STAC3 stably interacts through its C1 domain with CaV1.1 in skeletal muscle triads

Figures

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  Figure 1. STAC3 is a stable component of the DHPR complex in skeletal muscle myotubes. (A) In dysgenic (CaV1.1−/−) myotubes the RyR1 is distributed in discrete clusters in the triads, while STAC3-GFP and the endogenous β1a are diffusely distributed in the cytosol. (B) On co-expression with CaV1.1, STAC3-GFP is colocalized in clusters with RyR1 and the DHPR subunits CaV1.1 and β1a (lower panel; N = 4, n = 120). Color overlay: 4X of area marked with blue rectangle. Scale bars: 10 μm and 5 μm. (C) Clusters of STAC3-GFP/CaV1.1 in live dysgenic myotubes were photobleached (within circles) and imaged up to 6 minutes. Representative high magnification images and the corresponding normalized FRAP recording (below) show little fluorescence recovery of STAC3-GFP in the bleached clusters (a–d, time points of example images; scale bars: 10 μm and 1 μm). The value of the fitted curve at 75 s after bleaching was used to calculate the fractional fluorescence recovery (R75). Average recovery curves (lower panel) reveal similar low fluorescence recovery of STAC3-GFP (red) and β1a-GFP (blue) in the presence of CaV1.1 and comparable recovery rates 75 s after bleaching (R75) (mean ± s.e., N = 3, n = 13;…

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  Figure 2. The NAM mutation does not affect the stability of STAC3 in the DHPR complex. (A) STAC3-NAM-GFP colocalizes in clusters with CaV1.1, albeit at a reduced extent compared to STAC3-GFP (Fig. 1B) (26.9 ± 0.1%, N = 3, n = 80; P = 0.001). Color overlay: 4X of blue rectangle. Scale bars: 10 μm and 5 μm. (B) Fluorescence of STAC3-NAM-GFP co-expressed with CaV1.1 did not recover within 6 min after bleaching. The mean FRAP curve and the R75 of STAC3-NAM-GFP were similar to that of STAC3-GFP (mean ± s.e., N = 3, n = 17; Student’s t-test, P = 0.46), suggesting that the NAM mutation does not alter the stability of STAC3 within DHPR complex. Scale bars: 10 μm and 1 μm.

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  Figure 3. STAC3 colocalizes also with heterologous CaV1.2 in skeletal muscle triads. (A) When co-expressed with CaV1.2, STAC3 is colocalized in clusters with the RyR and the CaV subunits CaV1.2 and β1a in dysgenic myotubes. Color overlay: 4X magnification of blue rectangle. Scale bars: 10 μm and 5 μm. (B) FRAP analysis of STAC3-GFP co-expressed with CaV1.2 shows very little recovery of fluorescence, very similar to when STAC3 is co-expressed with CaV1.1. Average recovery curves reveal similar low fluorescence recovery of STAC3-GFP co-expressed with CaV1.2 (red) and with CaV1.1 (blue) and comparable recovery rates 75 s after bleaching (R75) (mean ± s.e., N = 4, n = 17, Student’s t-test, P = 0.47). Upper scale bar: 10 μm. Lower scale bar: 1 μm.

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  Figure 4. STAC1 and STAC2 display different degrees of colocalization with CaV1.1 and CaV1.2 in skeletal myotubes. (A) Domain structures of STAC1, STAC2, and STAC3. (B) STAC1 fails to show triad targeting in dysgenic myotubes expressing CaV1.1, but co-clusters with the channel in about half of the myotubes expressing CaV1.2 (51.8 ± 4.2%, N = 4, n = 120). Color overlay: 4X of blue rectangle. Scale bars: 10 μm and 5 μm. (C) Only STAC3 co-clusters with CaV1.1, while the three STAC proteins display different degrees of co-clustering with CaV1.2 (mean ± s.e.; N = 4, n = 120).

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  Figure 5. The C1 domain of STAC3 is necessary for the colocalization with L-type calcium channels. (A,B) Domain structure of the STAC2 and STAC3 isoforms and chimeras with STAC2 sequences in red and STAC3 sequences in blue. The bar graphs on the right show the percentage of transfected myotubes in which the expressed STAC isoform/chimera co-clustered with CaV1.2/β1a. The two C-terminal SH3 domains (A; N = 3, n = 90) or the N-terminal pE and C1 domains (B; N = 4, n = 120) of STAC3 were inserted in the corresponding STAC2 sequences individually and jointly. (C) STAC2 showed triad targeting in none of the myotubes expressing CaV1.2 but insertion of the C1 domain of STAC3 conferred to STAC2 triad targeting in about three fourths of the myotubes (for complete set of micrographs see Fig S3). Color overlay: 4X of blue rectangle. Scale bars: 10 μm and 5 μm.

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  Figure 6. Residue V104 of STAC3 is important for its association with L-type calcium channels in triads of dysgenic myotubes. (A) Amino acid sequences of the C1 domain of all three mouse STAC proteins: Yellow, residues conserved in all three paralogs; green, conserved between STAC1 and STAC3; blue, conserved between STAC1 and STAC2; purple, conserved between STAC2 and STAC3. Red dots indicate STAC3 residues mutated to the corresponding residues of STAC2 (here and in Fig. 7). Numbers indicate the amino acid position of STAC3. (B) Domain structure of the STAC isoforms and chimeras with STAC2 sequences in red and STAC3 sequences in blue. Bar graphs on the right give the percentage of transfected myotubes in which the expressed STAC isoform/chimera co-clustered with CaV1.2/β1a. Entire or partial substitution (indicated A and B in panel A) of the C1 domain of STAC3 by the corresponding sequences of STAC2 caused a total failure to colocalize with CaV1.2. The isolated C1 domain of STAC3 was also not able to colocalize with CaV1.2. (C) Single amino acid residues of STAC3 were systematically mutated to the corresponding residues of STAC2. Of these only STAC3-V104L-GFP resulted in a significantly…

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  Figure 7. A binding pocket of STAC3 containing residues V104 and Y133 is critical for its association with L-type calcium channels in triads of dysgenic myotubes. (A) Homology structure model of the C1 domain of STAC3. The enlargement reveals the side chains of V104 and Y133 protruding into a pocket of the C1 domain. (B) V104 and Y133 were mutated individually and together to the corresponding residues of STAC2. STAC3-V104L and STAC3-Y133E showed reduced co-clustering with CaV1.2 (46.7 ± 4.0% and 63.3 ± 3.3%, respectively), while the combined mutant STAC3-V104L/Y133E showed a further co-clustering reduction (22.2 ± 4.8%) compared to STAC3 (97.8 ± 2.2%; N = 3, n = 90). Anova F(3,8) = 71.4; P < 0.001 (P in the figures are for post-hoc analysis; ***P < 0.001; **P = 0.001). (C) FRAP analysis of STAC3-V104/Y133E-GFP co-expressed with CaV1.2 shows substantial recovery of fluorescence. Average recovery curves reveal an approximately threefold higher recovery rate of STAC3-V104/Y133E-GFP compared to that of STAC3-GFP (mean ± s.e., N = 3, n = 18, Student’s t-test, P < 0.001). Upper scale bar: 10 μm. Lower scale bar: 1 μm. (D,E) When co-expressed with CaV1.1, the double STAC3-V104L/Y133E mutant…