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Circulation Research. 2007;101:465-474
Published online before print July 12, 2007, doi: 10.1161/CIRCRESAHA.107.156976
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(Circulation Research. 2007;101:465.)
© 2007 American Heart Association, Inc.

Molecular Medicine

Protein Kinase G Phosphorylates Cav1.2 {alpha}1c and ß2 Subunits

Lin Yang, Guoxia Liu, Sergey I. Zakharov, Andrew M. Bellinger, Marco Mongillo, Steven O. Marx

From the Division of Cardiology, Department of Medicine (L.Y., G.L., S.I.Z., S.O.M.), the Department of Pharmacology (S.O.M.), and the Wu Center for Molecular Cardiology (L.Y., G.L., S.I.Z., A.M.B., M.M., S.O.M.), Columbia University College of Physicians and Surgeons, New York.

Correspondence to Steven O. Marx, Department of Medicine, Division of Cardiology, Columbia University College of Physicians and Surgeons, 630 W 168th Street, P & S 9-401, New York, NY 10032. E-mail sm460{at}columbia.edu


*    Abstract
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 *Abstract
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 down arrowMaterials and Methods
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Voltage-dependent Ca2+ channel function (Cav1.2, L-type Ca2+ channel) is required for cardiac excitation-contraction (E-C) coupling. Cav1.2 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 Cav1.2 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 Cav1.2 function, it is still unclear whether Cav1.2 subunits are PKG substrates. We have identified phosphorylation sites within the {alpha}1c and ß2a subunits that are phosphorylated by PKGI{alpha} 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 ß2a 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 ß2a subunits caused an inhibition of Cav1.2 whole-cell current. PKG-mediated inhibition of Cav1.2 current was significantly reduced by coexpression of an alanine-substituted Cav1.2 ß2a subunit (Ser496). Our results identify a molecular mechanism by which cGMP-PKG regulates Cav1.2 phosphorylation and function.


Key Words: Cav1.2 • calcium channel • protein kinase G • phosphorylation


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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The cyclic nucleotides cAMP and cGMP play critical roles in the regulation of heart function by the sympathetic and parasympathetic nervous systems, nitric oxide (NO), and natriuretic peptides. cGMP, like cAMP, regulates cardiac contractility, acting on proteins required for myocyte excitation-contraction coupling. In the heart, NO has been reported to have variable effects (activation, inhibition, or both) on Cav1.2 current in part because the effects depend on species, cardiac tissue, age, and myoglobin content.1–8 Cav1.2 is the L-type, voltage-gated calcium (Ca2+) channel present in the sarcolemma of cardiomyocytes. It is required for excitation-contraction (E-C) coupling9 and also contributes to the plateau phase of the cardiac action potential, pacemaker activity in nodal cells and modulation of gene expression.10

The molecular basis for Cav1.2 regulation by PKG has been explored by several groups but is not completely elucidated. Cardiac Cav1.2 consists of 3 subunits, the pore-forming {alpha}1 subunit, the intracellular ß subunit, and the largely extracellular {alpha}2/{delta} subunit.10 Direct PKG phosphorylation of Cav1.2 has not been observed in a cellular context, although Wray and colleagues reported that Ser533, in the {alpha}1 I-II loop, was a PKG-target (based on electrophysiological analysis).11 To determine the phosphorylation sites within the {alpha}1c and ß2 subunits, we used glutathione S-transferase (GST) fusion proteins to screen potential phosphorylation sites. Here, we demonstrate that PKG phosphorylates the previously reported PKA12–14 and PKC15 phosphorylation site (Ser1928) on the {alpha}1c subunit and Ser496 on the ß2a subunit. Using phospho-epitope specific antibodies designed to specifically recognize PKG-phosphorylated Cav1.2 residues, we demonstrate that PKG phosphorylates these Cav1.2 residues in intact cardiomyocytes. Using heterologous expression of rabbit {alpha}1c and ß2a in HEK cells, we found that activation of PKG inhibits Cav1.2 current, as previously shown.11 Ala-substitution of the Cav1.2 ß2a PKG-phosphorylation site significantly reduced PKG-mediated Cav1.2 current inhibition. The results identify a molecular mechanism mediating PKG-regulation of the Cav1.2 current.


*    Materials and Methods
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 *Materials and Methods
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Additional details can be found in the online data supplement, available at http://circres.ahajournals.org.

