Physical interaction of junctophilin and the CaV1.1 C terminus is crucial for skeletal muscle contraction
Physical interaction of junctophilin and the CaV1.1 C terminus is crucial for skeletal muscle contraction
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DOI：
10.1073/pnas.1716649115
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发表时间：
2018-04-24
影响因子：
11.1
通讯作者：
Yamada, Mitsuhiko
中科院分区：
文献类型：
--
作者：
Nakada, Tsutomu;Kashihara, Toshihide;Yamada, Mitsuhiko
肌膜连接膜钙通道(LTCCs)与肌浆网兰尼定受体(RyRs)的紧密结合是骨骼肌兴奋收缩偶联的关键。然而，JM靶向LTCC的分子机制尚不清楚。连接蛋白1(JP1)和JP2通过连接肌膜和SR膜来稳定JM。在这里，我们研究了JP在LTCC定位和功能中的作用。在培养的肌管中敲除jp1或jp2可抑制JM处的LTCC聚集，并在不破坏JM结构的情况下抑制诱发的钙瞬变。免疫共沉淀和GST下拉实验表明，JPs与CaV1.1近端C末端的12-AA残基发生物理相互作用。缺失跨膜区的Jp1突变体(Jp1 Delta CT)与肌膜/T管膜相互作用，但不与SR膜相互作用。该突变体在体内成年小鼠肌肉中的表达对内源JPs产生显性-负性效应，在不破坏JM形态的情况下削弱三联体上的LTCC-RyR偶联，并显著减少钙瞬变而不影响肌浆网钙含量。此外，与对照组相比，Jp1 Delta CT表达的肌收缩力显著降低。综上所述，太平绅士通过物理互动将LTCC招募到JM，并确保骨骼肌中三联体的强大ECC。
Close physical association of Ca(V)1.1 L-type calcium channels (LTCCs) at the sarcolemmal junctional membrane (JM) with ryanodine receptors (RyRs) of the sarcoplasmic reticulum (SR) is crucial for excitation-contraction coupling (ECC) in skeletal muscle. However, the molecular mechanism underlying the JM targeting of LTCCs is un-explored. Junctophilin 1 (JP1) and JP2 stabilize the JM by bridging the sarcolemmal and SR membranes. Here, we examined the roles of JPs in localization and function of LTCCs. Knockdown of JP1 or JP2 in cultured myotubes inhibited LTCC clustering at the JM and suppressed evoked Ca2+ transients without disrupting JM structure. Coimmunoprecipitation and GST pull-down assays demonstrated that JPs physically interacted with 12-aa residues in the proximal C terminus of the CaV1.1. A JP1 mutant lacking the C terminus including the transmembrane domain (JP1 Delta CT) interacted with the sarcolemmal/T-tubule membrane but not the SR membrane. Expression of this mutant in adult mouse muscles in vivo exerted a dominant-negative effect on endogenous JPs, impairing LTCC-RyR coupling at triads without disrupting JM morphology, and substantially reducing Ca2+ transients without affecting SR Ca2+ content. Moreover, the contractile force of the JP1 Delta CT-expressedmuscle was dramatically reduced compared with the control. Taken together, JPs recruit LTCCs to the JM through physical interaction and ensure robust ECC at triads in skeletal muscle.