Receptor-operated Ca2+ entry (ROCE) via transient receptor potential canonical channel 6 (TRPC6) is important machinery for an increase in intracellular Ca2+ concentration triggered by the activation of Gq protein-coupled receptors. TRPC6 is phosphorylated by various protein kinases including protein kinase A (PKA). However, the regulation of TRPC6 activity by PKA is still controversial. The purpose of this study was to elucidate the role of adenylate cyclase/cAMP/PKA signaling pathway in the regulation of Gq protein-coupled endothelin type A receptor (ETAR)-mediated ROCE via TRPC6. For this purpose, human embryonic kidney 293 (HEK293) cells stably coexpressing human ETAR and TRPC6 (wild type) or its mutants possessing a single point mutation of putative phosphorylation sites for PKA were used to analyze ROCE and amino acids responsible for PKA-mediated phosphorylation of TRPC6. Ca2+ measurements with thapsigargin-induced Ca2+-depletion/Ca2+-restoration protocol to estimate ROCE showed that the stimulation of ETAR induced marked ROCE in HEK293 cells expressing TRPC6 compared with control cells. The ROCE was inhibited by forskolin and papaverine to activate the cAMP/PKA pathway, whereas it was potentiated by Rp-8-bromoadenosine-cAMP sodium salt, a PKA inhibitor. The inhibitory effects of forskolin and papaverine were partially cancelled by replacing Ser28 (TRPC6S28A) but not Thr69 (TRPC6T69A) of TRPC6 with alanine. In vitro kinase assay with Phos-tag biotin to determine the phosphorylation level of TRPC6 revealed that wild-type and mutant (TRPC6S28A and TRPC6T69A) TRPC6 proteins were phosphorylated by PKA, but the phosphorylation level of these mutants was lower (approximately 50%) than that of wild type. These results suggest that TRPC6 is negatively regulated by the PKA-mediated phosphorylation of Ser28 but not Thr69.
Ca2+ signaling regulates various important physiological and pathophysiological events, including cell constriction, cell proliferation, cell differentiation, and activation of immune cells. Increased Ca2+ influx via transient receptor potential canonical channel 6 (TRPC6), a voltage-independent, Ca2+-permeable nonselective cation channel, is particularly a major stimulus for the development of cardiovascular diseases, such as idiopathic pulmonary arterial hypertension (IPAH), which is associated with the continued stimulation of endothelin type A receptor (ETAR) resulting from excessive production of endothelin-1 (ET-1) (Abramowitz and Birnbaumer, 2009). TRPC6 has been identified as a potential candidate for a receptor-operated Ca2+ channel (ROCC) rather than a store-operated Ca2+ channel, and it is operated by phospholipase C-mediated diacylglycerol production after stimulation of Gq protein-coupled receptors (GqPCRs) such as α1-adrenergic and angiotensin type I receptors (Watanabe et al., 2008). However, it remains unclear whether the activation of Gq protein-coupled ETAR with its agonist, ET-1, triggers ROCE through TRPC6.
The activity of TRPC6 is positively and negatively regulated by various protein kinases: the channel is activated by Ca2+/calmodulin-dependent protein kinase II and the Src tyrosine kinase family (Hisatsune et al., 2004; Shi et al., 2004), whereas it is inactivated by protein kinase C and protein kinase G (PKG) (Kim and Saffen, 2005; Kinoshita et al., 2010; Nishida et al., 2010). TRPC6 is also phosphorylated by protein kinase A (PKA), which is a downstream target of cAMP, whereas PKA-mediated phosphorylation of TRPC6 is reported not to affect channel function (Hassock et al., 2002). However, Nishioka et al. (2011) have shown that PKA-mediated phosphorylation of TRPC6 at Thr69 is essential for the vasorelaxant effects of phosphodiesterase type 3 (PDE3) inhibition against the angiotensin II-induced constriction of vascular smooth muscle cells. In contrast to the negative regulation of TRPC6 activity by PKA-mediated phosphorylation, cAMP is reported to activate TRPC6 via the phosphoinositide 3-kinase/protein kinase B/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 signaling pathway (Shen et al., 2011). Thus, post-translational modification of TRPC6 by protein kinases plays a critical role in the regulation of channel activity.
