抄録
Close physical association of CaV1.1 L-type calcium channels (LTCCs) at the sarcolemmal junctional membrane (JM) with ryanodine receptors of the sarcoplasmic reticulum (SR) is crucial for excitation-contraction coupling (ECC) in skeletal muscle. However, molecular mechanism underlying the JM-targeting of LTCCs is unexplored. Junctophilins (JPs) are a molecule known to stabilize the JM complex by bridging the sarcolemma and SR membranes. Here we examined whether JPs contribute to the JM-targeting and proper function of LTCC. We first suppressed JPs by siRNA in cultured myotubes. Suppression of JPs disturbed the JM-targeting of LTCC and a robust calcium transient in response to electrical stimulation. Co-immunoprecipitation and GST pull down assays demonstrated that JPs physically interacted with 12 amino acid residues in proximal C-terminus of the CaV1.1. We prepared and transfected a JP1 mutant lacking the C-terminal transmembrane domain (JP1ΔTM) into cultured myotubes. JP1ΔTM was not specifically clustered to JM but diffusely localized over the entire plasma membrane in the myotubes. Transient expression of JP1ΔTM inhibited the JM-targeting of CaV1.1, indicating a dominant negative effect of the mutant. To examine the in vivo effect of JP1, JP1ΔTM was transduced in the flexor digitorum profundus (FDB) or tibialis anterior (TA) muscle of living mice by using an adeno-associated virus (AAV)-mediated gene delivery system. Ca2+ imaging assay in the FDB fibers showed that expression of JP1ΔTM significant decreased the peak amplitude of calcium transients elicited by electrical stimulations. Significant change in SR Ca2+ content was not observed between control and JP1ΔTM -expressed FDB fibers. Immunocytochemical analysis revealed that JP1ΔTM was localized more strongly to the sarcolemmal than the T-tubule membrane. Interestingly, abundant CaV1.1 signals were observed in the sarcolemma of JP1ΔTM -expressing fibers but not control fibers. The proximity ligation assay revealed that the physical coupling between LTCC and RyR in the FDB fibers was significantly decreased by JP1ΔTM. Moreover, the specific force of the TA muscle was dramatically reduced compared to the control. From these results, we conclude that the physical interaction between JPs and the C-terminus of CaV1.1 is crucial for the excitation-contraction coupling of the skeletal muscle.