Preparation of Phospho-Specific Antibodies
The {alpha}1c phospho-Ser1928, phospho-Ser528, phospho-Ser533, and ß2a phospho-Ser496 antibodies were prepared at Zymed Laboratories Inc, using the peptides: Ser1928 (pS1928): NH2- LGRRApSFHLECLK-COOH; Ser528 (pS528): NH2- RLAHRIpSKS-COOH; Ser533 (pS533): NH2- KFpSRYWRRW-COOH; Ser496 (pS496): NH2- GTSRGLpSRQE-COOH.

The phospho-epitope–specific antibodies were purified and sensitivity and specificity tested using ELISA before use.


*    Results
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 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
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Cav1.2 {alpha}1 Subunit Is Phosphorylated by PKG
Although prior work has established that cGMP can modulate Cav1.2 activity, the targets for PKG within the Cav1.2 complex have not been established. To determine whether the {alpha}1c subunit is phosphorylated by PKG, we performed in vitro kinase assays. Immune complexes were isolated with anti-{alpha}1c subunit antibody or preimmune serum and subjected to kinase assays with [{gamma}-32P] ATP in the absence or presence of cGMP and PKGI{alpha} (Figure 1A; n=3). The signal at {approx}220 kDa indicates that the {alpha}1c subunit is phosphorylated by exogenous PKG in vitro. The signal at {approx}220 kDa is specific because no signal was detected in the preimmune serum lane. The relatively weak 32P labeling of {alpha}1c in the absence of exogenous PKG may represent the phosphorylation of {alpha}1c by an associated kinase (Cav1.2 associates directly with endogenous PKC{alpha} in HEK cells15).


Figure 1
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  		Figure 1. PKG I{alpha} phosphorylates Cav1.2 {alpha}1c in vitro. A, {alpha}1c and ß2a subunits were expressed in HEK293 cells, and cell extracts were prepared. Immunoprecipitated {alpha}1c was phosphorylated with or without exogenous PKG and [{gamma}-32P] ATP. {alpha}1c was phosphorylated by PKG. B, Schematic is shown of the {alpha}1c subunit with the intracellular segments used to construct GST fusion proteins. {alpha}1c subunit-GST fusion proteins bound to glutathione-sepharose were subjected to PKG kinase reaction with [{gamma}-32P] ATP. PKG phosphorylated GST-fused {alpha}1c fragments I-II loop and 1906 to 2170. The amount of GST fusion protein used is shown in the Coomassie-stained gel below. C, {alpha}1c subunit-GST fusion proteins bound to glutathione-sepharose were subjected to PKA kinase reaction with [{gamma}-32P] ATP. PKA phosphorylated GST-fused {alpha}1c 1906 to 2170. The amount of GST fusion protein utilized is shown in the Coomassie-stained gel below.

To map the PKG phosphorylation sites, we used GST fusion proteins containing nonoverlapping regions of the {alpha}1c intracellular regions as substrates for the in vitro PKG kinase assay. PKG phosphorylated the GST fusion proteins containing the I-II loop and the distal C terminus (1906–2170) (Figure 1B; n=4). PKA also phosphorylated the distal C terminus (1906–2170) but phosphorylated the I-II loop to a lesser extent (Figure 1C; n=5).

{alpha}1c Ser528 and Ser,533 Located Within the I-II Loop, Are Potential PKG Phosphorylation Sites
An examination of the amino acid sequence within the I-II loop revealed several potential phosphorylation sites. Based on electrophysiological experiments, a potential phosphorylation site (Ser533) within the I-II loop of the {alpha}1c was previously identified.11 However, biochemical evidence supporting the phosphorylation of this site was not performed. We created, by site-directed mutagenesis, single and double alanine (Ala) substitution mutants of the potential PKG phosphorylation residues within the I-II loop. Radiolabeling of the GST I-II loop fusion protein was significantly reduced by Ala-substitution of either Ser528 or Ser533, and was completely abrogated by the double Ala mutation (Figure 2B). In contrast, Ala-substitution of Ser530 alone caused an increase in the radiolabeling of the GST fusion protein. We developed phospho-epitope specific–antibodies, designed to report the phosphorylation of either Ser528 (pS528) or Ser533 (pS533). The antibodies immunodetected the appropriate phosphorylated residue (Figure 2C). In contrast, no anti–phospho-Ser528 antibody immunoreactivity was detected when a single substitution (S528A) was introduced into the substrate sequence. The pS533 antibody weakly recognized the I-II loop, under nonphosphorylated conditions, but the signal did not substantially increase after PKG phosphorylation of an alanine-substituted GST fusion protein. These results suggest that both Ser528 and Ser533 are potential {alpha}1c PKG phosphorylation sites, and that we have developed appropriate reagents to detect the phosphorylation of these residues in full-length channel and in intact cells.


