Mitsugumin 53 regulates extracellular Ca2+ entry and intracellular Ca2+ release via Orai1 and RyR1 in skeletal muscle

Abstract

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.

Figure 1. Interaction between MG53 and Orai1.

(a) HEK293 cells were co-transfected with constructs of HA-MG53 along with Orai1-myc, STIM1-myc, annexin V-myc, or annexin I-myc. 24 h after the transfection, cell lysates were immunoprecipitated with anti-HA antibody, and were immunobloted with anti-HA or anti-myc antibodies. Three independent experiments were conducted. Lysate refers to a simple immunoblot, and IP to immunoprecipitation. The asterisks indicate the artifact band for the heavy or light chains of anti-HA antibody (about 50 or 25 kDa) due to the cross-reactivity of the anti-myc antibody to the chains. Quantitative analysis for the band intensity of co-immunoprecipitated Orai1 with MG53 is presented in the bar graphs in the right-hand panel. Annexin V was used as a negative control, and the normalized value to MG53 was normalized to those of Orai1. *Significant difference compared with Annexin V (p < 0.05). (b) The triad vesicle sample obtained from rabbit skeletal muscle (30 μg of total proteins) was subjected to co-immunoprecipitation assay with anti-Orai1 (upper panel) or anti-MG53 antibody (lower panel), and was immunobloted with anti-Orai1, anti STIM1, or anti-MG53 antibodies. Input (triad) indicates the simple immunoblot of the triad vesicle sample (5 μg of total proteins). Without Ab indicates a reaction without anti-Orai1 or anti-MG53 antibody. Three independent experiments per each were conducted. The reaction without anti-Orai1 or anti-MG53 antibody was used as a negative control. The asterisks in blots indicate the artifact band for the light chains of anti-Orai1 or anti-MG53 antibody (about 25 kDa) due to the cross-reactivity of the anti-Orai1 antibody to the chains. Full-length blots are presented in Supplementary Figs 1 and 2. Degree of co-immunoprecipitated MG53 to total MG53 (upper panel) or degree of co-immunoprecipitated Orai1 or STIM1 to the corresponding total protein (lower panel) is presented in the bar graphs in the right-hand panel. *Significant difference compared with Without anti-Orai1 Ab or Without anti-MG53 Ab (p < 0.05). (c) HEK293 cells (left-hand panel) or C2C12 mouse skeletal myotubes (right-hand panel) co-transfected with GFP-MG53 and Orai1-myc constructs were subjected to immunocytochemistry with anti-GFP and anti-myc antibodies. The data are representative images from three independent experiments (ten images for each group).

Figure 2. Binding of the PRY-SPRY region of MG53 to Orai1.

(a) Schematic diagrams of full-length MG53 and MG53 domains. Numbers indicate the sequence of amino acids. TRIM, tripartite motif; CC, coiled-coil; four black dots, a zinc-binding leucine zipper motif; a triangle, a cysteine between the two CC domains; PRY, a domain associated with SPRY domains; SPRY, a sequence repeat in the dula-specificity kinase splA and ryanodine receptor. (b) Various GST-MG53 domains expressed in E. coli (indicated by white asterisks) were separated on a SDS–PAGE gel (10%) and were stained with Coomassie Brilliant Blue staining. (c) The bound proteins obtained from the binding assays of GST-MG53 domains with the triad vesicle sample from rabbit skeletal muscle were separated on SDS–PAGE gels (12%) and were subjected to immunoblot assays with anti-Orai1 or anti-GST-antibodies. GST was used as a negative control. Three independent experiments were conducted. Full-length blots are presented in Supplementary Fig. 3. The relative amount of Orai1 to the corresponding amount of MG53 domain is presented in bar graphs in the right-hand panel. The value for the relative amount of Orai1 to full-length MG53 (GST-MG53) was regarded as 1, and others were normalized by this value. *Significant difference compared with GST control (p < 0.05).

Figure 3. Enhanced SOCE by MG53.

C2C12 skeletal myotubes over-expressing MG53 were loaded with 5 μM fura-2-AM, and the SR Ca²⁺ storage was depleted with the addition of TG (2.5 μM) in the absence of extracellular Ca²⁺. The addition of extracellular Mn²⁺ (0.5 mM) led to the quenching of intracellular fura-2 fluorescence. A representative trace for each group is shown. The rate of Mn²⁺ influx was presented as bar graphs in the right-hand panel. The rate of Mn²⁺ influx was determined from the variable (i.e., slope) of a linear equation obtained from a linear fitting of the traces from the initial 10 seconds. A steeper slope indicates a more active SOCE. The results are presented as the mean ± S.E. of six (for Vector) or eight independent experiments (for MG53). Triton was used as an indicator that cell membranes are intact. *Significant difference compared with Vector controls (p < 0.05).

