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J Biol Chem. 2010 Dec 24; 285(52): 40534–40543.
Published online 2010 Oct 20. doi:  10.1074/jbc.M110.160051
PMCID: PMC3003352

Protein Kinase C-dependent Phosphorylation of Transient Receptor Potential Canonical 6 (TRPC6) on Serine 448 Causes Channel Inhibition*An external file that holds a picture, illustration, etc.
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TRPC6 is a cation channel in the plasma membrane that plays a role in Ca2+ entry following the stimulation of a Gq-protein coupled or tyrosine kinase receptor. A dysregulation of TRPC6 activity causes abnormal proliferation of smooth muscle cells and glomerulosclerosis. In the present study, we investigated the regulation of TRPC6 activity by protein kinase C (PKC). We showed that inhibiting PKC with GF1 or activating it with phorbol 12-myristate 13-acetate potentiated and inhibited agonist-induced Ca2+ entry, respectively, into cells expressing TRPC6. Similar results were obtained when TRPC6 was directly activated with 1-oleyl-2-acetyl-sn-glycerol. Activation of the cells with carbachol increased the phosphorylation of TRPC6, an effect that was prevented by the inhibition of PKC. The target residue of PKC was identified by an alanine screen of all canonical PKC sites on TRPC6. Unexpectedly, all the mutants, including TRPC6S768A (a residue previously proposed to be a target for PKC), displayed PKC-dependent inhibition of channel activity. Phosphorylation prediction software suggested that Ser448, in a non-canonical PKC consensus sequence, was a potential target for PKCδ. Ba2+ and Ca2+ entry experiments revealed that GF1 did not potentiate TRPC6S448A activity. Moreover, activation of PKC did not enhance the phosphorylation state of TRPC6S448A. Using A7r5 vascular smooth muscle cells, which endogenously express TRPC6, we observed that a novel PKC isoform is involved in the inhibition of the vasopressin-induced Ca2+ entry. Furthermore, knocking down PKCδ in A7r5 cells potentiated vasopressin-induced Ca2+ entry. In summary, we provide evidence that PKCδ exerts a negative feedback effect on TRPC6 through the phosphorylation of Ser448.

Keywords: Calcium Channels, Calcium Imaging, Ion Channels, Protein Kinase C (PKC), TRP Channels, Calcium Entry, Phosphorylation, TRPC


Ca2+ is a second messenger in all cell types. While the intracellular concentration of Ca2+ ([Ca2+]i) is tightly controlled and normally maintained at low levels, increases modulate cellular functions such as secretion, gene transcription, and the activation of a variety of effectors (1). TRPC6 allows Ca2+ to enter cells from the extracellular medium when it is stimulated by the phospholipase C/inositol-1,4,5-trisphosphate (IP3)2 pathway. This channel is a member of the TRPC family (transient receptor potential canonical), which includes seven members (TRPC1 to TRPC7). Many pathophysiologies arise when TRPC6 expression or activity is dysregulated. Focal segmental glomerulosclerosis (FSGS) is a channelopathy associated with TRPC6 and is caused by missense mutations, including gain-of-function mutations (2, 3). In some cases of idiopathic pulmonary arterial hypertension, the expression of TRPC6 has been reported to be higher in pulmonary artery smooth muscle cells (4, 5). An up-regulation of TRPC6 in hepatocytes has also been linked to liver cancer (6). These examples stress the importance of understanding the mechanisms responsible for the regulation of TRPC6.

The exact mechanism by which TRPC6 is activated is not clear, but it occurs when a GqPCR or tyrosine kinase receptor is activated, leading to IP3 and diacylglycerol (DAG) formation from phosphatidyl-4,5-bisphosphate (PIP2) hydrolysis by phospholipase C. IP3 binds to its receptor, IP3R, on the endoplasmic reticulum leading to Ca2+ release, which is the first phase of Ca2+ mobilization. The second phase takes place as Ca2+ channels, including TRPCs and Orai (7, 8), are activated at the plasma membrane and maintain high intracellular Ca2+ levels as long as the stimulation is sustained. DAG, along with Ca2+, activates protein kinase C (PKC), which regulates the activity of many proteins involved in Ca2+ signaling, including IP3R (9, 10), L-type Ca2+ channels (11), and TRP proteins (12). Trebak et al. showed that TRPC3, the closest relative of TRPC6, is phosphorylated on residue Ser712 by PKC (13), and that the activation of PKC by phorbol 12-myristate 13-acetate (PMA) inhibits 1-oleyl-2-acetyl-sn-glycerol (OAG)-mediated TRPC3 channel activation (13).