Figure 2
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  		Figure 2. PKG phosphorylates {alpha}1c Ser528, Ser533, and Ser1928. A, The schematic demonstrates the PKG phosphorylation sites in the I-II loop and 1906 to 2170 fragments. B, upper panel, Shown is autoradiogram of PKG in vitro kinase reaction performed with [{gamma}-32P] ATP and GST-fused WT, S528A, S530A, S533A, S530A/S533A, and S528/S533A I-II loop. PKG phosphorylated Ser528 and Ser.533 Lower panel, Coomassie-staining of autoradiogram demonstrating amount of fusion protein used. C, upper panels, GST fusion proteins (WT, S528A, or S533A) were phosphorylated with PKG, size-fractionated on SDS-polyacrylamide gel, transferred to nitrocellulose, and immunoblotted using anti–phospho-Ser528 and Ser533 antibodies (pS528 and pS533, respectively). PKG phosphorylates Ser528 and Ser533 in the GST fusion protein I-II loop. Lower panels, Coomassie staining indicates equivalent loading of GST fusion proteins. D, Shown is an autoradiogram of PKG in vitro kinase reaction performed using [{gamma}-32P] ATP and GST-fused WT and S1928A 1906 to 2170. PKG phosphorylated Ser1928. E, GST fusion proteins (WT and S1928A 1906 to 2170 fragment) were phosphorylated by PKG, size fractionated, transferred to nitrocellulose, and immunoblotted with a phospho-specific antibody recognizing phosphorylated Ser1928 (pS1928).

{alpha}1c Ser1928 Is a PKG Phosphorylation Site
Previous studies have mapped PKA12,13 and PKC15 phosphorylation sites on the {alpha}1c subunit to a consensus site (RRAS1928) within the distal C-terminus. Ser1928 also represents a consensus PKG phosphorylation site. PKG induced significant 32P-ATP incorporation into the GST-fused wild-type fragment, but radiolabeling was completely abrogated by a single Ala substitution (S1928A; Figure 2D). Similarly, using a phospho-epitope–specific antibody that we have previously developed,15 prominent immunoreactive bands were detected (with a range of mobilities corresponding to full-length and truncated fragments; Figure 2E). No anti–phospho-Ser1928 antibody immunoreactivity was detected when a single substitution (S1928A) was introduced into the substrate sequence.

Ser1928, but not Ser528 and Ser533, Are PKG Phosphorylation Sites in Full-Length {alpha}1c
Having validated the specificity of the antibodies pS528, pS533, and pS1928, as reagents to track the PKG-dependent phosphorylation of the {alpha}1c subunit, we used them to examine the phosphorylation status of full-length recombinant wild-type and Ala-substituted mutants {alpha}1c, coexpressed with the ß2a subunit in HEK cells. Wild-type and Ala-substituted {alpha}1c mutants were immunoprecipitated by anti-{alpha}1c antibody from the HEK cells extracts (Figure 3A) or rat heart (Figure 3B) and subjected to the PKG in vitro kinase assay. In contrast to the GST fusion protein results, only Ser1928 demonstrated significant PKG phosphorylation (Figure 3A). Possible explanations for the lack of PKG phosphorylation of Ser528 and Ser533 in full-length recombinant {alpha}1c, as compared with the GST fusion proteins, is the coexpression of the ß2a subunit, which may sterically block access to PKG. Cav1.2 ß subunits bind to the {alpha}1c I-II loop.16 The {alpha}-interaction domain (AID), however, does not overlap with the identified PKG phosphorylation residues. We found that Ser528 or Ser533 was not phosphorylated in full-length channel expressed in HEK cells in the absence of ß2 subunit (data not shown). Moreover, PKG failed to phosphorylate Ser528 and Ser533 in {alpha}1c immunoprecipitated from rat heart (Figure 3B). It is possible that the I-II loop adopts a different conformation in full-length channel, which alters the PKG phosphorylation of these residues.