Drug therapy designed to elevate intracellular contents of cAMP and cGMP with Gs protein-coupled prostaglandin I2 receptor agonists (e.g., beraprost) and cGMP-specific PDE5 inhibitors (e.g., sildenafil), respectively, is highly effective against IPAH, which is closely correlated with either the prolonged activation of ETAR or the augmentation of Ca2+ influx through up-regulated TRPC6 (Kunichika et al., 2004; Yu et al., 2004). Many lines of evidence indicate that the PKA- and PKG-dependent phosphorylation of TRPC6 at Thr69 inhibits channel activity, leading to vasorelaxant and antihypertrophic effects, respectively (Kinoshita et al., 2010; Nishida et al., 2010; Nishioka et al., 2011). In terms of the substrate specificity of PKA, a study with an oriented peptide library has demonstrated that PKA is an arginine-directed serine/threonine protein kinase, and arginine residue is preferred at positions −4 to −1 amino terminal to the phosphorylation site (Songyang et al., 1994). In addition, the greatest selectivity was observed at residues −3 and −2, that is, R2-X-S/T, and the lysine residue at the −2 position was the second preferred amino acid (R-L-X-S/T) (Songyang et al., 1994). Searching for the primary sequence of TRPC6, we have found that, in addition to the R-R-Q-T sequence surrounding Thr69, potential sequences for PKA-mediated phosphorylation are present within the TRPC6 sequence, namely R-R-G-G-S at Ser14, R-R-N-E-S at Ser28, and R-K-L-S at Ser321. Unlike Thr69, which is responsible for PKA-mediated negative regulation of TRPC6 activity (Nishioka et al., 2011), there is no conclusive evidence for the functional role of these serine residues in the regulation of TRPC6 activity by PKA.
In the present study, we tried to clarify whether TRPC6 functions as ETAR-operated Ca2+ channels by using Ca2+ measurements with a thapsigargin (TG)-induced Ca2+-depletion/Ca2+-restoration protocol. In addition, we made and used TRPC6 mutants possessing a single point mutation of putative phosphorylation sites for PKA to identify key amino acids responsible for the regulation of TRPC6 activity by PKA-mediated phosphorylation. For this purpose, we improved an in vitro kinase assay by using Phos-tag biotin (a phosphate-specific ligand with biotin tag) to detect specifically phosphorylated proteins (Kinoshita et al., 2006). We here show that, although PKA can phosphorylate TRPC6 on Ser28 and Thr69, ETAR-operated Ca2+ entry through TRPC6 is negatively regulated by the activation of the AC/cAMP/PKA signaling pathway, via phosphorylation of TRPC6 on Ser28 but not on Thr69 of the N terminus.
Materials and Methods
The following drugs and reagents were used in the present study: synthetic human ET-1 (Peptide Institute Inc., Osaka, Japan); fura-2/acetoxymethyl ester (fura-2/AM) and Pluronic F-127 (Dojindo Laboratories, Kumamoto, Japan); gadolinium (III) chloride, G418, TG, probenecid, aprotinin, leupeptin, pepstatin, sodium deoxycholate, SDS, phenylmethylsulfonyl fluoride, Na3VO4, NaF, puromycin dihydrochloride, forskolin, 1,9-dideoxyforskolin, 8-bromoadenosine-cAMP, papaverine hydrochloride, ATP, and bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO); Rp-8-bromoadenosine-cAMP sodium salt (Rp-8-Br-cAMP) and 8-(4-chlorophenylthio)-2′-O-methyladenosine-cAMP sodium salt (Enzo Life Sciences Inc., Plymouth Meeting, PA); SQ-22,536 [9-(tetrahydro-2-furanyl)-9H-purin-6-amine] (Calbiochem, San Diego, CA), cAMP-dependent protein kinase catalytic subunit (Promega, Madison, WI); and rapid alkaline phosphatase (Roche Applied Science, Mannheim, Germany). All cell culture media and supplements, except fetal calf serum (Invitrogen, Carlsbad, CA), were obtained from Sigma-Aldrich. Antibodies for FLAG peptide, a green fluorescent protein (GFP), glyceraldehyde-3-phosphate dehydrogenase, horseradish peroxidase (HRP)-conjugated FLAG peptide (HRP-FLAG), and HRP-conjugated streptavidine (HRP-SA) were obtained from Sigma-Aldrich, Clontech (Mountain View, CA), Santa Cruz Biotechnology Inc. (Santa Cruz, CA), Medical and Biological Laboratories Co., Ltd. (Aichi, Japan), and Thermo Fisher Scientific (Waltham, MA), respectively. Phos-tag biotin was obtained from NARD Institute, Ltd. (Hyogo, Japan). The other reagents used were of the highest grade in purity.
Construction of Retrovirus Vectors.
The pCI-neo mammalian expression vector encoding TRPC6 (pCI-neo-TRPC6) was generously provided by Dr. Yasuo Mori (Kyoto University, Kyoto, Japan). The insert cDNA of wild-type TRPC6 was generated from the pCI-neo-TRPC6 as a template by a polymerase chain reaction with specific primers containing the restriction enzyme sites, which are BamHI at the 5′ end and AgeI at the 3′ end, for subcloning into the pCR-Blunt II-TOPO vector (Invitrogen). The resulting pCR-Blunt II-TOPO vector and the pEGFP-N1 vector encoding a red-shifted variant of GFP (Clontech) were digested with two restriction enzymes (BamHI/AgeI, AgeI/NotI, or BamHI/NotI) simultaneously. The cDNA fragments were ligated into the BamHI/NotI-treated pMXrmv5 retrovirus vector to yield the pMXrmv5 vectors encoding GFP and TRPC6 fused with GFP at the C terminus (TRPC6-GFP). All of the constructs were verified by DNA sequencing.
