De novo reconstitution reveals the proteins required for skeletal muscle voltage-induced Ca2+ release
De novo reconstitution reveals the proteins required for skeletal muscle voltage-induced Ca2+ release
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DOI：
10.1073/pnas.1716461115
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发表时间：
2017-12-26
影响因子：
11.1
通讯作者：
Beam, Kurt G.
中科院分区：
文献类型：
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作者：
Perni, Stefano;Lavorato, Manuela;Beam, Kurt G.
骨骼肌收缩是由肌浆网（SR）响应质膜（PM）兴奋释放Ca 2+触发的。在脊椎动物中，这取决于SR中RyR 1 Ca 2+孔的激活，在Ca（V）1.1的构象变化的控制下，位于PM中12 nm处。在过去的30年中，基因敲除揭示了Ca（V）1.1/RyR 1偶联需要额外的蛋白质，但留下了目前未测试的蛋白质也是必要的可能性。在这里，我们证明了在tsA 201细胞的构象偶联的重建表达的Ca（V）1.1，β 1a，Stac 3，RyR 1，和junctophilin 2。在肌肉中，去极化引起的Ca 2+瞬变独立于外部Ca 2+进入，并具有饱和依赖于电压的幅度。此外，冷冻断裂电子显微镜表明，这五种蛋白质足以在Ca（V）1.1和RyR 1之间建立物理联系。因此，这些蛋白质构成骨骼肌兴奋-收缩偶联的关键要素。
Skeletal muscle contraction is triggered by Ca2+ release from the sarcoplasmic reticulum (SR) in response to plasma membrane (PM) excitation. In vertebrates, this depends on activation of the RyR1 Ca2+ pore in the SR, under control of conformational changes of Ca(V)1.1, located similar to 12 nm away in the PM. Over the last similar to 30 y, gene knockouts have revealed that Ca(V)1.1/RyR1 coupling requires additional proteins, but leave open the possibility that currently untested proteins are also necessary. Here, we demonstrate the reconstitution of conformational coupling in tsA201 cells by expression of Ca(V)1.1, beta 1a, Stac3, RyR1, and junctophilin2. As in muscle, depolarization evokes Ca2+ transients independent of external Ca2+ entry and having amplitude with a saturating dependence on voltage. Moreover, freeze-fracture electron microscopy indicates that the five identified proteins are sufficient to establish physical links between Ca(V)1.1 and RyR1. Thus, these proteins constitute the key elements essential for excitation-contraction coupling in skeletal muscle.