Fig. 2.

Fig. 2.

Loss-of-function β_1a/β₄ chimeras revealed the importance of the β_1a C terminus in skeletal muscle DHPR–RyR1 coupling. (A) Block schemes of domain organization of putative loss-of-function β_1a/β₄ chimeras with systematic exchange of N terminus (N), SH3 domain (SH3), HOOK region (H), GK domain (GK), or C terminus (C) of β_1a (blue) by β₄ sequences (orange). Homologous SH3 and GK domains are represented by hatched boxes. (B, Left) Analyses of voltage dependence of integrated outward gating currents normalized to cell capacitance exhibited maximum charge movement (Q_max) values indistinguishable (P > 0.05) between relaxed myotubes expressing β_1a (n = 16), β₄ (n = 12), β_1a/β₄(N) (n = 21), β_1a/β₄(SH3) (n = 19), β_1a/β₄(H) (n = 24), β_1a/β₄(GK) (n = 15), or β_1a/β₄(C) (n = 13). Q_max values from untransfected relaxed myotubes were slightly above detection level (P < 0.001, n = 11). (Right) Representative Q recordings from relaxed myotubes expressing either β_1a or β₄. (Scale bars, 5 ms [horizontal], 3 pA/pF [vertical].) (C and D) Cytoplasmic Ca²⁺ transient restoration was comparable (P > 0.05) between relaxed myotubes expressing β_1a (n = 9), β_1a/β₄(SH3) (n = 17), β_1a/β₄(GK) (n = 11), β_1a/β₄(N) (n = 14), or β_1a/β₄(H) (n = 16). By contrast, ΔF/F₀ values were significantly lower (P < 0.001) for chimera β_1a/β₄(C) (n = 12) and similar (P > 0.05) to those of β₄ (n = 13). Exemplar Ca²⁺ transient recordings from relaxed myotubes expressing β_1a/β₄(SH3) (C, Right) or β_1a/β₄(C) (D, Right). (Scale bars, 50 ms [horizontal], ΔF/F₀ = 1 [vertical].) Error bars indicate SEM. P determined by unpaired Student’s t test, ***P < 0.001.