TRPC6 mutations associated with focal segmental glomerulosclerosis cause constitutive activation of NFAT-dependent transcription

Keywords: calcium, calcineurin, phosphatidylinositol 3-kinase, podocyte, canonical transient receptor potential channel

Reagents and plasmids.

Full-length human TRPC6 cDNA containing an amino-terminal FLAG tag sequence was cloned into pcDNA3.1 and pcDNA4/TO/myc-His B (Invitrogen) using standard PCR-based techniques. Various FSGS-associated mutations (46, 58), as well as the dominant-negative pore mutation changing amino acids LFW to AAA (21), were introduced into this construct using the QuikChange II XL site-directed mutagenesis kit from Stratagene. All plasmids were sequenced to confirm the presence of mutations and exclude the presence of unwanted mutations. The angiotensin II type I receptor (AT₁R) expression plasmid (16) was kindly provided by M. Lopez-Ilasaca. Green fluorescent protein-tagged NFATc3 expression plasmid (NFATc3-GFP) was the gift of F. McKeon (56). The dual luciferase assay kit and reporter vectors pGL4.30 and pGL4.74 were obtained from Promega.

Angiotensin II, carbachol, cyclosporine A, FK506, KN-93, and poly-l-lysine were obtained from Sigma-Aldrich. Genistein, LY-294002, PP2, and U-73122 were purchased from Calbiochem; 1-oleoyl-2-acetyl-sn-glycerol (OAG) was obtained from Avanti Polar Lipids; and EZ-Link Sulfo-NHS-biotin was from Pierce. Affinity-purified rabbit anti-TRPC6 polyclonal antibody was purchased from Chemicon, and FLAG M2 and anti-actin monoclonal antibodies were purchased from Sigma. Fura-2 AM and probenecid were obtained from Invitrogen.

Cell culture and luciferase assays.

Human embryonic kidney (HEK)-293T cells were obtained from American Type Culture Collection (ATCC) and maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum and penicillin G, streptomycin, and amphotericin B. T-Rex-293 cells (Invitrogen) stably expressing the M1 acetylcholine receptor (M1R cells) were maintained in the same media as the 293T cells supplemented with 5 μg/ml blasticidin and 1 mg/ml G418. M1R cells were transfected with pcDNA4/TO/myc-His containing FLAG-tagged wild-type or mutant TRPC6 cDNA using Fugene 6 (Roche) following the manufacturer's protocol. After 24 h, the cells were passaged and placed in selection media containing 200 μg/ml Zeocin (Invitrogen). Individual clones were selected and expanded. FLAG-TRPC6 expression in individual clones was assessed by Western blot after the cells had been exposed to 5 μg/ml tetracycline, or carrier, for 24–48 h. SYF cells (murine embryonic fibroblasts lacking the Src-family kinases Src, Yes, and Fyn) were obtained from ATCC and cultured as directed by the provider (30). Conditionally immortalized human podocytes were the kind gift of M. Saleem and were maintained under permissive conditions as previously detailed (47).

For luciferase assays, cells were plated in 24-well plates and transfected with Fugene 6 (Roche) following the manufacturer's protocol. Cells were transfected with a 50:1 mixture of pGL4.30 (NFAT-response element) and pGL4.74 (Renilla luciferase under a thymidine kinase promoter), and an equal amount of control vector (pcDNA4) or TRPC6 expression vector. AT₁R expression plasmid was included when indicated. Twenty-four hours after transfection, cells were serum starved overnight, followed by a further 6-h incubation in the presence or absence of various stimuli and inhibitors. Cells were washed once in PBS and then lysed in 100 μl of passive lysis buffer (Roche). The dual luciferase reporter assay (DLR; Roche) was performed with 20 μl of lysate using the manufacturer's protocol and a Veritas microplate luminometer (Turner Biosystems). All results were normalized to the thymidine kinase-Renilla luciferase internal control and are presented as a fold increase relative to the control condition.

Western blot analysis.

Residual cell lysates from the dual luciferase assay were spun at 15,000 g for 15 min. The resulting supernatant was mixed with sample loading buffer, separated by SDS-PAGE, and transferred to polyvinylidene difluoride membrane (Bio-Rad). The membrane was blocked with 5% nonfat milk in PBS with 0.05% Tween 20 (PBST) for 1 h at room temperature, followed by overnight incubation in 1:1,000 anti-TRPC6 polyclonal antibody in 5% nonfat milk PBST. After three washes in PBST, blots were incubated in 1:3,000 goat anti-rabbit IgG conjugated to horseradish peroxidase (Pierce) in PBST at room temperature, followed by detection with SuperSignal West Pico chemiluminescent substrate (Pierce).

NFATc3-GFP localization.