Figure 3
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  		Figure 3. Ser1928 is phosphorylated by PKG in full-length recombinant and native cardiac channels. Extracts from HEK cells transfected with WT or S1928A, S528A, or S533A (A) or heart (B) were prepared, followed by preimmune or {alpha}1c immunoprecipitation and kinase reaction as indicated. Samples were size-fractionated on SDS-polyacrylamide gel, transferred to nitrocellulose membrane, and probed with anti–phospho-Ser1928, anti–phospho-Ser528, and anti–phospho-Ser533 antibodies (upper panel) or {alpha}1c antibody (lower panel).

Cav1.2 ß2 Subunit Is Phosphorylated by PKG
We divided the rabbit ß2a into amino terminus (NT; amino acid residues 1 to 412) and carboxy terminus (CT; amino acid residues 413 to 606) fragments, expressed each as GST fusion proteins in E coli and purified the protein on glutathione sepharose. GST-NT-ß2 and GST-CT-ß2 fusion proteins were subjected to in vitro kinase assays with [{gamma}-32P] ATP. Figure 4A shows that both proteins were labeled in the in vitro kinase assay by PKG. However, the CT fusion protein contained {approx}90% of the total 32P incorporation (based on densitometry of the 32P incorporated into the CT protein divided by the total 32P incorporated into both NT [PKG autophosphorylation subtracted] and CT proteins normalized for the amount of protein on Coomassie stain; n=3). To identify the phosphorylation site within the CT fragment, MALDI analysis was performed of the trypsin-digested phosphorylated GST-CT ß2 fusion protein. We identified a phosphorylated residue within the ß2 subunit peptide GLSRQETFDSETQESR. LC-MS/MS analysis of the trypsin-digested fusion protein identified Ser496 (GLSR) as the phosphorylated residue. We mutated Ser496 to Ala and expressed the mutant fusion protein in E coli. Wild-type and S496A CT and full-length (FL) ß2 fusion proteins were subjected to in vitro kinase reaction; radiolabeling was nearly completely abolished by the alanine (Ala) substitution at Ser496 (Figure 4B and 4C). In agreement with Figure 4A, Ala substitution of Ser496 decreased 32P incorporation by nearly 90% (n=3), confirming that Ser496 is the primary PKG phosphorylation site within the rabbit ß2a subunit. It is possible, however, that several other minor sites are present within the ß2a subunit that are not stoichiometrically PKG phosphorylated in vitro. We developed a phospho-epitope–specific antibody to Ser496 which specifically detected the PKG phosphorylation of Ser496 in full-length GST-ß2a fusion protein (Figure 4D). The antibody is specific, as immunoreactivity was not present in PKG-phosphorylated full-length alanine-substituted ß2a subunit (Figure 4D). Ser496 is not phosphorylated by PKA (Figure 4D), consistent with a prior report demonstrating that the PKA phosphorylation sites in Cav1.2 ß2a are Ser459, Ser478, and Ser479.17 The Ser496 phosphorylation site is conserved in all ß2 isoforms in many species (human, rat, mouse, rabbit).


Figure 4
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  		Figure 4. PKG phosphorylates Cav1.2 ß2 subunit. A, upper panel, N terminus (NT)- and C terminus (CT)- GST fusion proteins bound to glutathione sepharose were subjected to PKG kinase reaction with [{gamma}-32P]ATP. The band at 75 kDa in the GST-NT-ß2 lane represents the convergence of 2 signals—PKG autophosphorylation and the phosphorylation of GST-NT-ß2. PKG phosphorylation of GST-CT-ß2 accounts for 90% of the 32P incorporation into the ß2 subunit. Lower panel, Coomassie-stained gel. B and C, upper panel, WT and S496A ß2 C-terminal (B) and full-length (C) GST fusion proteins were subjected to PKG kinase reaction with [{gamma}-32P]ATP. PKG phosphorylates Ser496 in vitro. Lower panel, Coomassie stained gel. D, upper panel, GST fusion proteins (WT and S496A full-length ß2) were phosphorylated by PKA or PKG, size-fractionated, transferred to nitrocellulose, and immunoblotted with a phospho-specific antibody recognizing phosphorylated Ser496 (pS496). Ser496 is phosphorylated by PKG, but not by PKA. Lower panel, Ponceau-stained membrane.

{alpha}1c Ser1928 and ß2 Ser496 Are Phosphorylated in Response to cGMP in HEK Cells
We determined Cav1.2 phosphorylation in HEK cells transfected with wild-type PKGI{alpha} or a catalytically inactive K390R-PKGI{alpha} mutant.18,19 Incubation of HEK cells transfected with {alpha}1c and ß2a subunits with 8 Br-cGMP, prior to lysis, led to phosphorylation of {alpha}1c Ser1928 and ß2a Ser496 (Figure 5A and 5B). Phosphorylation of {alpha}1c and ß2a is dependent on cGMP-induced activation of PKGI{alpha}, because cGMP-induced phosphorylation above basal levels could not be demonstrated with the expression of a dominant negative catalytic-inactive PKGI{alpha} mutant (K390R; Figure 5A and 5B). Exposure of the cells to a relatively low concentration of calyculin A (10 nmol/L) slightly increased the phosphorylation of {alpha}1c Ser1928, as previously reported15 (Figure 5A). In contrast, exposure of the cells to calyculin A (10 nmol/L) alone did not increase the phosphorylation of ß2a Ser496 (Figure 5B). The detection of {alpha}1c Ser1928 and ß2a Ser496 was specific, as Ala-substitution of either residue caused loss of immunoreactivity (Figure 5A and 5B).


Figure 5
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  		Figure 5. Reconstitution of PKG-mediated phosphorylation of {alpha}1c and ß2 in HEK293 cells. A, WT and S1928A {alpha}1c + ß2a were coexpressed with PKG or a catalytically inactive PKG (PKG-K390R). Cells were exposed to 8 Br-cGMP (0.5 mmol/L), in the absence or presence of calyculin A (Cal: 10 nmol/L) for 10 minutes. Nitrocellulose membranes were blotted with anti–phospho-Ser1928 (pS1928; upper panel) or {alpha}1c (lower panel) antibodies. Bar graph of densitometric quantification of S1928 phosphorylation (normalized to wild-type {alpha}1c in absence of cGMP or calyculin) (n=3 to 7, error bars represent SEM). B, Recombinant WT and S496A ß2a were transiently coexpressed with {alpha}1c. Methodology is identical to A except nitrocellulose membranes were blotted with anti–phospho-Ser496 (pS496; upper panel) or ß2 (lower panel) antibodies. Bar graph of densitometric quantification of S496 phosphorylation (normalized to wild-type ß2a in absence of cGMP or calyculin; n=3 to 8, error bars represent SEM). **P<0.01.

PKG expression is developmentally regulated in the heart, with highest expression in the newborn compared with adult ventricular myocytes.20 Several studies have shown that PKG is expressed in the adult rat heart and in isolated adult rat ventricular myocytes.21–23 Immunoblotting experiments with homogenates prepared from isolated neonatal and adult rat ventricular myocytes showed that PKG is present in appreciable amounts in isolated rat adult and neonatal ventricular myocytes (Figure 6A). Exposure of neonatal cardiomyocytes to cGMP analogues increased the phosphorylation of Ser1928 by 1.7-fold and Ser496 by 2.2-fold (Figure 6B and 6C). Protein phosphatase 2A (PP2A) associates directly with the C terminus of the {alpha}1c subunit, near the Ser1928 phosphorylation site.24 The close association of PP2A with the {alpha}1c C terminus may reduce the cGMP-induced phosphorylation Ser1928 relative to ß2a Ser496. Similarly, 8Br-cGMP+calyculin significantly increased the phosphorylation of Ser1928 and Ser496 in isolated adult rat ventricular myocytes (Figure 6D and 6E). Exposure of the cells to calyculin had no significant effect on the phosphorylation of {alpha}1c Ser1928 and ß2a Ser496. Ser1928 can be phosphorylated by PKA,25 PKC,15 and PKG. Although cyclic nucleotides bind to and activate specific cyclic nucleotide-dependent protein kinases, studies have demonstrated that high concentrations of cGMP can "crossover" and activate PKA.26,27 To assess whether PKG mediated the cGMP-induced Ser1928 phosphorylation, we applied the PKA inhibitor H-89 (1 µmol/L; Ki for PKA=48 nmol/L) to isolated cardiac myocytes 30 minutes before stimulation with 8-Br-cGMP. H-89 had no effect on the cGMP-induced phosphorylation of Ser1928, indicating that cGMP-induced cross-activation of PKA did not account for the phosphorylation. Recent work by Curran and colleagues demonstrated that this concentration of H-89 effectively blocked isoproterenol-PKA enhancement of the Ca2+ transient amplitude and rate of Ca2+ transient decline in isolated cardiomyocytes.28 Taken together, these findings demonstrate that {alpha}1c Ser1928 and ß2a Ser496 are phosphorylated by PKG in cardiomyocytes.