To identify the target sites of TRPC6 for phosphorylation by PKA, serine residues at positions 14, 28, and 321, and threonine at 69 were replaced with alanine by using a KOD-Plus- Mutagenesis Kit (Toyobo Co., Ltd., Osaka, Japan). The sequences of the resulting mutants tagged with GFP or FLAG peptide at the C terminus were confirmed by DNA sequencing.
Human embryonic kidney 293 (HEK293) cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum, penicillin (100 units · ml−1), and streptomycin (100 μg · ml−1) at 37°C in humidified air with 5% CO2.
Stable Expression of Human ETAR in HEK293 Cells.
The pDisplay mammalian expression vector containing cDNA of human ETAR fused with an influenza hemagglutinin (HA) epitope tag at the N terminus (HA-ETAR) was transfected into HEK293 cells by using a TransIT-293 transfection kit (Mirus Bio Corporation, Madison, WI) according to the manufacturer's instructions. Stable transformants were selected in medium containing 800 μg · ml−1 G418 for 3 weeks. Clonal cell lines were obtained by limiting dilution. Clones were expanded and screened for expression levels by whole-cell radioligand binding assay and Western blot analysis. The resulting suitable clone (HA-ETAR/HEK293 cells) was grown up for further experiments.
Stable Expression of TRPC6 and Its Mutants.
To generate HA-ETAR-positive HEK293 cells stably expressing GFP, TRPC6-GFP, TRPC6-FLAG, or their mutants, these genes were introduced into HA-ETAR/HEK293 cells by retroviral gene transfer. In brief, retroviruses were produced by triple transfection of HEK293T cells with retroviral constructs along with gag-pol and vesicular stomatitis virus G glycoprotein expression constructs (Yee et al., 1994). The supernatants containing virus were collected 24 h after transfection and added to HA-ETAR/HEK293 cells. The HA-ETAR/HEK293 cells were then centrifuged at 900g for 45 min at 25°C followed by incubation for 6 h at 37°C in 5% CO2 and 95% air. Then, fresh culture media were added to dilute supernatants containing virus. GFP- or TRPC6-positive HA-ETAR/HEK293 cells were selected for growth in medium containing 5 μg · ml−1 puromycin for 1 week.
Measurement of Intracellular Free Ca2+ Concentration.
Intracellular free Ca2+ concentration ([Ca2+]i) was monitored by using a fluorescent Ca2+ indicator, fura-2/AM, as described previously (Horinouchi et al., 2009; Higa et al., 2010). In brief, HEK293 cells grown in 3.5-cm dishes were incubated with 4 μM fura-2/AM admixed with 2.5 mM probenecid and 0.04% Pluronic F-127 at 37°C for 45 min under reduced light. After collecting and washing cells, the cells were suspended in Ca2+-free Krebs-HEPES solution (140 mM NaCl, 3 mM KCl, 1 mM MgCl2·6H2O, 11 mM d-(+)-glucose, and 10 mM HEPES, adjusted to pH 7.3 with NaOH) at 4 × 105 cells · ml−1. CaCl2 was added to 0.5-ml aliquots of the cell suspension at a final concentration of 2 mM, when necessary. Changes of [Ca2+]i in cells were measured at 30°C by using a CAF-110 spectrophotometer (Jasco, Tokyo, Japan) with the excitation wavelengths of 340 and 380 nm and emission wavelength of 500 nm.
Confocal microscopy was carried out by using a FluoView FV300 microscope (Olympus, Tokyo, Japan) with a 63× oil-immersion lens.
In Vitro Kinase Assay.