M1R cells were plated on collagen I-coated coverslips and transfected with Fugene 6 and a 1:3 ratio of NFATc3-GFP and TRPC6 expression plasmid (or control vector). After 18–30 h, cells were fixed in 4% paraformaldehyde in PBS for 10 min at room temperature, stained with 1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI; Sigma) for 5 min, washed several times with PBS, and mounted on slides with Fluoromount-G (Southern Biotech). Cells were scored for GFP localization using a Nikon E-1000 microscope. GFP localization was categorized as either cytoplasmic (C), cytoplasmic greater than nuclear (C>N), nuclear greater than cytoplasmic (N>C), or exclusively nuclear (N). At least 100 GFP positive cells over several random high-power fields were scored for each transfection.

Cell surface biotinylation.

Cells were processed either 36 to 48 h after transient transfection or 24 to 36 h after TRPC6 expression was induced by the addition of tetracycline. Cells were washed and incubated in ice-cold PBS containing 1 mM calcium and magnesium for 10 min, followed by a 30-min incubation in ice-cold PBS supplemented with 0.5 mg/ml Sulfo-NHS-biotin. The biotin solution was removed and the cells were incubated for an additional 10 min in ice-cold Tris-buffered saline supplemented with 10 mM glycine to quench any remaining reactive biotin cross-linker. Cells were then lysed in PBS with 1% (vol/vol) Nonidet P-40 and Complete protease inhibitor (Roche). Lysates were briefly sonicated and cleared by centrifugation. An aliquot of lysate was set aside and mixed with sample loading buffer. The remaining lysate was incubated with 20 μl of a 50% slurry of streptavidin beads (Pierce) in lysis buffer and incubated at 4°C for 2 h. The beads were washed three times with 1 ml of lysis buffer, and bound material was eluted by boiling in sample loading buffer. Total TRPC6 and biotinylated TRPC6 were detected by Western blot analysis as above. The relative intensities of total and biotinylated TRPC6 were determined using ImageJ software (National Institutes of Health, Bethesda, MD).

Deglycosylation.

M1R cells transiently transfected with various TRPC6 expression constructs were lysed 36–48 h after transfection. Cleared lysates were either mock treated or treated with peptide N-glycosidase F (PNGase F; New England Biolabs) as per the manufacturer's recommendations, followed by SDS-PAGE and detection of TRPC6 by Western blot analysis.

Electrophysiology.

HEK-293 cells were transiently transfected with 1 μg of the wild-type or mutant TRPC6 cDNAs and 0.1 μg of GFP. The cells were used for electrophysiological recordings 24–72 h after transfection.

Macroscopic currents were recorded in the whole cell configuration of the patch-clamp technique using an EPC-9 amplifier and Patchmaster software (HEKA). Patch pipettes had a resistance of 1.5–3 MΩ and were filled with a solution consisting of (in mM) 140 CsAsp, 10 EGTA, 10 HEPES, 2.27 MgCl₂, and 1.91 CaCl₂, pH 7.2 with CsOH. The bath solution contained (in mM) 145 NaCl, 5 KCl, 1 MgCl₂, 2 CaCl₂, 10 HEPES, and 10 glucose, pH 7.4 with NaOH. For all of the experiments, the holding potential was −30 mV, and 400-ms ramps from −100 mV to 100 mV were applied every 4 or 5 s. The data were collected at 10 kHz and filtered at 2.9 kHz. Liquid junction potential correction was not used. A saturating concentration of a TRPC6 blocker (34) was used to determine the TRPC6-specific component of the basal currents plotted in Fig. 5. Experiments were performed at room temperature.

Calcium imaging.

M1R cells were plated on poly-l-lysine-coated coverslips and transfected with Fugene 6 and plasmids encoding for wild-type or mutant TRPC6 in addition to GFP downstream of an internal ribosome entry site sequence. In the case of tetracycline-inducible stable cells, cells were plated on coverslips in the presence of 2.5 μg/ml tetracycline. Sixteen to twenty-four hours after transfection or addition of tetracycline, cells were loaded in DME media without serum containing 2 μM Fura-2 and 2.5 μM probenecid. Cells were washed once in physiological saline solution (in mM: 135 NaCl, 5 KCl, 1.8 CaCl₂, 0.8 MgCl₂, 10 HEPES, and 5.5 glucose, pH 7.4) and then imaged in physiological saline solution at room temperature. Transiently transfected cells were identified by the presence of GFP. Fura-2 ratios (F₃₄₀/F₃₈₀) were determined using an Olympus IX70 inverted microscope, and image analysis was performed using Slidebook 4.2 (Intelligent Imaging Solutions).

RT-PCR.

RNA was isolated from M1R cells and human podocytes maintained under permissive conditions using the RNeasy Plus Mini kit (Qiagen). Complementary DNA was generated from 1.5 μg of RNA using the Transcriptor First Strand cDNA synthesis kit (Roche) with the oligo(dT) primer; control reactions were performed by omitting the reverse transcriptase. Primer sequences to detect human GAPDH, TRPC1, and TRPC3-7 (PrimerBank IDs 7669492a2, 4507685a1, 4507687a1, 7706747a1, 6912736a1, 5730102a1, and 9966865a1, respectively) were obtained from PrimerBank (57). Previously published primer pairs were used to detect NFATc1-NFATc4 transcripts (41). Semiquantitative PCR was performed using the indicated primers for 30 cycles with an annealing temperature of 55°C. PCR products were resolved on a 2% agarose gel and detected with ethidium bromide.

