title: Truncation of the carboxyl terminus of the dihydropyridine receptor beta1a subunit promotes Ca2+ dependent excitation-contraction coupling in skeletal myotubes.
abstract: We investigated the contribution of the carboxyl terminus region of the beta1a subunit of the skeletal dihydropyridine receptor (DHPR) to the mechanism of excitation-contraction (EC) coupling. cDNA-transfected beta1 KO myotubes were voltage clamped, and Ca(2+) transients were analyzed by confocal fluo-4 fluorescence. A chimera with an amino terminus half of beta2a and a carboxyl terminus half of beta1a (beta2a 1-287/beta1a 325-524) recapitulates skeletal-type EC coupling quantitatively and was used to generate truncated variants lacking 7 to 60 residues from the beta1a-specific carboxyl terminus (Delta7, Delta21, Delta29, Delta35, and Delta60). Ca(2+) transients recovered by the control chimera have a sigmoidal Ca(2+) fluorescence (DeltaF/F) versus voltage curve with saturation at potentials more positive than +30 mV, independent of external Ca(2+) and stimulus duration. In contrast, the amplitude of Ca(2+) transients expressed by the truncated variants varied with the duration of the pulse, and for Delta29, Delta35, and Delta60, also varied with external Ca(2+) concentration. For Delta7 and Delta21, a 50-ms depolarization produced a sigmoidal DeltaF/F versus voltage curve with a lower than control maximum fluorescence. Moreover, for Delta29, Delta35, and Delta60, a 200-ms depolarization increased the maximum fluorescence and changed the shape of the DeltaF/F versus voltage curve, from sigmoidal to bell-shaped, with a maximum at approximately +30 mV. The change in voltage dependence, together with the external Ca(2+) 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 Ca(2+) current. Analyses of d(DeltaF/F)/dt showed that the rate of cytosolic Ca(2+) increase during the Ca(2+) transient was fivefold faster for the control chimera than for the severely truncated variants (Delta29, Delta35, and Delta60) and was consistent with the kinetics of the DHPR Ca(2+) current. In summary, absence of the beta1a-specific carboxyl terminus (last 29 to 60 residues of the control chimera) results in a loss of the fast component of the Ca(2+) transient, bending of the DeltaF/F versus voltage curve, and emergence of EC coupling triggered by the Ca(2+) current. The studies underscore the essential role of the carboxyl terminus region of the DHPR beta1a subunit in fast voltage dependent EC coupling in skeletal myotubes.
authors.affiliation: Department of Physiology, University of Wisconsin School of Medicine, Madison, 53706, USA.
authors.forename: David C
authors.initials: DC
authors.lastname: Sheridan
authors.forename: Weijun
authors.initials: W
authors.lastname: Cheng
authors.forename: Chris A
authors.initials: CA
authors.lastname: Ahern
authors.forename: Lindsay
authors.initials: L
authors.lastname: Mortenson
authors.forename: Dania
authors.initials: D
authors.lastname: Alsammarae
authors.forename: Paola
authors.initials: P
authors.lastname: Vallejo
authors.forename: Roberto
authors.initials: R
authors.lastname: Coronado
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D015220:Calcium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D002245:Carbon Dioxide / Q000737:chemistry / Q000502:physiology; D002478:Cells, Cultured; D003001:Cloning, Molecular; D006614:Hindlimb / Q000196:embryology / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009119:Muscle Contraction / Q000235:genetics / Q000502:physiology*; D018485:Muscle Fibers, Skeletal / Q000737:chemistry / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000502:physiology; D012680:Sensitivity and Specificity; D014170:Transformation, Genetic
chemical_list: D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D021122:Protein Subunits; D002245:Carbon Dioxide; C041069:carboxyl radical; D002118:Calcium