Protein kinase G phosphorylates Cav1.2 alpha1c and beta2 subunits.
Voltage-dependent Ca(2+) channel function (Ca(v)1.2, L-type Ca(2+) channel) is required for cardiac excitation-contraction (E-C) coupling. Ca(v)1.2 plays a key role in modulating cardiac function in response to classic signaling pathways, such as the renin-angiotensin system and sympathetic nervous system. Regulation of cardiac contraction by neurotransmitters and hormones is often correlated with Ca(v)1.2 current through the actions of cAMP and cGMP. Cardiac cGMP, which activates protein kinase G (PKG), is regulated by nitric oxide (NO), and natriuretic peptides. Although PKG has been reported to activate or inhibit Ca(v)1.2 function, it is still unclear whether Ca(v)1.2 subunits are PKG substrates. We have identified phosphorylation sites within the alpha(1c) and beta(2a) subunits that are phosphorylated by PKGIalpha in vitro. We demonstrate that a subset of these phosphorylation sites is modulated, in a cGMP-PKG-specific manner, in intact HEK cells heterologously expressing alpha(1c) and beta(2a) subunits. Using phospho-epitope-specific antibodies, we show that the phosphorylation of these residues is enhanced by PKG in intact cardiac myocytes. Activation of PKG in HEK cells transfected with alpha(1c) and beta(2a) subunits caused an inhibition of Ca(v)1.2 whole-cell current. PKG-mediated inhibition of Ca(v)1.2 current was significantly reduced by coexpression of an alanine-substituted Ca(v)1.2 beta(2a) subunit (Ser(496)). Our results identify a molecular mechanism by which cGMP-PKG regulates Ca(v)1.2 phosphorylation and function.
Yang
Lin
L
Division of Cardiology, Department of Medicine, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.
Liu
Guoxia
G
Zakharov
Sergey I
SI
Bellinger
Andrew M
AM
Mongillo
Marco
M
Marx
Steven O
SO
D000200:Action Potentials / Q000502:physiology; D000818:Animals; D020746:Calcium Channels, L-Type / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D004594:Electrophysiology; D005786:Gene Expression Regulation; D006801:Humans; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism; D010766:Phosphorylation; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection
C504728:Cacna1c protein, rat; D020746:Calcium Channels, L-Type; C419385:L-type calcium channel alpha(1C); D021122:Protein Subunits; D012694:Serine; D017869:Cyclic GMP-Dependent Protein Kinases