Figure 6
Expression of C-terminal fragment of α1S is essential for EC coupling. A) Voltage dependence of peak Ca2+ during the Ca2+ transient for dysgenic myotubes transfected with the indicated constructs. The sigmoidal curve is a Boltzmann fit with parameters ΔF/Fo max = 2.45; V1/2 = 15.4 mV; k = 9.3 mV for 5 cells coexpressing fs-α1SM701I + α1SΔ1–700. Absence of response is shown for 9 cells expressing fs-α1SM701I alone. B) ΔF/Fo max (mean ± SEM) obtained from a depolarization to +90 mV is shown for the indicated number of cells. NT denotes non-transfected dysgenic myotubes. Compared to wt-α1S (control), the statistical significance in unpaired t-Student test was p = 1.6 × 10-6 (non-transfected, NT); 0.014 (fs-α1S); 0.0002 (fs-α1SM701I); 0.0008 (α1SΔ1–700); 0.671 (fs-α1SM701I + α1SΔ1–700). Compared to fs-α1S, the statistical significance was p = 1 × 10-6 (non-transfected, NT); 0.014 (wt-α1S); 0.00019 (fs-α1SM701I); 0.0008 (α1SΔ1–700); 0.013 (fs-α1SM701I + α1SΔ1–700). C) Immunoblots using a polyclonal antibody directed to the II-III loop epitope Ala739-Ile752 [34] in cultures of dysgenic myotubes expressing fs-α1S and fs-α1SM701I. Indicated are 3 of 7 molecular weight markers run in the same gel. D) Confocal images of cells transfected with the CD8 cDNA plus T7-tagged fs-α1SM701I or T7-tagged fs-α1SM701I + untagged α1SΔ1–700. Cells were incubated with CD8 antibody beads, fixed, and stained with T7 primary/ fluorescein-conjugated secondary antibodies Pixel intensity was converted to a 16-level inverted gray scale with high-intensity pixels in black color. NT indicates a non-transfected myotube. CD8 antibody beads have a diameter of 4.5 microns. Calibration bar is 10 microns.