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Abstract：
In skeletal muscle, Ca(2+) release from sarcoplasmic reticulum (SR) following the action potential relies on the delicate architecture of the T-Tubule/SR junction. The T-Tubule/SR junction comprises two membrane systems: the integral proteins DHPR and RyR1 and the Ca(2+)-buffering apparatus within the SR lumen. The arrangement and interactions of the components have remained elusive due to technological limitations. Here, we determined whether electron tomography is effective fort the in situ determination of the relationships between RyR1 and DHPR, the SR membrane and other involved structures. First, we visually confirmed that RyR1 and DHPR are close neighbors that are mutually staggered with each other and directly interact with one of RyR1 subunits. Second, the Ca(2+) storage network within the SR lumen is directly correlated with RyR1. These results suggest that the excitation of the T-Tubule may induce Ca(2+) release through a direct interaction among DHPR, RyR1 and the Ca(2+) buffering apparatus. These results indicate that electron tomography has potential as an efficient method for the in situ characterization of the complex architecture and arrangement of two integral-integral-membrane proteins in the context of the surrounding phospholipid-bilayer and proteins.
摘要：
在骨骼肌中，动作电位后从肌浆网（SR）释放的Ca（2）依赖于T-Tubule/SR连接的精致结构。T-Tubule/SR连接包含两个膜系统：SR腔内的整合蛋白DHPR和RyR1以及Ca（2）缓冲装置。由于技术限制，组件的排列和相互作用仍然难以捉摸。这里,我们确定电子层析成像是否是有效的依据RyR1和DHPR之间的关系的原位确定，SR膜和其他涉及的结构。首先,我们在视觉上证实RyR1和DHPR是相互交错的近邻,并且直接与RyR1亚基之一相互作用.第二,SR管腔内的Ca(2+)存储网络与RyR1直接相关。这些结果表明，T-小管的激发可能通过DHPR之间的直接相互作用诱导Ca（2）释放，RyR1和Ca(2+)缓冲装置。这些结果表明，在周围的磷脂双层和蛋白质的背景下，电子层析成像具有作为原位表征两种完整-完整膜蛋白的复杂结构和排列的有效方法的潜力。