Wild-type and mutant TRPC6-FLAG protein-expressed HA-ETAR/HEK293 cells grown in 10-cm dishes were washed twice with ice-cold PBS and lysed in radioimmunoprecipitation assay buffer (150 mM NaCl, 1.5 mM MgCl2, 50 mM Tris-HCl, pH 6.8, 1% nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 20 mM NaF, 10 μg · ml−1 leupeptin, 10 μg · ml−1 aprotinin, and 10 μg · ml−1 pepstatin) supplemented with EDTA-free, protease inhibitor cocktail (Thermo Fisher Scientific). The cell lysates were sonicated for 10 s on setting 10 of a handy sonicator (UR-20P; Tomy Seiko Co., Ltd., Tokyo, Japan) and centrifuged at 20,000g for 20 min at 4°C. Protein content of supernatant was measured according to the method of Bradford (1976) using BSA as standard. Immunoprecipitation was carried out with an immunoprecipitation kit (Dynabeads Protein G; Invitrogen). In brief, the Dynabeads were incubated with a primary antibody (anti-FLAG, 1:100 dilution) for 1 h at room temperature. The Dynabeads-antibody complex was washed twice with washing buffer (150 mM NaCl, 1.5 mM MgCl2, 50 mM Tris-HCl, pH 6.8, 1% nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS) by gentle pipetting, and then incubated with lysates containing equal protein amounts for 1 h at room temperature. Western blot analysis showed that the expression of TRPC6 proteins relative to total protein was quantitatively similar between wild-type and mutant proteins (Supplemental Fig. S1). The resulting Dynabeads were washed three times with the washing buffer. The Dynabeads-binding TRPC6 proteins were then incubated with alkaline phosphatase for 1 h at 37°C to reduce basal phosphorylation levels. After being rinsed with the washing buffer three times, the dephosphorylated TRPC6 proteins were incubated in phosphorylation buffer [20 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 0.1 mM ATP, and cAMP-dependent protein kinase catalytic subunit (40 units per reaction)] at 37°C for 30 min. This amount of cAMP-dependent protein kinase catalytic subunit produced approximately 50% response of the maximum TRPC6 phosphorylation (Supplemental Fig. S2). The TRPC6 proteins bound to the Dynabeads were eluted in the phosphorylation buffer by adding SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 5% 2-mercaptoethanol, 2.5% SDS, and 0.1% bromphenol blue) followed by incubation at 37°C for 30 min. The phosphorylation levels of TRPC6 protein were analyzed by Western blotting.
Western Blot Analysis.
The proteins in immunoprecipitated samples and whole-cell lysates were separated on a 5 to 20% polyacrylamide gel (SuperSep; Wako Pure Chemicals, Osaka, Japan) and electrotransferred to a polyvinylidene fluoride membrane (Immobilon-P; pore size 0.45 μm; Millipore Corporation, Billerica, MA) with a semidry electroblotter. After transfer, the membranes were washed three times for 5 min with Tris-buffered saline-Tween 20 (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 0.1% Tween 20) followed by blocking (2% BSA in Tris-buffered saline-Tween 20) of nonspecific binding for 1 h at room temperature. The membranes were incubated with anti-FLAG-HRP antibody or Phos-tag biotin-bound HRP-SA complex (which was prepared according to the instructions of the manufacturer, Nard Institute, Ltd.) at room temperature for 6 h or with a monoclonal antibody for GFP or glyceraldehyde-3-phosphate dehydrogenase as a primary antibody overnight at 4°C. The anti-FLAG-HRP antibody and Phos-tag biotin-bound HRP-SA complex were detected with an ECL Western blotting Analysis System (GE Healthcare, Little Chalfont, Buckinghamshire, UK). The primary antibody was detected with a secondary horseradish peroxidase-conjugated anti-mouse IgG antibody and enhanced chemiluminescence (GE Healthcare). The blots were exposed to Amersham Hyperfilm ECL (GE Healthcare). Phosphorylation levels of wild-type and mutant TRPC6 proteins were analyzed with Image J1.37 software (National Institutes of Health, Bethesda, MD).
Data regarding change in [Ca2+]i were collected and analyzed by using a MacLab/8s with Chart (v. 3.5) software (ADInstruments Japan, Tokyo, Japan). All data are presented as means ± S.E.M. where n refers to the number of experiments. The significance of the difference between mean values was evaluated with Prism (version 3.00; GraphPad Software Inc., San Diego, CA) by Student's paired or unpaired t test. A P value < 0.05 was considered to indicate significant differences.
Characterization of ETAR-Operated Ca2+ Entry through TRPC6.
To determine whether the stimulation of ETAR with its agonist, ET-1, induces ROCE via TRPC6, we used the TG-induced Ca2+-depletion/Ca2+-restoration protocol to measure store-operated Ca2+ entry (SOCE) followed by ETAR stimulation to measure ROCE (Boulay, 2002). Gd3+ (10 μM) in the extracellular medium was used throughout the experiments to inhibit the endogenous SOCE (capacitative Ca2+ entry) that masks the ROCE via TRPC3 and TRPC7 in HEK293 cells (Okada et al., 1999). In GFP- and TRPC6-GFP-expressing HA-ETAR/HEK293 cells, SOCE induced by TG-induced Ca2+ depletion/Ca2+ restoration was significantly inhibited by the addition of 10 μM Gd3+ (210.2 ± 11.5 to 46.0 ± 2.1 nM for GFP and 177.2 ± 4.8 to 41.2 ± 1.9 nM for TRPC6-GFP; n = 6 for each). Figure 1 shows that in nominally Ca2+-free solution containing 10 μM Gd3+ 2 μM TG evoked Ca2+ release from the endoplasmic reticulum (ER), causing a transient increase in [Ca2+]i that promptly returned to near baseline. In the TG-treated HA-ETAR/HEK293 cells expressing GFP as a control, stimulation with 10 nM ET-1 after restoration of extracellular Ca2+ to 2 mM did not produce a further increase in [Ca2+]i (Fig. 1A), indicating that under this condition ETAR stimulation cannot elicit either Ca2+ release from Ca2+ store, SOCE, or ROCE. On the other hand, the stimulation of ETAR with 10 nM ET-1 elicited a Gd3+-insensitive transient increase in [Ca2+]i resulting from ROCE through TRPC6 in the TG-treated HA-ETAR/HEK293 cells expressing TRPC6-GFP (Fig. 1B; Table 1). These results clearly suggest that the activation of ETAR is able to elicit ROCE mediated through TRPC6-GFP.