It has been shown that TRPC6 is negatively regulated after phosphorylation of threonine 69 by PKG (14). This phosphorylation event is also essential for the anti-hypertrophic effects of phosphodiesterase 5 inhibitors (15,17). Because phosphorylation events play an important role in TRPC6 activity, we verified, in the present study, whether TRPC6 was a substrate for PKC. We showed that PKC can phosphorylate TRPC6 and that the inhibition of PKC by bisindolylmaleimide I (GF1) or Gö 6983 enhances agonist-induced Ca2+ entry into TRPC6-expressing cells. Mutagenesis studies showed that none of the twelve canonical phosphorylation sites for PKC exposed to the intracellular environment is involved in the phosphorylation of TRPC6. Further analysis of the TRPC6 sequence suggested that PKCδ can phosphorylate TRPC6 on Ser448, a non-canonical phosphorylation site for PKC. The mutation of Ser448 to Ala abolished the ability of PKC to phosphorylate TRPC6. In addition, agonist-induced Ca2+ entry into cells expressing TRPC6S448A was not modified by GF1. Furthermore, we showed that PKCδ regulates vasopressin (AVP)-induced Ca2+ entry into A7r5 cells, which endogenously express TRPC6. In summary, we demonstrated that TRPC6 is phosphorylated on a non-canonical site by PKC in cellulo and that this phosphorylation down-regulates the activity of TRPC6.



Cell culture media, serum, Hepes, trypsin, Opti-MEM I, Lipofectamine 2000, Stealth RNAiTM siRNA Negative Control Med GC, and Zero Blunt Topo PCR cloning kits were purchased from Invitrogen (Burlington, ON, Canada). Phosphate-free culture media and G418 were from Wisent (St-Bruno, QC, Canada). Nonidet P-40 was from Roche (Laval, QC, Canada). GF1, Gö 6976, Gö 6983 carbachol (CCh), arginine vasopressin (AVP), phosphatase mixture inhibitor set 2 and Fura-2/AM were from Calbiochem (San Diego, CA). PKCδ siRNA (ON-TARGETplus siRNA duplex, 5′-GCAACGCUGCCAUCCAUAAUU-3′ (sense) and 5′-CUUAUGGAUGGCAGCGUUGCUU-3′ (antisense)) were from Dharmacon (Chicago, IL). Rabbit polyclonal and mouse monoclonal anti-hemagglutinin (HA)-specific antibodies were from Covance (Berkeley, CA). Rabbit polyclonal anti-TRPC6 was from Chemicon (Temecula, CA). Rabbit polyclonal anti-PKCδ (C-17) was from Santa Cruz Biotechnology (Santa Cruz, CA). Peroxidase-conjugated donkey anti-rabbit antibodies, peroxidase-conjugated sheep anti-mouse antibodies, protein A-Sepharose CL-4B, and Biomax MR films were from GE Healthcare (Baie d'Urfé, QC, Canada). Western Lightning Chemiluminescence Reagent Plus, 0.2 μm nitrocellulose membrane, and 32P-orthophosphoric acid were from Perkin-Elmer Life Sciences (Woodbridge, ON, Canada). All primers and oligonucleotides were from Integrated DNA Technologies (Coralville, IA). Phusion High-Fidelity DNA polymerase was from Finnzymes (Espoo, Finland). Restriction enzymes and T4 DNA ligase were from New England Biolab (Pickering, ON, Canada). Unless otherwise stated, all other reagents were from Sigma-Aldrich (Oakville, ON, Canada) or Laboratoire MAT (Quebec City, QC, Canada).

Molecular Biology

Standard molecular biology techniques were used for to isolate, analyze, and clone DNA (18, 19). Point mutations in mouse TRPC6 were introduced using a PCR-based site-directed mutagenesis strategy. The PCR fragment was subcloned into the pCR-Blunt II-TOPO vector using a Zero Blunt TOPO PCR cloning kit. The fragment was sequenced and was then introduced into HA-tagged TRPC6 in pcDNA3.1. All constructs were confirmed by sequencing from double-stranded DNA templates using the dideoxynucleotide termination method (20).

Cell Culture and Transfection

HEK293T cells and A7r5 vascular myocytes were maintained at subconfluence in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 50 units/ml of penicillin, and 50 μg/ml streptomycin at 37 °C in a humidified atmosphere containing 5% CO2. T6.11 cells (HEK293 stably transfected with mouse TRPC6) were cultured in the same medium supplemented with G418 (400 μg/ml). For transient transfections, six-well plates were treated with 0.1 mg/ml poly-l-lysine for 30 min, rinsed with PBS (137 mm NaCl, 3.5 mm KCl, 10 mm sodium phosphate buffer, pH 7.4), and air-dried. A total of 1 μg of plasmid DNA per well was added in 250 μl of Opti-MEM I, to which 2.5 μl of Lipofectamine 2000 diluted in 250 μl of Opti-MEM I was added and thoroughly mixed. After a 20 min of incubation, 8 × 105 HEK293T cells diluted in 1.5 ml of culture medium without antibiotics were added per well. The cells were incubated for 16 h at 37 °C in a humidified atmosphere containing 5% CO2. After a 24-h transfection period, the cells from one well were transferred onto 3 poly-l-lysine coated coverslips for [Ca2+]i measurements or into a 60-mm Petri dish for metabolic labeling assays. Transfection of A7r5 cells with siRNA were performed 24 h after the cells were plated at 1.5 × 105 cells/poly-l-lysine-treated wells (6-well plate). siRNA were diluted in 250 μl of Opti-MEM I and added to each well before adding 5 μl of Lipofectamine 2000 diluted in 250 μl of Opti-MEM I. Final concentration of siRNA was 50 nm. The mixture was incubated for 20 min at room temperature before addition to the A7r5 cells. The siRNA-Lipofectamine 2000 complex was incubated with A7r5 cells for 48 h at 37 °C in a humidified atmosphere containing 5% CO2.

Metabolic Labeling

Stably or transiently transfected cells grown in 60-mm Petri dishes were washed once with phosphate-free DMEM and incubated for 4 h in phosphate-free DMEM supplemented with 250 μCi/ml 32P-inorganic phosphate. The stimulations were performed by adding directly the agonists at 100× in the medium. After the appropriate incubation time, the cells were washed twice with ice-cold PBS prior to being lysed.


The cells were lysed with 1 ml of ice-cold lysis buffer (1.25% Nonidet P-40, 1.25% sodium deoxycholate, 2 mm EDTA, 12.5 mm sodium phosphate, pH 7.2, 1 μg/ml of soybean trypsin inhibitor, 5 μg/ml of leupeptin, 100 μm phenylmethylsulfonyl fluoride) supplemented with a phosphatase inhibitor mixture for 30 min on ice with gentle agitation. They were then scraped from the surface of the Petri dish and centrifuged at 15,000 × g for 15 min at 4 °C. The supernatant was collected and immunoprecipitated with 50 μl of protein A-Sepharose beads (50% slurry) and anti-HA mouse antibody (1:1000) for 2 h at 4 °C. Samples were then centrifuged for 1 min at 4 °C at 800 × g and washed twice with 500 μl of ice-cold lysis buffer. Immunoprecipitated proteins were dissolved in 40 μl of 2× Laemmli buffer and boiled for 5 min before being separated on 7% SDS-polyacrylamide gels. The gels were then either dried and exposed to a film for autoradiography, or the protein bands were transferred to a 0.2-μm nitrocellulose membrane (400 mA for 2 h or 100 mA overnight in 150 mm glycine, 20 mm Tris-base, and 20% methanol) for immunoblotting.


The nitrocellulose membranes to which the whole cell lysates and immunoprecipitated proteins had been transferred were stained with Ponceau S (0.1% in 5% acetic acid) to visualize the marker proteins, destained in TBST (20 mm Tris-HCl, pH 7.5, 137 mm NaCl, 0.1% Tween 20) and blocked in TBST containing 5% (w/v) nonfat dry milk for either 1 h at room temperature or overnight at 4 °C. The membranes were then washed and incubated in TBST for either 2.5 h at room temperature or overnight at 4 °C with specific primary antibodies (rabbit anti-HA (1:1000) or mouse anti-actin (1:10 000)). After three washes with TBST, the membranes were incubated for 1.5 h at room temperature in TBST containing peroxidase-conjugated donkey anti-rabbit-IgG (1:30,000) or peroxidase-conjugated sheep anti-mouse-IgG (1:10,000). The blots were washed three times with TBST and the immune complexes were detected with the Western Lightning Chemiluminescence Reagent Plus kit using the manufacturer's protocol.

Measurement of [Ca2+]i

We used the method described by Zhu et al. (21) to measure [Ca2+]i. Briefly, A7r5, T6.11, or transfected HEK293T cells grown on poly-l-lysine-treated coverslips were washed twice with HBSS (120 mm NaCl, 5.3 mm KCl, 0.8 mm MgSO4, 10 mm glucose, 1.8 mm CaCl2, 20 mm Hepes, pH 7.4) and loaded with Fura-2/AM (1.5 μm in HBSS) for 20 min at room temperature in the dark. After washing and de-esterifying in fresh HBSS for 20 min at room temperature, the coverslips were inserted in a circular open-bottom chamber and placed on the stage of a Zeiss Axovert microscope fitted with an Attofluor Digital Imaging and Photometry System (Attofluor Inc., Rockville, MD). The [Ca2+]i in selected Fura-2-loaded cells was measured by fluorescence videomicroscopy at room temperature using alternating excitation wavelengths of 334 and 380 nm (10 nm bandpass filters), and emitted fluorescence was monitored through a 510 nm dichroic mirror and a 520 nm long pass filter set. Free [Ca2+]i was calculated from the 334/380 fluorescence ratios using the method of Grynkiewicz et al. (22). Reagents were diluted to their final concentrations in HBSS and applied to the cells by surface perfusion. Ca2+-free HBSS was supplemented with 0.5 mm EGTA to chelate any remaining extracellular Ca2+. For the Ba2+ entry assays, 1 mm Ba2+ was added to Ca2+-free, EGTA-free HBSS. For the transient transfections, the cells were co-transfected with cDNA encoding the M5 muscarinic receptor, and only those responding to carbachol (CCh) were analyzed. [Ca2+]i values were recorded every 3 s.


We first investigated whether the inhibition of PKC could modulate CCh-induced Ca2+ mobilization in HEK 293 cells stably transfected with mTRPC6 (T6.11 cells) (23). As shown in Fig. 1A, in the presence of extracellular Ca2+, 50 μm CCh caused a robust increase in [Ca2+]i from a basal value of ~70 nm to a peak value of ~210 nm. The [Ca2+]i then declined slowly and remained above the basal level for several minutes, which constituted a plateau phase. When the cells were pretreated with 100 nm GF1, a highly selective PKC inhibitor, the CCh-induced increase in [Ca2+]i reached a peak value similar to that of untreated cells. However, the [Ca2+]i remained elevated at a plateau of ~220 nm. In control HEK293 cells expressing angiotensin II receptor type 1 instead of TRPC6, the CCh-induced increase in [Ca2+]i was similar to that of the T6.11 cells, but the [Ca2+]i declined quickly to a plateau level of 130 nm within 45 s after the stimulation (Fig. 1B). Under the same conditions, the [Ca2+]i in the T6.11 cells after 45 s was 165 nm (Fig. 1A). In HEK293 cells expressing the angiotensin II receptor type 1, GF1 slightly increased the peak [Ca2+]i, but Ca2+ levels quickly returned to values similar to those of untreated cells (Fig. 1B).

Inhibition of PKC with GF1 potentiates CCh-induced Ca2+ entry into TRPC6-expressing cells. A, monitoring of [Ca2+]i in T6.11 cells loaded with Fura-2. Cells were treated with 100 nm GF1 (filled circles) or not (open circles) for 30 s before stimulating ...

To discriminate between CCh-induced Ca2+ release and CCh-induced Ca2+ entry, we used a Ca2+ depletion/Ca2+ readdition protocol. Cells were incubated for 30 s in Ca2+-free medium containing 0.5 mm EGTA before depleting the intracellular Ca2+ stores with 5 μm CCh. Once the [Ca2+]i had returned to the basal level (2 min after the addition of CCh), the extracellular medium was replaced with medium containing 1.8 mm CaCl2. As shown in Fig. 2, in the absence of extracellular Ca2+, CCh-induced Ca2+ release was similar in TRPC6- and mock-transfected cells. When 1.8 mm CaCl2 was added to TRPC6-transfected cells, Ca2+ entry raised the [Ca2+]i to a plateau level of ~190 nm (Fig. 2A). In the mock-transfected cells, the Ca2+ entry raised the [Ca2+]i to a lower plateau level of ~160 nm. Pretreating the cells with GF1 increased CCh-induced net Ca2+ entry by 52.4 ± 13.1 nm and 21.9 ± 10.0 nm in TRPC6- and mock-transfected cells, respectively (Fig. 2B). Net Ca2+ entry was calculated by subtracting the basal [Ca2+]i from the maximal [Ca2+]i recorded once the Ca2+ had been restored. These results suggested that the inhibition of PKC causes increased TRPC6 activity and that PKC exerts an inhibitory effect on TRPC6. We next investigated how the activation of PKC with PMA could influence TRPC6-mediated Ca2+ entry. Because PMA greatly decreased CCh-induced IP3 production in the T6.11 cells (data not shown), we performed the Ca2+ depletion/Ca2+ readdition protocol by stimulating the cells with 100 μm ATP. As shown in Fig. 2C, ATP caused a net Ca2+ entry of 38.4 ± 16.5 nm while a pretreatment of the cells with GF1 increased ATP-induced net Ca2+ entry to 78.7 ± 28.7 nm. The presence of PMA before the stimulation with ATP reduced Ca2+ entry by 75% to a net Ca2+ entry of 9.2 ± 3.7 nm. These results further suggested that PKC decreases the activity of TRPC6.

PKC modulates agonist-induced Ca2+ entry. A, [Ca2+]i values were recorded in HEK293T transiently transfected with pcDNA3 (square) or TRPC6 (circle). Cells were incubated in the absence of extracellular Ca2+ (in the presence of 0.5 mm EGTA) for 30 s before ...

