Loss-of-function β1a/β4 chimeras revealed the importance of the β1a C terminus in skeletal muscle DHPR–RyR1 coupling. (A) Block schemes of domain organization of putative loss-of-function β1a/β4 chimeras with systematic exchange of N terminus (N), SH3 domain (SH3), HOOK region (H), GK domain (GK), or C terminus (C) of β1a (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 (Qmax) values indistinguishable (P > 0.05) between relaxed myotubes expressing β1a (n = 16), β4 (n = 12), β1a/β4(N) (n = 21), β1a/β4(SH3) (n = 19), β1a/β4(H) (n = 24), β1a/β4(GK) (n = 15), or β1a/β4(C) (n = 13). Qmax values from untransfected relaxed myotubes were slightly above detection level (P < 0.001, n = 11). (Right) Representative Q recordings from relaxed myotubes expressing either β1a 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 β1a (n = 9), β1a/β4(SH3) (n = 17), β1a/β4(GK) (n = 11), β1a/β4(N) (n = 14), or β1a/β4(H) (n = 16). By contrast, ΔF/F0 values were significantly lower (P < 0.001) for chimera β1a/β4(C) (n = 12) and similar (P > 0.05) to those of β4 (n = 13). Exemplar Ca2+ transient recordings from relaxed myotubes expressing β1a/β4(SH3) (C, Right) or β1a/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.
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