Regulation of cloned cardiac L-type calcium channels by cGMP-dependent protein kinase.
We have studied the effect of 8-bromo-cyclic GMP (8-Br-cGMP) on cloned cardiac L-type calcium channel currents to determine the site and mechanism of action underlying the functional effect. Rabbit cardiac alpha(1C) subunit, in the presence or absence of beta(1) subunit (rabbit skeletal muscle) or beta(2) subunit (rat cardiac/brain), was expressed in Xenopus oocytes, and two-electrode voltage-clamp recordings were made 2 or 3 days later. Application of 8-Br-cGMP caused decreases in calcium channel currents in cells expressing the alpha(1C) subunit, whether or not a beta subunit was co-expressed. No inhibition of currents by 8-Br-cGMP was observed in the presence of the protein kinase G inhibitor KT5823. Substitutions of serine residues by alanine were made at residues Ser(533) and Ser(1371) on the alpha(1C) subunit. As for wild type, the mutant S1371A exhibited inhibition of calcium channel currents by 8-Br-cGMP, whereas no effect of 8-Br-cGMP was observed for mutant S533A. Inhibition of calcium currents by 8-Br-cGMP was also observed in the additional presence of the alpha(2)delta subunit for wild type channels but not for the mutant S533A. These results indicate that cGMP causes inhibition of L-type calcium channel currents by phosphorylation of the alpha(1C) subunit at position Ser(533) via the action of protein kinase G.
Jiang
L H
LH
School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom.
Gawler
D J
DJ
Hodson
N
N
Milligan
C J
CJ
Pearson
H A
HA
Porter
V
V
Wray
D
D
D000470:Alkaloids / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000235:genetics / Q000378:metabolism*; D002227:Carbazoles; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007211:Indoles; D008845:Microinjections; D008969:Molecular Sequence Data; D009154:Mutation; D009206:Myocardium / Q000378:metabolism*; D009865:Oocytes; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011817:Rabbits; D014981:Xenopus
D000470:Alkaloids; D020746:Calcium Channels, L-Type; D002227:Carbazoles; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D007211:Indoles; C073601:KT 5823; C016276:8-bromocyclic GMP; D011494:Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP; D002118:Calcium