Negative Regulation of ETAR-Operated, TRPC6-Mediated Ca2+ Entry by the Activation of Adenylate Cyclase/cAMP/Protein Kinase A Signaling Pathway.
Next, we examined the effects of cAMP and PKA on TRPC6-mediated ROCE in response to ETAR stimulation. The ROCE was markedly suppressed by either 10 μM forskolin, an AC activator (Fig. 2B), or 10 μM papaverine, a nonselective PDE inhibitor (Fig. 2D), both of which increase the intracellular cAMP level, thereby activating PKA. The inhibitory effect of forskolin on TRPC6-mediated ROCE was cancelled by treatment with 1 mM SQ-22,536, a membrane-permeable AC inhibitor, that also weakly but significantly augmented the ROCE (Supplemental Fig. S3). Because it is well known that forskolin can exhibit pleiotropic effects in an AC-independent manner (Laurenza et al., 1989), we examined the effect of 1,9-dideoxyforskolin, an inactive analog of forskolin as a negative control (Pinto et al., 2008, 2009), on the ETAR-operated Ca2+ influx via TRPC6. We were surprised to find that 10 μM 1,9-dideoxyforskolin as well as 10 μM forskolin inhibited the TRPC6-mediated Ca2+ entry (Supplemental Fig. S3). The inhibitory effect of 10 μM 1,9-dideoxyforskolin was cancelled by 1 mM SQ-22,536, indicating the possibility that 1,9-dideoxyforskolin was able to directly activate AC. Rp-8-Br-cAMP (100 μM), a membrane-permeable PKA inhibitor, enhanced ETAR-operated Ca2+ influx (Fig. 2C), whereas 500 μM 8-bromoadenosine-cAMP, a membrane-permeable direct PKA activator, suppressed the Ca2+ response to ET-1 (Supplemental Fig. S3). The effects of these drugs on ETAR-induced ROCE via TRPC6 are summarized in Fig. 2E and Supplemental Fig. S3. Our findings indicate the possibility that the activity of TRPC6 is negatively regulated by its phosphorylation by PKA, resulting in the inhibition of ETAR-operated Ca2+ entry via TRPC6.
Effect of Activation of cAMP/PKA Signaling Pathway on Subcellular Localization of TRPC6.
Wild-type TRPC6 protein expressed in HEK293 cells is reported to be mainly present on the plasma membrane (Lussier et al., 2008; Graham et al., 2010). We confirmed the subcellular localization of TRPC6-GFP expressed in HA-ETAR/HEK293 cells under basal conditions by using a confocal laser-scanning microscopic approach. Although GFP expressed in HA-ETAR/HEK293 cells as a control was localized in the cytosol and nucleus (Fig. 3A), wild-type TRPC6-GFP was predominantly targeted to the plasma membrane (Fig. 3B). The intracellular distribution of GFP and TRPC6-GFP after treatment with 10 μM forskolin (Fig. 3, C and D), and 10 μM papaverine (Fig. 3, E and F) was similar to that in the control cells. Likewise, pharmacological inhibition of AC and PKA with 1 mM SQ-22,536 and 100 μM Rp-8-Br-cAMP, respectively, did not seem to affect the intracellular distribution of GFP and TRPC6-GFP (Supplemental Fig. S4).
Determination of the Amino Acid Residue Responsible for the Inhibition of TRPC6-Mediated Ca2+ Influx by Forskolin and Papaverine.
To determine the critical amino acid residues involved in the inhibition of ROCE via TRPC6 by forskolin and papaverine, we searched for the PKA phosphorylation candidate sites in the TRPC6 sequence. As a result, we found three serine residues at positions 14 (R-R-G-G-S), 28 (R-R-N-E-S), and 321 (R-K-L-S) and a single threonine residue at 69 (R-R-Q-T): all of these sequences were present on the intracellular N-terminal region of TRPC6. To identify the target residues of PKA phosphorylation, we made HA-ETAR/HEK293 cells stably expressing GFP-tagged TRPC6 mutants carrying an alanine substitution for these serine/threonine residues. Functional study with Ca2+ measurement demonstrated that there was no significant difference in the magnitude of ROCE between wild type and these mutants (Table 1). Replacement of Ser28 (S28A) but not other residues (Ser14, Thr69, and Ser321) by alanine (S14A, T69A, and S321A) attenuated the inhibitory effects of 10 μM forskolin (Fig. 4, A and B) and 10 μM papaverine (Fig. 5, A and B). The effects of these drugs on ETAR-induced ROCE via wild-type and mutant TRPC6 proteins are summarized in Figs. 4C and 5C.
