Truncation of the Carboxyl Terminus of the DihydropyridineReceptor β1a Subunit Promotes Ca2+ Dependent Excitation-Contraction Coupling in Skeletal Myotubes

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.