Differential regulation of skeletal muscle L-type Ca2+ current and excitation-contraction coupling by the dihydropyridine receptor beta subunit.
The dihydropyridine receptor (DHPR) of skeletal muscle functions as a Ca2+ channel and is required for excitation-contraction (EC) coupling. Here we show that the DHPR beta subunit is involved in the regulation of these two functions. Experiments were performed in skeletal mouse myotubes selectively lacking a functional DHPR beta subunit. These beta-null cells have a low-density L-type current, a low density of charge movements, and lack EC coupling. Transfection of beta-null cells with cDNAs encoding for either the homologous beta1a subunit or the cardiac- and brain-specific beta2a subunit fully restored the L-type Ca2+ current (161 +/- 17 pS/pF and 139 +/- 9 pS/pF, respectively, in 10 mM Ca2+). We compared the Boltzmann parameters of the Ca2+ conductance restored by beta1a and beta2a, the kinetics of activation of the Ca2+ current, and the single channel parameters estimated by ensemble variance analysis and found them to be indistinguishable. In contrast, the maximum density of charge movements in cells expressing beta2a was significantly lower than in cells expressing beta1a (2.7 +/- 0.2 nC/microF and 6.7 +/- 0. 4 nC/microF, respectively). Furthermore, the amplitude of Ca2+ transient measured by confocal line-scans of fluo-3 fluorescence in voltage-clamped cells were 3- to 5-fold lower in myotubes expressing beta2a. In summary, DHPR complexes that included beta2a or beta1a restored L-type Ca2+ channels. However, a DHPR complex with beta1a was required for complete restoration of charge movements and skeletal-type EC coupling. These results suggest that the beta1a subunit participates in key regulatory events required for the EC coupling function of the DHPR.
Beurg
M
M
Department of Physiology, University of Wisconsin School of Medicine, Madison, Wisconsin 53706, USA.
Sukhareva
M
M
Ahern
C A
CA
Conklin
M W
MW
Perez-Reyes
E
E
Powers
P A
PA
Gregg
R G
RG
Coronado
R
R
D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020746:Calcium Channels, L-Type; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics; D017136:Ion Transport; D007700:Kinetics; D008564:Membrane Potentials; D051379:Mice; D009119:Muscle Contraction / Q000502:physiology; D018482:Muscle, Skeletal / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits; D014162:Transfection
D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D018076:DNA, Complementary; D002118:Calcium