Identification of PKA Phosphorylation Sites on TRPC6 by Using In Vitro Kinase Assay with Phos-Tag Biotin.
To directly demonstrate that TRPC6 is a substrate for PKA, PKA-mediated phosphorylation of TRPC6 was estimated by using Phos-tag biotin, which is a biotinylated phosphate-specific ligand that can specifically detect phosphorylated proteins (Kinoshita et al., 2006). Figure 6A shows that immunoblotting with Phos-tag biotin-bound HRP-SA complex detected wild-type and mutant phosphorylated TRPC6 proteins under basal conditions, which were reduced by treatment of immunoprecipitated TRPC6 proteins with phosphatase, indicating the ability of Phos-tag biotin to specifically identify phosphorylated proteins. The basal phosphorylation level of wild-type TRPC6 was similar to that of mutant TRPC6 proteins (Fig. 6A). To analyze a change in the levels of TRPC6 phosphorylation by PKA in vivo, the HA-ETAR/HEK293 cells stably expressing wild-type or mutant TRPC6-FLAG protein were treated with a combination of 10 μM forskolin and 10 μM papaverine to enhance cAMP production and inhibit cAMP breakdown by PDE, respectively. However, there was little or no change in the phosphorylation levels of wild-type and mutant TRPC6 after the activation of the AC/cAMP/PKA signaling pathway (Fig. 6B). We considered the possibility that the high basal phosphorylation levels masked the effects of cAMP-elevating agents. Therefore, we next attempted to perform an in vitro kinase assay on immunoprecipitated TRPC6 proteins, which were pretreated with a phosphatase to reduce their basal phosphorylation levels. Incubation with an exogenous PKA catalytic subunit induced phosphorylation of wild-type TRPC6 protein dephosphorylated with a phosphatase pretreatment (Fig. 6C, top), demonstrating that the target sites for PKA-mediated phosphorylation are present within the TRPC6 sequence. There was no significant difference in an increase in the phosphorylation level of TRPC6 protein by PKA between wild type and TRPC6S14A mutant. It is noteworthy that an in vitro kinase assay using TRPC6 mutants with Ser28 (TRPC6S28A) and Thr69 (TRPC6T69A) replaced to alanine showed significant loss of phosphorylation, suggesting that PKA can phosphorylate TRPC6 on these sites and the Ser28 and Thr69 residues contributed to approximately 50% of the phosphorylation of TRPC6 by PKA.
Activation of GqPCR and tyrosine kinase receptor induces formation of the second messengers such as inositol 1,4,5-trisphosphate and diacylglycerol via phospholipase C. Binding of inositol 1,4,5-trisphosphate to its receptor on ER triggers Ca2+ release from ER, resulting in store-operated (capacitative) Ca2+ entry mediated through voltage-independent Ca2+-permeable cation channels including TRPC and Orai proteins (Lee et al., 2010). In addition, ROCE is activated, and it is mediated via certain TRPCs categorized as ROCCs that operate independently of store depletion. TRPC6 is reported to be involved in ROCE triggered by the stimulation of GqPCRs such as muscarinic (Bousquet et al., 2010) and α1-adrenergic receptors (Suzuki et al., 2007). In the present study, we showed that activation of Gq protein-coupled ETAR induced TRPC6-mediated ROCE but not SOCE. That is, an increase in [Ca2+]i induced by TG-induced Ca2+-depletion/Ca2+-restoration in GFP-expressing cells was not significantly different from that in TRPC6-GFP-expressing cells, indicating that TRPC6 does not contribute to SOCE. Different from no significant Ca2+ influx in response to ET-1 after TG-induced Ca2+-depletion/Ca2+-restoration in the GFP-expressing cells, 10 nM ET-1 was capable of producing additional Ca2+ influx in the TRPC6-expressing cells where SOCE had been maximally activated by TG-induced Ca2+ depletion/Ca2+ restoration. In addition, our fluorescent confocal microscopic observations were consistent with the previous report of Graham et al. (2010) showing that, unlike diffuse distribution of GFP, TRPC6-GFP is predominantly present in the plasma membrane or subplasma membrane of the cells. These data, taken together, indicate that TRPC6 located in plasma membrane functions as an ETAR-activated ROCC but not a store-operated Ca2+ channel.
