1 定位于心脏 T 小管并与 Cav1.2 聚集在那里。研究涉及使用互补免疫细胞化学、双免疫金标记的电子显微镜和共免疫沉淀法对新获得的人源和鼠源成年心肌细胞进行研究。此外，我们使用表面生物素化和活细胞共聚焦和全内荧光显微镜成像在心肌细胞和细胞系中探索 Cav1.2 向 BIN1 结构的递呈。我们通过视觉和定量发现，动态微管被固定在由 BIN1 支撑的膜上，允许 Cav1.2 从微管靶向递呈到相关膜上。由于 Cav1.2 递呈到 BIN1 发生在简化的非心肌细胞系中，我们发现其他心肌细胞特异性结构不是必需的，并且微管基 Cav1.2 递呈与其 BIN1 支架之间存在内在关系。在分化的小鼠心肌细胞中，BIN1 的敲低减少了表面 Cav1.2 的表达，并延迟了钙瞬变的发生，表明 Cav1.2 靶向 BIN1 对心脏钙信号转导具有功能重要性。我们已经确定，膜相关的 BIN1 不仅诱导膜弯曲，而且可以指导微管转运的膜蛋白的特定顺行递呈。此外，该范例提供了 Cav1.2 递呈到 T 小管的微管和 BIN1 依赖性机制。这种新的 Cav1.2 运输途径应该作为 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。
