We investigated the contribution of the carboxyl terminus region of the β1a subunit of the skeletal dihydropyridine receptor (DHPR) to the mechanism of excitation-contraction (EC) coupling, cDNA-transfected β1 KO myotubes were voltage clamped, and Ca2+ transients were analyzed by confocal fluo-4 fluorescence. A chimera with an amino terminus half of β2a and a carboxyl terminus half of β1a (β2a 1-287 / β1a 325-524) recapitulates skeletal-type EC coupling quantitatively and was used to generate truncated variants lacking 7 to 60 residues from the β1a-specific carboxyl terminus (Δ7, Δ21, Δ29, Δ35, and Δ60). Ca2+ transients recovered by the control chimera have a sigmoidal Ca2+ fluorescence (ΔF/F) versus voltage curve with saturation at potentials more positive than +30 mV, independent of external Ca2+ and stimulus duration. In contrast, the amplitude of Ca2+ transients expressed by the truncated variants varied with the duration of the pulse, and for Δ29, Δ35, and Δ60, also varied with external Ca2+ concentration. For Δ7 and Δ21, a 50-ms depolarization produced a sigmoidal ΔF/F versus voltage curve with a lower than control maximum fluorescence. Moreover, for Δ29, Δ35, and Δ60, a 200-ms depolarization increased the maximum fluorescence and changed the shape of the ΔF/F versus voltage curve, from sigmoidal to bell-shaped, with a maximum at ∼+30 mV. The change in voltage dependence, together with the external Ca2+ dependence and additional controls with ryanodine, indicated a loss of skeletal-type EC coupling and the emergence of an EC coupling component triggered by the Ca2+ current. Analyses of d(ΔF/F)/dt showed that the rate of cytosolic Ca2+ increase during the Ca2+ transient was fivefold faster for the control chimera than for the severely truncated variants (Δ29, Δ35, and Δ60) and was consistent with the kinetics of the DHPR Ca2+ current. In summary, absence of the β1a-specific carboxyl terminus (last 29 to 60 residues of the control chimera) results in a loss of the fast component of the Ca2+ transient, bending of the ΔF/F versus voltage curve, and emergence of EC coupling triggered by the Ca2+ current. The studies underscore the essential role of the carboxyl terminus region of the DHPR β1a subunit in fast voltage dependent EC coupling in skeletal myotubes.
Sheridan, D. C., Cheng, W., Ahern, C. A., Mortenson, L., Alsammarae, D., Vallejo, P., & Coronado, R. (2003). Truncation of the carboxyl terminus of the dihydropyridine receptor β1a subunit promotes Ca2+ dependent excitation-contraction coupling in skeletal myotubes. Biophysical Journal, 84(1), 220–237. https://doi.org/10.1016/S0006-3495(03)74844-9