Functional regulation of L-type calcium channels via protein kinase A-mediated phosphorylation of the beta(2) subunit.
Activation of protein kinase A (PKA) through the beta-adrenergic receptor pathway is crucial for the positive regulation of cardiac L-type currents; however it is still unclear which phosphorylation events cause the robust regulation of channel function. In order to study whether or not the recently identified PKA phosphorylation sites on the beta(2) subunit are of functional significance, we coexpressed wild-type (WT) or mutant beta(2) subunits in tsA-201 cells together with an alpha(1C) subunit, alpha(1C)Delta1905, that lacked the C-terminal 265 amino acids, including the only identified PKA site at Ser-1928. This truncated alpha(1C) subunit was similar to the truncated alpha(1C) subunit isolated from cardiac tissue not only in size ( approximately 190 kDa), but also with respect to its failure to serve as a PKA substrate. In cells transfected with the WT beta(2) subunit, voltage-activated Ba(2+) currents were significantly increased when purified PKA was included in the patch pipette. Furthermore, mutations of Ser-478 and Ser-479 to Ala, but not Ser-459 to Ala, on the beta(2) subunit, completely abolished the PKA-induced increase of currents. The data indicate that the PKA-mediated stimulation of cardiac L-type Ca(2+) currents may be at least partially caused by phosphorylation of the beta(2) subunit at Ser-478 and Ser-479.
Bünemann
M
M
Department of Molecular Pharmacology, Northwestern University Medical School, Chicago, Illinois 60611, USA.
Gerhardstein
B L
BL
Gao
T
T
Hosey
M M
MM
D000818:Animals; D017609:Barium Compounds / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002461:Cell Line, Transformed; D002712:Chlorides / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000494:pharmacology*; D004558:Electric Stimulation; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D018408:Patch-Clamp Techniques; D010766:Phosphorylation / Q000187:drug effects; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism
D017609:Barium Compounds; D020746:Calcium Channels, L-Type; D002712:Chlorides; D011993:Recombinant Fusion Proteins; C024986:barium chloride; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases