STIM1 (stromal interaction molecule 1) mediates SOCE (store-operated Ca²⁺ entry) in skeletal muscle. However, the direct role(s) of STIM1 in skeletal muscle, such as Ca²⁺ release from the SR (sarcoplasmic reticulum) for muscle contraction, have not been identified. The times required for the maximal expression of endogenous STIM1 or Orai1, or for the appearance of puncta during the differentiation of mouse primary skeletal myoblasts to myotubes, were all different, and the formation of puncta was detected with no stimulus during differentiation, suggesting that, in skeletal muscle, the formation of puncta is a part of the differentiation. Wild-type STIM1 and two STIM1 mutants (Triple mutant, missing Ca²⁺-sensing residues but possessing the intact C-terminus; and E136X, missing the C-terminus) were overexpressed in the myotubes. The wild-type STIM1 increased SOCE, whereas neither mutant had an effect on SOCE. It was interesting that increases in the formation of puncta were observed in the Triple mutant as well as in wild-type STIM1, suggesting that SOCE-irrelevant puncta could exist in skeletal muscle. On the other hand, overexpression of wild-type or Triple mutant, but not E136X, attenuated Ca²⁺ releases from the SR in response to KCl [evoking ECC (excitation-contraction coupling) via activating DHPR (dihydropyridine receptor)] in a dominant-negative manner. The attenuation was removed by STIM1 knockdown, and STIM1 was co-immunoprecipitated with DHRP in a Ca²⁺-independent manner. These results suggest that STIM1 negatively regulates Ca²⁺ release from the SR through the direct interaction of the STIM1 C-terminus with DHPR, and that STIM1 is involved in both ECC and SOCE in skeletal muscle.

Background: Junctophilin-2 (JP2) contributes to the formation of junctional membrane complexes (JMC) in striated muscle. Results: Different from the S165F mutant of JP2, Y141H induces hypertrophy in skeletal myotubes involving abnormal JMC and altered Ca 2ϩ signaling due to the increased store-operated Ca 2ϩ entry (SOCE) via Orai1. Conclusion: JP2 is linked to muscle hypertrophy via various Ca 2ϩ signaling pathways. Significance: SOCE is a novel factor in understanding muscle hypertrophy.

Stromal interaction molecule 1 (STIM1) mediates Ca2+ movements from the extracellular space to the cytosol through a store-operated Ca2+ entry (SOCE) mechanism in various cells including skeletal muscle cells. In the present study, to reveal the unidentified functional role of the STIM1 C terminus from 449 to 671 amino acids in skeletal muscle, binding assays and quadrupole time-of-flight mass spectrometry were used to identify proteins binding in this region along with proteins that mediate skeletal muscle contraction and relaxation. STIM1 binds to sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1a (SERCA1a) via this region (called STIM1-SBR). The binding was confirmed in endogenous full-length STIM1 in rabbit skeletal muscle and mouse primary skeletal myotubes via co-immunoprecipitation assay and immunocytochemistry. STIM1 knockdown in mouse primary skeletal myotubes decreased Ca2+ uptake from the cytosol to the sarcoplasmic reticulum (SR) through SERCA1a only at micromolar cytosolic Ca2+ concentrations, suggesting that STIM1 could be required for the full activity of SERCA1a possibly during the relaxation of skeletal muscle. Various Ca2+ imaging experiments using myotubes expressing STIM1-SBR suggest that STIM1 is involved in intracellular Ca2+ distributions between the SR and the cytosol via regulating SERCA1a activity without affecting SOCE. Therefore, in skeletal muscle, STIM1 could play an important role in regulating Ca2+ movements between the SR and the cytosol.

Mitsugumin 53 (MG53) participates in the membrane repair of various cells, and skeletal muscle is the major tissue that expresses MG53. Except for the regulatory effects of MG53 on SERCA1a, the role(s) of MG53 in the unique functions of skeletal muscle such as muscle contraction have not been well examined. Here, a new MG53-interacting protein, Orai1, is identified in skeletal muscle. To examine the functional relevance of the MG53-Orai1 interaction, MG53 was over-expressed in mouse primary or C2C12 skeletal myotubes and the functional properties of the myotubes were examined using cell physiological and biochemical approaches. The PRY-SPRY region of MG53 binds to Orai1, and MG53 and Orai1 are co-localized in the plasma membrane of skeletal myotubes. MG53-Orai1 interaction enhances extracellular Ca2+ entry via a store-operated Ca2+ entry (SOCE) mechanism in skeletal myotubes. Interestingly, skeletal myotubes over-expressing MG53 or PRY-SPRY display a reduced intracellular Ca2+ release in response to K+-membrane depolarization or caffeine stimulation, suggesting a reduction in RyR1 channel activity. Expressions of TRPC3, TRPC4, and calmodulin 1 are increased in the myotubes, and MG53 directly binds to TRPC3, which suggests a possibility that TRPC3 also participates in the enhanced extracellular Ca2+ entry. Thus, MG53 could participate in regulating extracellular Ca2+ entry via Orai1 during SOCE and also intracellular Ca2+ release via RyR1 during skeletal muscle contraction.

Stromal interaction molecule 1 (STIM1) along with Orai1 mediates extracellular Ca2+ entry into the cytosol through a store-operated Ca2+ entry (SOCE) mechanism in various tissues including skeletal muscle. However, the role(s) of STIM2, a homolog of STIM1, in skeletal muscle has not been well addressed. The present study, first, was focused on searching for STIM2-binding proteins from among proteins mediating skeletal muscle functions. This study used a binding assay, quadrupole time-of-flight mass spectrometry, and co-immunoprecipitation assay with bona-fide STIM2- and SERCA1a-expressing rabbit skeletal muscle. The region for amino acids from 453 to 729 of STIM2 binds to sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1a (SERCA1a). Next, oxalate-supported 45Ca2+-uptake experiments and various single-myotube Ca2+ imaging experiments using STIM2-knockdown mouse primary skeletal myotubes have suggested that STIM2 attenuates SERCA1a activity during skeletal muscle contraction, which contributes to the intracellular Ca2+ distribution between the cytosol and the SR at rest. In addition, STIM2 regulates Ca2+ movement through RyR1 during skeletal muscle contraction as well as SOCE. Therefore, via regulation of SERCA1a activity, STIM2 regulates both intracellular Ca2+ distribution and Ca2+ movement in skeletal muscle, which makes it both similar to, yet different from, STIM1.