Figure 6
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  		Figure 6. PKG phosphorylates {alpha}1c Ser1928 and ß2a Ser496 in isolated cardiomyocytes. A, PKG immunoblot of homogenates (30 µg) obtained from isolated adult and neonatal rat cardiomyocytes (CM), HEK cells, and HEK cells transfected with PKGI{alpha}. Lower panel is the Ponceau stain of nitrocellulose membrane. B, Membranes were prepared from rat neonatal cardiomyocytes exposed to either no agonist or membrane-permeable cGMP analogue for 10 minutes. Proteins (100 µg) were size-fractionated on SDS-polyacrylamide gel, transferred to nitrocellulose membranes, and blotted with {alpha}1c anti-phospho-Ser1928 (pS1928; upper panel) or anti-{alpha}1c (lower panel) antibodies. Bar graph of densitometric quantification of S1928 phosphorylation (normalized to pS1928 signal in absence of cGMP; n=3, *P<0.05). C, Membranes were prepared from rat neonatal cardiomyocytes exposed to no agonist or 2 membrane-permeant cGMP analogues. Nitrocellulose membranes were immunoblotted with anti–phospho-Ser496 (upper panel) or anti-ß2 (lower panel) antibodies. Bar graph of densitometric quantification of S496 phosphorylation (normalized to pS496 signal in absence of cGMP; n=3, P<0.05). D, Membranes were prepared from isolated rat adult cardiomyocytes exposed to either no agonist, calyculin A (Cal), 8-Br-cGMP+Cal or 8Br-cGMP+Cal+H-89 (1 µmol/L). Nitrocellulose membranes were blotted with {alpha}1c anti–phospho-Ser1928 (pS1928; upper panel) or anti-{alpha}1c (lower panel) antibodies. Bar graph of densitometric quantification of S1928 phosphorylation (normalized to pS1928 signal in absence of cGMP; n=3, *P<0.05). E, Membranes were prepared from isolated rat adult cardiomyocytes exposed to no agonist, Cal, or 8-Br-cGMP+Cal. Nitrocellulose membranes were immunoblotted with anti–phospho-Ser496 (upper panel) or anti-ß2 (lower panel) antibodies. Bar graph of densitometric quantification of S496 phosphorylation (normalized to pS496 signal in absence of cGMP; n=3, *P<0.05).

Electrophysiological Analysis in HEK Cells
There is a lack of consensus on the precise role of PKGI in modulating Cav1.2 function in cardiac myocytes, with reports suggesting that cGMP-induced activation of PKG causes Cav1.2 current facilitation, inhibition, or even both.1–4,7,29–34 cGMP-PKG has been shown to inhibit both basal Cav1.2 current and ß-adrenergic–mediated upregulation of Cav1.2 current.35–37 To explore the effect of PKG phosphorylation of Cav1.2, we coexpressed wild-type (WT) {alpha}1c, ß2a, and PKGI{alpha} in HEK293 cells. After obtaining a stable baseline peak Ba2+ current (assessed at +10 mV every 10 sec) in whole cell configuration, we exposed the cell to calyculin A or 8Br-cGMP+calyculin A. Peak Cav1.2 current amplitude (at +10 mV) remained relatively stable during the experimental period in the absence of cGMP+calyculin, with <10% run-down during the first 3 minutes (Figure 7A, 7D, and 7G). Activation of PKG significantly reduced the amplitude of peak Cav1.2 current to 88.5±3% of control at 1 minute, to 81±3.8% of control at 2 minutes, and 75±4.1% of control at 3 minutes (Figure 7C, 7F, and 7I; n=8; P<0.01 for 1 and 2 minutes, P<0.05 for 3 minutes compared with control and calyculin-treated cells). PKG-modulation of Cav1.2 did not shift the current-voltage relationship (supplemental Figure IB).