To confirm that the observed effect of PKC was due to a direct regulation of TRPC6, we exploited two characteristics that are distinctive of TRPC6. First, it has been shown that CCh generates a marked increase in receptor-operated Ca2+ entry (ROCE) in addition to store-operated Ca2+ entry (SOCE) in HEK293 cells overexpressing TRPC6 (23). The depletion of the intracellular Ca2+ store was induced with 1 μm thapsigargin, an irreversible inhibitor of the SERCA that causes a rapid leak of Ca2+ from the endoplasmic reticulum and triggers store-operated channels. Fig. 3A shows that after an incubation of T6.11 cells with thapsigargin for 5 min, which completely depleted the intracellular Ca2+ store (24, 25), 50 μm CCh caused an increase in [Ca2+]i to a peak value of 245 nm, which slowly declined to a value of 140 nm 2 min after the addition of CCh. In the presence of GF1, CCh still caused an increase in [Ca2+]i to a peak value similar to that of control cells, but the [Ca2+]i remained at a plateau value of 265 nm for at least 2 min after the addition of CCh. The net Ca2+ entry measured 2 min after the addition of CCh was 18.2 ± 11.5 nm in untreated cells and 136.6 ± 14.1 in GF1-treated cells (Fig. 3B).

PKC inhibits ROCE. A, Fura-2-loaded T6.11 cells were stimulated with 1 μm thapsigargin (Tg) in the presence of extracellular Ca2+ to induce store depletion and SOCE. CCh (50 μm) induced ROCE after 330 s. Cells were co-incubated with 100 ...

Another distinctive characteristic of TRPC6 is that it can be activated by high concentrations of OAG, a non-metabolizable DAG analog (26,28). Fig. 4A shows that 100 μm OAG caused a slow elevation of [Ca2+]i that reached a value of 122 nm in 1 min in T6.11 cells. In the presence of 100 nm PMA, OAG-induced Ca2+ entry decreased to 33.3 ± 11.0% of that of control cells (Fig. 4B). In the presence of 100 nm GF1, OAG-induced Ca2+ entry increased to 144 nm (Fig. 4A), which corresponds to a relative elevation of 162.3 ± 24.1% of that of control cells (Fig. 4B). These results further suggested that PKC down-regulates the activity of TRPC6.

PKC inhibits OAG-induced Ca2+ entry. A, Fura-2-loaded T6.11 cells were pretreated with 100 nm PMA (open triangles), 100 nm GF1 (filled circles), or not (open circles) for 60 s and were then stimulated with 100 μm OAG in the presence of extracellular ...

To determine whether PKC phosphorylates TRPC6 in cellulo, HEK293 cells expressing TRPC6 were metabolically labeled using [32P]orthophosphate. PKC was then activated through the stimulation of the muscarinic receptor with 50 μm CCh. Fig. 5A shows that the immunoprecipitated TRPC6 was weakly phosphorylated under basal conditions. CCh increased the phosphorylation level of TRPC6 by 1.34 ± 0.17-fold, compared with the basal level (Fig. 5B). To further determine whether CCh-induced TRPC6 phosphorylation was due to PKC, cells were pre-incubated for 5 min with GF1 before the stimulation with CCh. GF1 did not modify the basal level of phosphorylation of TRPC6 (0.97 ± 0.05 times the basal level), but it completely inhibited the effect of CCh on TRPC6 phosphorylation (0.98 ± 0.13 times the basal level) (Fig. 5, A and B). These results suggested that CCh-induced TRPC6 phosphorylation occurs through a PKC-dependent pathway.

PKC phosphorylates TRPC6 following a stimulation with CCh. A, metabolic labeling was carried out as described under “Experimental Procedures.” T6.11 cells were pretreated or not with 500 nm GF1 5 min prior to stimulation with 50 μ ...

It has previously been shown that the phosphorylation of Ser712 in TRPC3 by PKC abolishes TRPC3 activity (13). Because Ser712 in TRPC3 is part of a highly conserved five-residue motif (PS712PKS) common to all TRPC members, we first substituted the equivalent residue (Ser768) of TRPC6 for alanine. Because Ba2+ is a poor substrate for ATPase pumps (29), and its influx is weak through endogenous channels in HEK293 cells, we measured the activity of TRPC6 and TRPC6S768A using a Ca2+ depletion/Ba2+ readdition protocol. Cells were incubated for 30 s in Ca2+-free medium containing 0.5 mm EGTA before depleting the intracellular Ca2+ stores with 5 μm CCh. Once the [Ca2+]i had returned to the basal level (2 min after the addition of CCh), 1.0 mm Ba2+ was added extracellularly. After CCh-induced Ca2+ store depletion, mock-transfected cells displayed very weak Ba2+entry that was not modified in the presence of GF1 (Fig. 6A, upper panel). In the case of TRPC6-expressing cells, CCh induced steady Ba2+ entry that was potentiated by GF1 (Fig. 6A, middle panel). Unexpectedly, in the case of TRPC6S768A-expressing cells, CCh induced steady Ba2+entry that was also potentiated by GF1 (Fig. 6A, lower panel). Fig. 6B shows the net fluorescence (ΔF340) increase recorded 2 min after the addition of Ba2+. These results indicated that Ser768 is not involved in the PKC-mediated inhibition of TRPC6.