Other studies have indicated that the negative regulation of TRPC6 by PKA in addition to PKG is an important mechanism underlying the protective effect of cAMP-elevating agent against cardiovascular diseases such as hypertension and cardiac hypertrophy (Kinoshita et al., 2010; Nishida et al., 2010; Nishioka et al., 2011). PKA is the major target of the intracellular second-messenger cAMP, which is synthesized from ATP via AC and inactivated by some members of the PDE superfamily (Boswell-Smith et al., 2006; Pearce et al., 2010). To elucidate the functional role of AC/cAMP/PKA signaling pathway in the regulation of ETAR-operated Ca2+ influx via TRPC6, we used pharmacological agents targeting this pathway. The TRPC6-mediated ROCE in response to ETAR stimulation was markedly reduced in the presence of cAMP-elevating agents, forskolin and papaverine. In contrast, the response was significantly potentiated by pretreatment with a membrane-permeant PKA inhibitor, Rp-8-Br-cAMP, that competitively binds to the cAMP-binding domain of the PKA regulatory subunit and inhibits dissociation of the catalytic subunit from the regulatory subunit (Schwede et al., 2000). Furthermore, either forskolin or papaverine did not change the membrane localization of TRPC6 protein (Fig. 3). cAMP can activate not only PKA but also exchange protein activated by cAMP (EPAC) that functions as guanine nucleotide exchange factors for both Rap1 and Rap2, members of the Ras family of small G proteins (Gloerich and Bos, 2010). However, the possibility that cAMP-dependent EPAC activation by forskolin or papaverine inactivates TRPC6 could be ruled out, because the selective EPAC agonist, 8-(4-chlorophenylthio)-2′-O-methyladenosine-cAMP sodium salt (200 μM), had no or little effect on the ROCE via TRPC6 (data not shown). Taken together, these findings suggest the inactivation of ETAR-activated TRPC6 by the AC/cAMP/PKA signaling pathway. This supports the observation by Nishioka et al. (2011) that cAMP-dependent PKA activation induced by cilostazol, a selective PDE3 inhibitor, results in the suppression of TRPC6-mediated Ca2+ entry. However, conflicting results have been reported on the role of cAMP-dependent signaling pathways in the regulation of TRPC6 activity. As opposed to the findings described in the present and previous studies (Nishioka et al., 2011), cAMP/PKA pathway did not affect SOCE-independent, nonselective cation entry (ROCE) via TRPC6 (Hassock et al., 2002), whereas TRPC6-mediated Ca2+ entry was triggered by cAMP-dependent activation of phosphoinositide 3-kinase/protein kinase B/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 signaling pathway (Shen et al., 2011). The reason for the discrepancy in these experimental results is not clear. However, the discrepancy may be attributed to the difference in the basal phosphorylation state of TRPC6, because TRPC6 is phosphorylated by several types of protein kinases under basal conditions (Fig. 6) (Bousquet et al., 2011).
It is noteworthy that we have found that 1,9-dideoxyforskolin, an inactive analog of forskolin (Pinto et al., 2008, 2009), also inhibited ETAR-operated Ca2+ influx via TRPC6 (Supplemental Fig. S3). The inhibition of the ROCE by 10 μM 1,9-dideoxyforskolin seems to be caused by the activation of AC, because the inhibitory effect is sensitive to 1 mM SQ-22,536, an AC inhibitor. Other studies with purified catalytic AC subunits clearly have suggested that 1,9-dideoxyforskolin binds to AC, whereas the stimulatory activity in AC is not observed (Pinto et al., 2008, 2009). Activation of AC requires both the binding of diterpenes, including forskolin and 1,9-dideoxyforskolin, to the catalytic subunits of AC and an additional conformational switch via a yet unidentified step (Pinto et al., 2009). In native systems such as intact cells the binding of 1,9-dideoxyforskolin to AC may trigger a second conformational switch that results in the activation of catalysis.
Does the activation of PKA via AC/cAMP signaling pathway induce phosphorylation of TRPC6? Hassock et al. (2002) have reported that pharmacological stimulation of cAMP/PKA signaling pathway actually phosphorylates an unidentified phosphorylation site of TRPC6 endogenously expressed in human platelets and exogenously overexpressed in QBI-293A cells (a subclone of HEK293 cells). Other studies have shown that a Thr69 residue within the TRPC6 sequence is phosphorylated by PKA in addition to PKG (Nishida et al., 2010; Nishioka et al., 2011). We analyzed the primary sequence of TRPC6 and found some serine/threonine residues other than Thr69, i.e., Ser14, Ser28, and Ser321, as potential sites for PKA phosphorylation. Both our site-directed mutagenesis and [Ca2+]i measurement experiments have provided the first functional evidence that Ser28 but not Thr69 is involved in the inhibition of TRPC6-mediated ROCE in response to ETAR stimulation by cAMP-elevating agents.