Figure 4. Enhanced SOCE by full-length MG53 or PRY-SPRY.

(a) Expression of full-length MG53 or PRY-SPRY in mouse primary skeletal myotubes was visualized by immunocytochemistry. Vector indicates GFP alone. Bar represents 50 μm. (b) The SR Ca²⁺ storage of myotubes over-expressing full-length MG53 or PRY-SPRY was depleted by the treatment of TG (2.5 μM) in the absence of extracellular Ca²⁺. Extracellular Ca²⁺ (2 mM) was applied to the myotubes to induce SOCE. A representative trace for each group is shown, and the results are summarized as bar graphs in the right-hand panel. *Significant difference compared with Vector controls (P < 0.05). The values are presented as the mean ± S.E. for the number of myotubes shown in the parentheses of Table 1. (c) Three different time intervals between the TG treatment (Ca²⁺ depletion from SR) and extracellular Ca²⁺ application (SOCE) were applied to the myotubes. The values were normalized to the mean value of those from vector controls at Ca²⁺-free for 6 min. The results are presented as the mean ± S.E. for the number of myotubes shown in the parenthesis in Table 1. *Significant difference versus corresponding Vector control (p < 0.05).

Figure 5. Increased expression levels of TRPC3 and TRPC4 by full-length MG53 or PRY-SPRY, and the binding of TRPC3 to MG53.

(a) Lysate from myotubes over-expressing full-length MG53 or PRY-SPRY was subjected to an immunoblot assay with one of the antibodies against six proteins that are known to be expressed and/or to mediate extracellular Ca²⁺ entry into skeletal muscle. α-Actin was used as a loading control. Three independent experiments per each protein were conducted. (b) The expression levels of TRPC3 and TRPC4 (indicated by asterisks in a) were presented as bar graphs. Bar graphs were presented as the mean ± S.E. for three independent experiments. *Significant difference versus corresponding Vector control (p < 0.05). Full-length blots are presented in Supplementary Figs 4 to 10. (c) The triad vesicle sample obtained from rabbit skeletal muscle (30 μg of total proteins) was subjected to a co-immunoprecipitation assay with anti-MG53 antibody, and the immunoprecipitant was subjected to immunoblot analysis with various antibodies. Input indicates the simple immunoblot of the triad vesicle sample (5 μg of total proteins). “Without Ab” indicates a reaction without anti-MG53 antibody. TRPC3 was co-immunoprecipitated with MG53 (indicated by an asterisk). Three independent experiments were conducted. IB or IP means immunoblot or immunoprecipitation. CaM1 refers to calmodulin1. Full-length blots are presented in Supplementary Fig. 11.

Figure 6. The increased resting cytosolic Ca²⁺ levels and the reduced response to KCl or caffeine by full-length MG53 or PRY-SPRY.

(a) The resting cytosolic Ca²⁺ levels in the myotubes over-expressing full-length MG53 or PRY-SPRY were measured. (b) To measure the releasable Ca²⁺ from the SR to the cytosol, TG (2.5 μM) was applied to the myotubes in the absence of extracellular Ca²⁺. The results are presented as the mean ± S.E. for the number of myotubes shown in the parenthesis in Table 1. KCl that is a membrane depolarizer and induces skeletal muscle contraction (c), or caffeine that is a specific and direct RyR1agonist (d), was applied to the myotubes over-expressing full-length MG53 or PRY-SPRY. A representative trace for each group is shown, and bar graphs of the peak amplitude normalized to the mean value of those from the vector controls are shown in the right-hand panels. The results are presented as the mean ± S.E. for the number of myotubes shown in the parenthesis in Table 1. *Significant difference versus Vector control (p < 0.05).

Figure 7. Increased expression level of CaM1 by full-length MG53 or PRY-SPRY.

(a) Lysate from the myotubes over-expressing full-length MG53 or PRY-SPRY was subjected to an immunoblot assay with one of the antibodies against nine proteins that are known to mediate skeletal EC coupling and/or the handling of Ca²⁺. α-Actin was used as a loading control. Three independent experiments per each protein were conducted. JP, junctophilin; CSQ, calsequestin. (b) Among them, the expression level of CaM1 (indicated by asterisks in a) is presented as bar graphs. Bar graphs are presented as the mean ± S.E. for three-independent experiments. *Significant difference versus Vector control (p < 0.05). Full-length blots are presented in Supplementary Figs 12 to 21.