The TRPC6S768A mutant is inhibited by PKC. A, HEK293T cells transiently transfected with pcDNA3 (upper panel), TRPC6 (middle panel), or TRPC6S768A (lower panel) were stimulated with 5 μm CCh as in Fig. 2A. However, rather than Ca2+, 1 mm BaCl ...

Because the amino acid sequence of TRPC6 contains 11 other intracellular consensus motifs ((S/T)X(R/K)) for PKC phosphorylation (supplemental Table S1), we next individually substituted all the serine and threonine residues for alanine and evaluated the functionality of the mutant channels and their sensitivity to PKC. Mutants TRPC6T629A and TRPC6S928A were poorly expressed and their activity could not be evaluated. Fig. 7 shows that GF1 could potentiate Ba2+ entry through wild-type TRPC6 as well as all the TRPC6 mutants. These results suggested that none of the conventional PKC phosphorylation motifs is involved in the modulation of TRPC6 by PKC.

All PKC consensus TRPC6 mutants remain potentiated by GF1. HEK293T cells transiently transfected with pcDNA3 (mock), TRPC6 (T6WT), or mutant TRPC6 were stimulated with 5 μm CCh, and Ba2+ entry was monitored as described in the legend of Fig. 6 ...

GPS2.1 computational software (30) predicts that PKCδ could phosphorylate TRPC6 on four additional putative phosphorylation sites (supplemental Table S2). We mutated two of these, Thr69 and Ser448, into Ala and investigated the impact on GF1-potentiated TRPC6 activity. GF1 potentiated CCh-induced Ba2+ entry into cells expressing TRPC6T69A (data not shown). Interestingly, however, CCh-induced Ba2+ entry was not potentiated by GF1 in cells expressing TRPC6S448A (Fig. 8A). The activity of TRPC6S448A was also investigated by measuring CCh-induced Ca2+entry, which was higher in cells expressing TRPC6S448A than in cells expressing TRPC6 (Fig. 8B). PKC (activated by CCh) likely did not exert its inhibitory effect on mutant TRPC6S448A. In support of this interpretation, Fig. 8B also shows that GF1 did not modify CCh-induced Ca2+entry into cells expressing TRPC6S448A whereas it potentiated CCh-induced Ca2+entry into cells expressing TRPC6. As shown in Fig. 2B, GF1 only slightly enhanced CCh-induced Ca2+ entry in mock-transfected cells whereas in cells expressing TRPC6, it potentiated CCh-induced Ca2+ entry 2.65 ± 1.57-fold compared with mock-transfected cells (Fig. 8C). However, in cells expressing TRPC6S448A, GF1 barely increased CCh-induced Ca2+ entry compared with mock-transfected cells (0.37 ± 0.09-fold) (Fig. 8C). These results demonstrated that Ser448 mutation abolishes the sensitivity of TRPC6 to PKC.

PKC does not inhibit TRPC6S448A. A, HEK293T cells transiently transfected with pcDNA3, TRPC6, or TRPC6S448A were treated or not with 100 nm GF1 and then stimulated with 5 μm CCh for 120 s before adding 1 mm BaCl2. Net GF1-induced potentiation ...

To investigate whether TRPC6S448A is a substrate for PKC, HEK293 cells were metabolically labeled using [32P]orthophosphate. Under these conditions, PMA increased the phosphorylation level of TRPC6 (Fig. 9A) 1.63 ± 0.20-fold over the basal phosphorylation level. In addition, the increase was prevented in the presence of GF1 (1.07 ± 0.22-fold over the basal level) (Fig. 9B). Under the same conditions, the basal phosphorylation level of TRPC6S448A was similar to that of TRPC6 (1.01 ± 0.19), while PMA had no effect (1.18 ± 0.23) (Fig. 9B). As expected, GF1 did not modify the phosphorylation level of TRPC6S448A in the presence of PMA (1.16 ± 0.27). These results suggested that Ser448 of TRPC6 is phosphorylated by PKC and is responsible for the PKC-mediated inhibition of TRPC6.

