Fig. 4.

Fig. 4.

The distal C terminus of β_1a is crucial for skeletal muscle EC coupling. (A) Block scheme of domain organization of gain-of-function chimera β₄/β_1a(C), where the the C terminus of β₄ (orange) was replaced by a corresponding β_1a sequence (blue). (B) Q_max values in relaxed myotubes expressing either chimera β₄/β_1a(C) (n = 18) or β_1a (n = 16) were comparable (P > 0.05). (C) Quantification of voltage dependence of cytoplasmic Ca²⁺ transients yielded significantly higher (P < 0.001) (ΔF/F₀)_max values for chimera β₄/β_1a(C) (n = 12) compared to β₄ (n = 13) but indistinguishable (P > 0.05) from that of β_1a (n = 9) expressing relaxed myotubes. (Right) Exemplar cytoplasmic Ca²⁺ transient recordings from relaxed myotubes expressing chimera β₄/β_1a(C). (Scale bars, 50 ms [horizontal], ΔF/F₀ = 1 [vertical].) (D) Amino acid sequence alignment of C termini of β_1a and β₄ depicting the homologous proximal C terminus (green box) and heterologous distal C terminus (blue box). Red bracket indicates the highly homologous sequence in the distal C terminus of β_1a revealed from sequence alignments of β_1a from several vertebrate species (fish to mammals) (SI Appendix, Fig. S2B). (E) Block scheme of domain organization of chimeras β₄/β_1a(prox.C) and β₄/β_1a(dist.C), where the proximal and distal C terminus of β₄ (orange) were exchanged by corresponding β_1a sequences (blue). (F) Q_max values were indistinguishable (P > 0.05) between relaxed myotubes expressing chimera β₄/β_1a(prox.C) (n = 11), β₄/β_1a(dist.C) (n = 19), or β_1a (n = 16). (G) Quantification of voltage dependence of cytoplasmic Ca²⁺ transients yielded (ΔF/F₀)_max values that were significantly lower (P < 0.001) for chimera β₄/β_1a(prox.C) (n = 15)- compared to β_1a (n = 9)-expressing relaxed myotubes. However, relaxed myotubes expressing chimera β₄/β_1a(dist.C) (n = 14) exhibited pronounced Ca²⁺ transients, equivalent (P > 0.05) to β_1a transfected myotubes (n = 13). (Right) Exemplar Ca²⁺ transient recordings from relaxed myotubes expressing chimera β₄/β_1a(dist.C) or β₄/β_1a(prox.C). (Scale bars, 50 ms [horizontal], ΔF/F₀ = 1 [vertical].) (H) Quantification of spontaneous or touch-evoked coiling of 27- to 30-hpf relaxed zebrafish injected with β_1a (n = 35), β₄ (n = 202), β₄/β_1a(C) (n = 79), and β₄/β_1a(dist.C) (n = 58) mRNA. Degree of motility was indistinguishable (P > 0.05) between relaxed zebrafish expressing β₄/β_1a(C) or β_1a. Relaxed zebrafish expressing β₄/β_1a(dist.C) displayed robust spontaneous coiling only slightly lower (P = 0.02) than β_1a. Conversely, β₄-injected relaxed zebrafish showed either no (n = 151) or very weak (n = 51) coiling following tactile stimulation and thus, highly significantly lower motility compared to (P < 0.001) β_1a-expressing relaxed zebrafish. Uninjected relaxed zebrafish displayed neither spontaneous nor tactile-induced motility (P < 0.001, n = 28). Error bars indicate SEM. P determined by unpaired Student’s t test, *P < 0.05; ***P < 0.001.