Figure 7
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  		Figure 7. PKG phosphorylation of Cav1.2 inhibits Ca2+ current. HEK cells were transiently transfected with WT {alpha}1c + ß2a. A through C, Exemplar whole-cell current traces recorded using voltage clamp pulses from a holding potential of –60 mV to +10 mV before and 3 minutes after exposure to vehicle, calyculin (10 nmol/L), or cGMP (0.5 mmol/L)+calyculin (10 nmol/L). D through F, Diary plot of normalized current recorded during repetitive stimulation by depolarizing pulses every 10 s to +10mV from a holding potential of –10 mV. Open symbols are the 3 pulses before exposure to vehicle, calyculin, or cGMP+calyculin (closed symbols). G through I, Bar graph depicting normalized current for depolarization to +10 mV at 1, 2, and 3 minutes after exposure to vehicle (G), calyculin (H), or cGMP+calyculin (I). Values are mean±SEM. *P<0.05; **P<0.01 compared with vehicle or calyculin-treated.

We studied the effect of Ala-substitution of the PKG phosphorylation sites on {alpha}1c and ß2a. Under basal conditions, the current density and the current-voltage relationship among the mutants were comparable (supplemental Figure IA through IF). A prior report suggested that the PKG-mediated inhibition of Cav1.2 expressed in Xenopus oocytes was dependent on the presence {alpha}1c Ser533.11 Consistent with the biochemical data showing that {alpha}1c Ser533 was not phosphorylated in full-length channel (Figure 3), Ala-substitution of Ser533 did not reduce PKG-mediated inhibition of peak Cav1.2 in HEK cells (Figure 8A, 8E, and 8I). Similarly, Ala-substitution of {alpha}1c Ser1928 did not reduce PKG-mediated inhibition (Figure 8B, 8F, and 8J), consistent with the premise that phosphorylation of Ser1928 may be responsible for a small increase in Cav1.2 current in response to ß-adrenergic stimulation.25 In contrast, coexpression of the mutant ß2a harboring an Ala-substitution at Ser496 prevented the PKG-mediated inhibition of Cav1.2 after exposure to 8Br-cGMP and calyculin (Figure 8C, 8G, and 8K; n=8, P<0.01 compared with cGMP+calyculin-treated wild-type at 1, 2, and 3 minutes). Similarly, coexpression of the {alpha}1c harboring an Ala-substitution of Ser1928 and ß2a harboring the Ala-substitution of Ser496 essentially eliminated the PKG-mediated inhibition of Cav1.2 (Figure 8D, 8H, and 8L; n=5; P<0.01 compared with cGMP+calyculin-treated wild-type at 1, 2, and 3 minutes).


Figure 8
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  		Figure 8. Phosphorylation of ß2a Ser496 is required for PKG-mediated inhibition of Cav1.2. HEK cells were transfected with {alpha}1c-S533A+WT ß2a, {alpha}1c-S1928A+WT ß2a, WT {alpha}1c+ß2a-S496A, or {alpha}1c-S1928A+ß2a-S496A. A through D, Exemplar whole-cell current traces recorded from –60 mV to +10 mV before and 3 minutes after exposure to 8-Br-cGMP (0.5 mmol/L)+calyculin (10 nmol/L). E through H, Diary plot of normalized current recorded during repetitive stimulation by depolarizing pulses every 10 s to +10mV from –60 mV. Open symbols are the 3 pulses before exposure to cGMP+calyculin (closed symbols). I through L, Bar graph depicting normalized current for depolarization to +10 mV at 1, 2, and 3 minutes after exposure to 8Br-cGMP+calyculin A. Values are mean±SEM. *P<0.05; **P<0.01 compared with vehicle- and calyculin-treated.


*    Discussion
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 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
Several approaches have been taken to elucidate the regulatory domains within Cav1.2, predominantly involving identification of potential consensus phosphorylation sites, and then studying the electrophysiological effects of phosphorylation site mutations. Although potentially informative, this approach does not prove that the site is actually phosphorylated in a cellular context, as unanticipated changes in the secondary structure of the channel may produce changes in the electrophysiological properties of the channel. The rabbit {alpha}1c sequence has several consensus PKG phosphorylation sites in extracellular domains, which complicates identification of phosphorylation sites using full-length protein. Using GST fusion proteins incorporating intracellular domains of the {alpha}1c subunit, we identified 3 residues, 2 within the I-II loop and 1 within the C terminus of the {alpha}1c subunit, which are phosphorylated by PKG in vitro. However, the 2 residues within the I-II loop (Ser528 and Ser533) were not phosphorylated in full-length channel. Although puzzling, the absence of PKG phosphorylation of these residues in full-length channel was not attributable to the ß subunit sterically blocking access to these residues. Other explanations for this finding are that access to the residues is blocked by other domains of the {alpha}1 subunit, or that a change in conformation of the I-II loop in full-length channel alters the ability of PKG to phosphorylate these residues.