TRPC6S448A is not phosphorylated following PKC activation. A, HEK293T cells transiently transfected with TRPC6 or TRPC6S448A were metabolically labeled, treated with 500 nm GF1 or not for 5 min, and then stimulated with 1 μm PMA or not for 5 min. ...

It has previously been shown that the A7r5 cells express high levels of TRPC6 and that knockdown of TRPC6 decreases AVP-induced Ca2+ entry by 50–70% (31,33). We investigated the influence of PKC on AVP-induced Ca2+ entry in A7r5 cells. Using the Ca2+ depletion/Ca2+ readdition protocol, we showed that 100 nm AVP caused a net Ca2+ entry of 82.6 ± 26.3 nm 2 min after extracellular Ca2+ was restored. PMA (100 nm) did not alter the Ca2+ release phase (Fig. 10A). However, net AVP-induced Ca2+ entry decreased to 21.2 ± 12.2 nm in cells pretreated with PMA (Fig. 10C). To evaluate the implication of novel PKC isoforms in the inhibition of AVP-induced Ca2+ entry, we used the two structurally similar PKC inhibitors, Gö 6983 and Gö 6976. Gö 6983 can inhibit conventional PKC and novel PKC with similar potencies whereas Gö 6976 is inactive on novel PKC (34, 35). Fig. 10, B and C show that 100 nm AVP caused a net Ca2+ entry (amplitude of 90.1 ± 17.4 nm) measured 2 min after extracellular Ca2+ was restored. In the presence of Gö 6976, AVP-induced net Ca2+ entry was similar to that of the control (75.3 ± 18.3 nm), whereas Gö 6983 potentiated AVP-induced net Ca2+ entry (153.3 ± 38.4 nm). These results suggest that a novel PKC isoform is involved in the inhibition of AVP-induced Ca2+ entry in A7r5 cells. The specific role of PKCδ was assessed by transfecting A7r5 cells with PKCδ siRNA. Treatment of cells with 50 nm PKCδ siRNA significantly reduced the expression of PKCδ to 47.0 ± 4.6% (Fig. 11, A and B). Nonspecific PKCδ siRNA showed no effect. The consequences of knocking down the expression of PKCδ on AVP-induced Ca2+ entry were evaluated using the Ca2+ depletion/Ca2+ readdition protocol. Fig. 11C shows that AVP-induced Ca2+ entry was higher in cells knocked down for PKCδ compared with control cells. In cells treated with siCtl, AVP-induced net Ca2+ entry was 122.8 ± 8.4 nm, whereas in cells treated with siPKCδ, AVP-induced net Ca2+ entry was 215.5 ± 30.8 nm (Fig. 11, C and D). These results demonstrated that the activity of endogenously expressed TRPC6 in A7r5 cells is modulated by PKCδ as with recombinant TRPC6 expressed in HEK293 cells.

AVP-induced Ca2+ entry in A7r5 cells is inhibited by PKC. A7r5 cells were incubated in the absence of extracellular Ca2+ (in the presence of 0.5 mm EGTA) for 30 s before being stimulated with 100 nm AVP. External Ca2+ (1.8 mm) was restored after 180 s. ...
Depletion of PKCδ in A7r5 enhances AVP-induced Ca2+ entry. A7r5 cells were transfected with 50 nm of siRNA specific for PKCδ (siPKCδ) or universal negative control (siCTL). A, After 48 h of transfection, cells were solubilized ...


PKC plays an important role in cellular functions by regulating many different signaling pathways, including Ca2+ entry (36,38). In the present study, we showed that PKC also inhibits the activity of TRPC6. These results are in agreement with those of Shi et al. (39), who showed that ionic currents in HEK293 cells transfected with TRPC6 are inhibited by PKC following the stimulation of the muscarinic receptor. We also observed an inhibitory effect of PKC on TRPC6 activity in cells endogenously expressing TRPC6. Stimulating mesenteric artery myocytes with angiotensin II activates two different types of current (40). One of these currents, which is inhibited by an intracellular application of an anti-TRPC6 antibody, is down-regulated by PKC. In PC12D cells, CCh-induced Ba2+ entry is abolished after an siRNA knockdown of TRPC6 (41). CCh-induced Ba2+ entry is also inhibited by PKC in these cells. All evidence to date indicates that the activity of TRPC6 is down-regulated by PKC.

In this study, we also showed that inhibition of PKC slightly increases the CCh-induced Ca2+ entry in HEK293 cells. HEK293 cells endogenously express TRPC1 and TRPC3 (21, 42). Previous studies have shown that PKC enhances the activity of TRPC1 (40, 43, 44) and inhibits the activity of TRPC3 (13, 45, 46), as well as TRPC4 (45), and TRPC5 (45, 47). Also, PKC negatively regulates the activity of Orai1 (48). Therefore, the effects observed with the mock-transfected cells could result from an assortment of PKC-induced inhibition and activation of channels activity.

