Inhibition of cardiac L-type calcium channels by protein kinase C phosphorylation of two sites in the N-terminal domain.
We have investigated the mechanism underlying the modulation of the cardiac L-type Ca(2+) current by protein kinase C (PKC). Using the patch-clamp technique, we found that PKC activation by 4-alpha-phorbol 12-myristate 13-acetate (PMA) or rac-1-oleyl-2-acetylglycerol (OAG) caused a substantial reduction in Ba(2+) current through Ca(v)1.2 channels composed of alpha(1)1.2, beta(1b), and alpha(2)delta(1) subunits expressed in tsA-201 cells. In contrast, Ba(2+) current through a cloned brain isoform of the Ca(v)1.2 channel (rbC-II) was unaffected by PKC activation. Two potential sites of PKC phosphorylation are present at positions 27 and 31 in the cardiac form of Ca(v)1.2, but not in the brain form. Deletion of N-terminal residues 2-46 prevented PKC inhibition. Conversion of the threonines at positions 27 and 31 to alanine also abolished the PKC sensitivity of Ca(v)1.2. Mutant Ca(v)1.2 channels in which the threonines were converted singly to alanines were also insensitive to PKC modulation, suggesting that phosphorylation of both residues is required for PKC-dependent modulation. Consistent with this, mutating each of the threonines individually to aspartate in separate mutants restored the PKC sensitivity of Ca(v)1.2, indicating that a change in net charge by phosphorylation of both sites is responsible for inhibition. Our results define the molecular basis for inhibition of cardiac Ca(v)1.2 channels by the PKC pathway.
McHugh
D
D
Department of Pharmacology, Mailstop 357280, University of Washington, Seattle, WA 98195-7280, USA.
Sharp
E M
EM
Scheuer
T
T
Catterall
W A
WA
D000595:Amino Acid Sequence; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009206:Myocardium / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013912:Threonine / Q000378:metabolism
D020746:Calcium Channels, L-Type; D013912:Threonine; D011493:Protein Kinase C