Finally, we have conducted immunoblotting analysis with Phos-tag biotin to clarify whether PKA can phosphorylate TRPC6 on Ser28 residue. Phos-tag biotin is a phosphate-specific ligand with a biotin tag and allows us to detect specifically phosphorylated proteins (Kinoshita et al., 2006). As shown in Fig. 6A, we have confirmed that Phos-tag biotin can discriminate between phosphorylated and unphosphorylated states of TRPC6 proteins. Bousquet et al. (2011) have reported that TRPC6 protein stably expressed in HEK293 cells is phosphorylated under basal conditions and the Ser814 residue contributes to 50% of the basal phosphorylation state, although its functional significance is unknown. Our site-directed mutagenesis approach has revealed that the contribution of Ser14, Ser28, and Thr69 residues to the basal phosphorylation of TRPC6 is minor (Fig. 6, A and B). In vivo treatment of wild-type and mutant cells with a combination of forskolin and papaverine to activate PKA via the AC/cAMP signaling pathway induced little or no significant change in the phosphorylation level, indicating that the high basal phosphorylation masks the relatively weak PKA-mediated phosphorylation of TRPC6. Therefore, we have carried out an in vitro kinase assay with immunoprecipitated wild-type and mutant TRPC6 proteins that are dephosphorylated by phosphatase treatment. We were surprised to find that an in vitro kinase assay revealed that PKA phosphorylates TRPC6 on not only Ser28 but also Thr69, although only Ser28 is involved in the negative regulation of ETAR-mediated ROCE via TRPC6 by the activation of the AC/cAMP/PKA signaling pathway. This discrepancy between [Ca2+]i measurement and in vitro kinase assays could result from the fact that the Thr69 mutant of TRPC6 is functionally resistant to increased PKA activity, because Thr69 but not Ser28 is maximally phosphorylated under basal conditions. Another possibility is that the PKA catalytic subunit at 40 units used in the present study nonspecifically phosphorylates Thr69 as a PKG phosphorylation site in addition to Ser28 as a PKA phosphorylation site, because PKA and PKG are known to have very similar consensus sites.
In summary, we have identified a new phosphorylation site (Ser28) on TRPC6 for PKA in addition to Thr69. We have provided the first evidence that the activation of the AC/cAMP/PKA signaling pathway inhibits ETAR-mediated ROCE via TRPC6 by phosphorylation of Ser28 but not Thr69, although both sites could be phosphorylated by PKA in vitro. In the treatment of IPAH attributable to excessive ETAR signaling and/or Ca2+ entry via TRPC6 (Kunichika et al., 2004; Yu et al., 2004), prostacyclin and its analogs have been used as cAMP-generating drugs to relax contracted pulmonary artery and inhibit the proliferation of pulmonary artery smooth muscle cells (Clapp et al., 2002). Taken together, our findings imply that the negative regulation of ETAR-activated TRPC6 via PKA phosphorylation may be an important therapeutic target for the treatment of PAH with cAMP-elevating agents.
Participated in research design: Horinouchi, Nishiya, and Miwa.
Conducted experiments: Horinouchi, Higa, Aoyagi, and Tarada.
Contributed new reagents or analytic tools: Horinouchi.
Performed data analysis: Horinouchi, Higa, and Aoyagi.
Wrote or contributed to the writing of the manuscript: Horinouchi, Nishiya, and Miwa.
We thank Dr. Yasuo Mori (Kyoto University, Kyoto, Japan) for kindly donating the vector encoding TRPC6 construct.
This study was supported in part by Grants-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Science [grant 21790236] (to T. Horinouchi); Grants-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science [grant 21390068] (to S.M.); and grants from the Smoking Research Foundation of Japan (to S.M.), the Mitsubishi Pharma Research Foundation (to T. Horinouchi), the Pharmacological Research Foundation, Tokyo (to T. Horinouchi), the Shimabara Science Promotion Foundation (to T. Horinouchi), and Actelion Pharmaceuticals Japan Ltd. (to T. Horinouchi).
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- transient receptor potential canonical
- adenylate cyclase
- bovine serum albumin
- intracellular free Ca2+ concentration
- enhanced chemiluminescence
- exchange protein activated by cAMP
- endoplasmic reticulum
- endothelin type A receptor
- fura-2/acetoxymethyl ester
- green fluorescent protein
- Gq protein-coupled receptor
- human embryonic kidney 293
- horseradish peroxidase
- idiopathic pulmonary arterial hypertension
- protein kinase A
- protein kinase G
- receptor-operated Ca2+ channel
- receptor-operated Ca2+ entry
- Rp-8-bromoadenosine-cAMP sodium salt
- store-operated Ca2+ entry
- wild type.
- Received August 31, 2011.
- Accepted October 13, 2011.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics