Figure 3.

Chimera GFP-SkLMS₄₅ restores skeletal-type EC coupling on expression in dysgenic myotubes. Action-potential-induced Ca²⁺ transients recorded from dysgenic myotubes expressing DHPR constructs, loaded with the fluorescent Ca²⁺ indicator Fluo-4 AM. Tick marks on the horizontal axes indicate 2 s. The skeletal GFP-α_1S (A) responded to 1-ms stimuli with Ca²⁺ transients that persisted after blocking currents with 0.5 mM Cd²⁺ and 0.1 mM La³⁺ (solid bar), whereas the cardiac GFP-α_1C Ca²⁺ transients (B) were blocked by the Cd²⁺/La³⁺ solution. Myotubes expressing GFP-SkLM (C) failed to restore action-potential-induced Ca²⁺ transients (n = 10 dishes) even though Ca²⁺ release could be induced with 6 mM caffeine (shaded bar). GFP-SkLMS₄₅ (D) fully restored action-potential-induced Ca²⁺ transients that were resistant to Cd²⁺/La³⁺ block of Ca²⁺ currents, indicating skeletal-type EC coupling. As for GFP-α_1S, the application of Cd²⁺/La³⁺ sometimes caused a modest reduction in the amplitude of the transient in cells expressing GFP-SkLMS₄₅. (E) Electrically evoked contractions (100 ms, 100 V) recorded in Cd²⁺/La³⁺ from dysgenic myotubes expressing either GFP-α_1S, GFP-SkLM, or GFP-SkLMS₄₅ indicated as percentage of myotubes stimulated.