Wray and colleagues11 attributed the PKG-mediated inhibition of Cav1.2 activity to the phosphorylation of Ser533 within the {alpha}1c I-II loop. Although Ser533 is PKG phosphorylated in a GST fusion protein comprising the I-II loop, we were unable to demonstrate its phosphorylation (using a phospho-epitope–specific antibody) in full-length recombinant channels or in native {alpha}1c, immunoprecipitated from rat heart, and Ala-substitution of Ser533 did not prevent cGMP+calcyculin–mediated reduction of peak current. In contrast, 2 residues, {alpha}1c-Ser1928 and ß2a-Ser,496 are phosphorylated in full-length recombinant channels and in protein isolated from heart, and these residues are modulated biochemically in a cellular context by 8Br-cGMP.

Using conditions that optimally permit phosphorylation of these residues (assessed biochemically), we sought to determine whether the residues identified in our biochemical screen were required for PKG-mediated inhibition of channel activity. We found that Ala-substitution of ß2a-Ser496 significantly reduced PKG-mediated inhibition of channel activity in HEK cells. Ser1928 is the only identified PKA phosphorylation site on {alpha}1c subunit, and its phosphorylation was felt to be responsible, in part, for the PKA-induced upregulation of channel activity in response to ß-adrenergic stimulation.38 However, the requirement for Ser1928 phosphorylation in mediating ß-adrenergic–mediated upregulation has been questioned,39 supported by the recent work from the O’Rourke group using virally-infected cardiomyocytes.40 In myocytes, transfected Ala-substituted Ser1928 Cav1.2 channels were still regulated by ß-adrenergic stimulation, although the stimulated Ca2+ current was 70% to 80% of that observed with wild-type channels.25,40 Assessing the role of Ser1928 phosphorylation in heterologous expression systems may be limited because {alpha}1c is not C-terminally truncated as in cardiomyocytes. Exploring the electrophysiological implications of PKG-phosphorylation of {alpha}1c-Ser1928 and ß2a-Ser496 in cardiomyocytes will be required to confirm our findings.

Studies on the effects of cGMP-PKG on Cav1.2 current have shown variable effects. Many studies have reported that cGMP opposes the ß-adrenergic receptor/cAMP-induced increase in Cav1.2 current and cardiac contractility.2,41–43 NO and acetylcholine were proposed to share a common cGMP-dependent signaling pathways, although studies using endothelial NO synthase knockout mice and PKGI knockout mice have suggested that perhaps effects of acetylcholine are not PKG-dependent.35,44,45 Using a PKGI transgenic mouse, PKGI was shown to be an important downstream target for NO/cGMP- but not muscarinic-induced inhibition of single Cav1.2 currents in cardiac myocytes.35 In wild-type cardiac myocytes, basal Cav1.2-gating properties were not controlled by NO and cGMP, whereas in the PKGI transgenic mice, basal Cav1.2 peak average current, mean open probability, and availability were reduced by NO and cGMP.35 This indicates that PKG-mediated modulation of basal Cav1.2 activity depends on the intracellular concentration of PKGI. In contrast, isoproterenol-stimulated Cav1.2 was suppressed by NO and cGMP in both wild-type and PKGI-TG cardiac myocytes.35

In conclusion, we have pinpointed the residues within the Cav1.2 ({alpha}1c+ß2) complex, which are modulated by cGMP-PKG signaling pathways. Using phospho-epitope specific antibodies designed to recognize the phosphorylation of specific Cav1.2 residues, we have demonstrated that Cav1.2 is directly phosphorylated by PKG.


*    Acknowledgments
 
We thank I.B. Weinstein for providing cDNA constructs for PKGI{alpha} and PKGI{alpha} K390R mutant and S.F. Steinberg for providing neonatal cardiomyocytes.

Sources of Funding

S.O.M. is supported by grants from the NIH (HL068093), the American Heart Association, and the Arlene and Arnold Goldstein Family Foundation. S.O.M. is an Established Investigator of the AHA.

Disclosures

None.


*    Footnotes
 
Original received January 24, 2007; resubmission received May 31, 2007; accepted June 28, 2007.


*    References
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 *References
 

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