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Department of Physiology, University of Wisconsin School of Medicine, Madison, Wisconsin 53706 USA
Correspondence: Address reprint requests to Roberto Coronado, Department of Physiology, University of Wisconsin, 1300 University Ave., Madison, WI 53706. Tel.: 608-263-7487; Fax: 608-265-5512; E-mail: coronado{at}physiology.wisc.edu.
We investigated the contribution of the carboxyl terminus region of the ß1a subunit of the skeletal dihydropyridine receptor (DHPR) to the mechanism of excitation-contraction (EC) coupling. cDNA-transfected ß1 KO myotubes were voltage clamped, and Ca2+ transients were analyzed by confocal fluo-4 fluorescence. A chimera with an amino terminus half of ß2a and a carboxyl terminus half of ß1a (ß2a 1-287/ß1a 325-524) recapitulates skeletal-type EC coupling quantitatively and was used to generate truncated variants lacking 7 to 60 residues from the ß1a-specific carboxyl terminus (
7, 21, 29, 35, and 60). Ca2+ transients recovered by the control chimera have a sigmoidal Ca2+ fluorescence (F/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 29, 35, and 60, also varied with external Ca2+ concentration. For 7 and 21, a 50-ms depolarization produced a sigmoidal F/F versus voltage curve with a lower than control maximum fluorescence. Moreover, for 29, 35, and 60, a 200-ms depolarization increased the maximum fluorescence and changed the shape of the F/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(F/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 (29, 35, and 60) and was consistent with the kinetics of the DHPR Ca2+ current. In summary, absence of the ß1a-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 F/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 ß1a subunit in fast voltage dependent EC coupling in skeletal myotubes.
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