We showed that TRPC6 expressed in HEK 293 cells is slightly phosphorylated under basal conditions and that the level of phosphorylation is increased by CCh. The CCh-induced TRPC6 phosphorylation was prevented by GF1, a specific PKC inhibitor. It has previously been shown that Ser712 in human TRPC3 is phosphorylated by PKC and that phosphorylation causes a loss of channel activity (13). For mouse TRPC6, the equivalent residue is Ser768. We showed that the activity of TRPC6S768A is similar to that of wild-type TRPC6, and that activity increased following the inhibition of PKC. Mutations of nine putative consensus phosphorylation sites for PKC ((S/T)X(R/K)) in TRPC6 revealed that none of them is involved in PKC-mediated inhibition of TRPC6. The phosphorylation prediction software, GPS 2.1, identifies four putative phosphorylation sites for PKCδ. One of the potential phosphorylation sites, Ser448, was in a sequence that was favorable for phosphorylation by PKCδ. An oriented peptide library was used to show that the optimal sequence for phosphorylation by PKCδ contains a Phe at position p + 1 and hydrophobic residues at p + 4 and p + 5. In addition, basic residues at p + 2, p + 3, and p + 4 are not favorable for phosphorylation (49, 50). The sequence surrounding Ser448 of TRPC6 contains Phe at p + 1, Thr at p + 2, Ile at p + 3, Phe at p + 4, and Leu at p + 5, which corresponds to the criterion for PKCδ phosphorylation. As expected, after being stimulated with CCh, mutant TRPC6S448A displayed greater activity than wild-type TRPC6. Moreover, GF1 did not potentiate CCh-induced Ba2+ or Ca2+ entry into cells expressing TRPC6S448A. More importantly, PMA did not increase the level of phosphorylation of TRPC6S448A. All these results suggested that Ser448 is phosphorylated by a non-conventional PKC. Further studies are needed to determine the involvement of PKCδ or other isoforms of PKC in the phosphorylation of TRPC6. Kim and Saffen (51) previously showed that, in PC12D cells, PKC phosphorylates TRPC6 on Ser768. In PC12D cells, TRPC6 forms a multiprotein complex that includes PKCα. However, the functional consequence of phosphorylating Ser768 on TRPC6 by PKC had not yet been investigated. The results of all these studies suggest that TRPC6 can be phosphorylated on at least two distinct sites, depending on which isoform of PKC is activated and on the cellular context.

In A7r5 cells, TRPC6 is a major component in the AVP-induced cation current (33, 52, 53). We showed that, in A7r5 cells, Gö 6983 strongly potentiated AVP-induced Ca2+ entry, whereas Gö 6976, which is inactive on novel PKC isoforms, did not potentiate AVP-induced Ca2+ entry. Furthermore, PMA nearly abolishes Ca2+ entry. This effect of PMA has also been reported by Soboloff et al. (32) who used OAG to activate TRPC6. Also, it has been shown that in smooth muscle cell from rat intrapulmonary arteries, sphingosylphosphorylcholine potentiated the contractile responses induced by prostaglandin F and U436619. This potentiation, which is attributed to enhancement of Ca2+ entry, was abolished by the broad spectrum PKC inhibitor Ro-31–8220, but not by Gö 6976 (54). Finally, it was observed that in PKCδ-deficient mast cells, a cell type expressing TRPC6 (55, 56), the intracellular Ca2+ concentration is elevated (57). Thus, all these results, which were obtained with recombinant cell models and cell models endogenously expressing TRPC6, suggest that PKCδ plays a major role in regulating Ca2+ entry.

In summary, our study demonstrated that PKC inhibits TRPC6 activity by phosphorylation of Ser448. Because both PKC and TRPC6 are activated following GqPCR signaling, PKC likely operates a negative feedback mechanism on TRPC6 activity to weaken Ca2+ entry.

Supplementary Material

Supplemental Table 2 (.pdf, 11 KB)


We thank Drs. Gaétan Guillemette and Emanuel Escher, Université de Sherbrooke, for the HEK293 cells expressing the angiotensin II receptor type 1.

*This work was supported by grants from the Canadian Institutes of Health Research, the Natural Sciences and Engineering Research Council of Canada, and the Quebec Heart & Stroke Foundation.

An external file that holds a picture, illustration, etc.
Object name is sbox.jpgThe on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and S2.

This work is part of S. M. Bousquet's PhD thesis at the Université de Sherbrooke.

2The abbreviations used are:

IP3 receptor
phorbol myristate acetate
G-protein-coupled receptor
human embryonic kidney cell clone 293
bisindolylmaleimide I
store-operated Ca2+ entry
receptor-operated Ca2+ entry
transient receptor potential canonical.


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