Cardiac calcium channels expressed in Xenopus oocytes are modulated by dephosphorylation but not by cAMP-dependent phosphorylation.
Enhancement of cardiac L-type Ca2+ channel activity by norepinephrine via phosphorylation by protein kinase A (PKA) underlines the positive inotropic effect of this transmitter and is a classical example of an ion channel modulation. However, it is not clear whether the channel protein itself (and which subunit) is a substrate for PKA. We have expressed various combinations of the cardiac Ca2+ channel subunits in Xenopus oocytes by injecting subunit mR-NAs. Expression of beta or alpha 2/delta + beta subunits potentiated the native (endogenous) Ca2+ channel currents in the oocyte (similar to T or N but not L-type). This potentiated endogenous current was enhanced by intracellular injection of cAMP or of the catalytic subunit of PKA, and this effect was reversed by the injection of a PKA inhibitor suggesting the presence of basal phosphatase activity. When a cardiac channel of alpha 1 + beta, alpha 1 + alpha 2/delta or alpha 1 + alpha 2/delta + beta composition was expressed at levels high enough that the contribution of the endogenous current became negligible, cAMP and PKA failed to increase the Ca2+ channel current, whereas PKA inhibitors and the catalytic subunit of protein phosphatase 1 reduced the amplitude of the current. Reduction of the current by PKA inhibitors was observed regardless of the presence of the beta subunit, suggesting a major role for the alpha 1 subunit in this process. These results suggest that, like in the heart, when expressed in Xenopus oocytes, the cardiac L-type Ca2+ channels are phosphorylated in basal state and dephosphorylation reduces their activity. However, unlike the situation in the heart, the activity of the channel cannot be enhanced by PKA-catalyzed phosphorylation, suggesting that the channel is already fully phosphorylated in its basal state.
Singer-Lahat
D
D
Department of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Israel.
Lotan
I
I
Biel
M
M
Flockerzi
V
V
Hofmann
F
F
Dascal
N
N
D000818:Animals; D016477:Artifacts; D001464:Barium / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D000242:Cyclic AMP / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000008:administration & dosage / Q000037:antagonists & inhibitors / Q000378:metabolism; D018076:DNA, Complementary / Q000008:administration & dosage / Q000235:genetics; D005260:Female; D008845:Microinjections; D008954:Models, Biological; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D009206:Myocardium / Q000737:chemistry*; D009638:Norepinephrine / Q000494:pharmacology; D009865:Oocytes; D009928:Organ Specificity; D010749:Phosphoprotein Phosphatases / Q000008:administration & dosage / Q000378:metabolism; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000008:administration & dosage / Q000235:genetics; D011817:Rabbits; D015854:Up-Regulation; D014982:Xenopus laevis
D015220:Calcium Channels; D018076:DNA, Complementary; D009124:Muscle Proteins; D012333:RNA, Messenger; D001464:Barium; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D002118:Calcium; D009638:Norepinephrine