Statistical analysis.

All statistical analysis was performed using GraphPad Prism4 (GraphPad Software). Luciferase data were analyzed by repeated-measures one-way ANOVA using log-transformed results and using the Bonferroni correction for multiple comparisons. Surface expression results were analyzed by one-way ANOVA using Dunnett's test. Fura-2 ratios were compared using the Kruskal-Wallis and Dunn's multiple-comparison tests.

A subset of FSGS-mutations in TRPC6 enhance NFAT activation.

To examine the effect of disease-associated mutations in TRPC6 on activation of the calcineurin-NFAT pathway, we used a luciferase reporter assay. An NFAT-response element driven firefly luciferase reporter plasmid and a thymidine kinase promoter-driven Renilla luciferase control plasmid were cotransfected with either wild-type TRPC6, one of the FSGS-associated TRPC6 mutants, or a known dominant-negative TRPC6 pore mutant. We used an HEK-293T cell line that overexpresses the M1 acetylcholine receptor (referred to as M1R cells); stimulation of the M1 receptor with carbachol in these cells leads to TRPC6 activation [(46) and data not shown]. NFAT-mediated transcription was assessed either in the absence of exogenous stimuli, or after stimulation with carbachol.

Overexpression of wild-type TRPC6 led to a modest increase in basal NFAT-mediated transcription (Fig. 1, A and B). Carbachol stimulation for 6 h led to a robust increase in NFAT-responsive reporter activity in the absence of overexpressed TRPC6, as has previously been described (4). This response was not significantly enhanced by the coexpression of wild-type TRPC6. Basal and carbachol-stimulated NFAT activation by amino terminal mutants N143S and S270T, as well as the carboxyl-terminal truncation mutant K874*, was not markedly different from wild-type TRPC6. In contrast, TRPC6 P112Q, R895C, and E897K, all of which have previously been shown to display enhanced current amplitudes or delayed current decay (46, 58), were able to markedly stimulate the NFAT-responsive reporter in the absence of stimulation, with a further increase in reporter activity upon carbachol treatment. This enhanced reporter activity was not due to increased channel expression of these three mutants compared with wild-type TRPC6 (Fig. 1C). Overexpression of a known dominant-negative TRPC6 mutant, generated by altering the conserved pore domain LFW sequence to AAA (21), was not able to suppress basal or carbachol-stimulated NFAT signaling compared with control transfected cells (Fig. 1A), indicating that while TRPC6 is capable of enhancing M1 receptor-mediated NFAT activation, it is not necessary.

To confirm the ability of TRPC6 and FSGS-associated mutants to activate the calcineurin-NFAT pathway, we examined the subcellular localization of a GFP-tagged fragment of NFATc3. This fusion protein, similar to wild-type NFAT proteins, is excluded from the nucleus under basal conditions, but it translocates to the nucleus upon calcineurin-mediated dephosphorylation (56). In unstimulated M1R cells not expressing TRPC6, NFATc3-GFP is exclusively present in the cytoplasm in >80% of cells, with no cells showing a predominantly nuclear GFP signal (Fig. 1, D and E). Cotransfection of wild-type TRPC6 led to a predominantly nuclear GFP signal in slightly more than 10% of cells. This increased to 40–50% when P112Q, R895C, or E897K mutant TRPC6 was expressed. Dominant-negative TRPC6 did not substantially affect NFATc3-GFP localization.

The ability of TRPC6 mutants to activate basal NFAT-mediated transcription was not dependent on the overexpression of M1 acetylcholine receptor, because the P112Q, R895C, and E897K mutants were more effective than wild-type TRPC6 in enhancing NFAT-driven luciferase activity in HEK-293T cells lacking the M1 receptor (data not shown) and in SYF murine embryonic fibroblasts and a human podocyte cell line (see below). Furthermore, the ability of the R895C mutant to enhance G protein-coupled receptor-mediated NFAT activation also extended to signaling via the AT₁R (data not shown), consistent with the role of TRPC6 in mediating angiotensin II-mediated NFAT activation and cardiac hypertrophy (44).

NFAT activation by TRPC6 in podocytes.

Although it has not been formally demonstrated that it is abnormal TRPC6 function in glomerular epithelial cells (also known as podocytes) per se that leads to FSGS, several lines of evidence are suggestive (22, 38, 46). To begin to address whether NFAT may represent a target of TRPC6 signaling in the podocyte, we assayed the ability of wild-type and mutant TRPC6 to activate an NFAT-responsive luciferase reporter in a cultured podocyte cell line. While the effect was more modest than in M1R cells, wild-type TRPC6 enhanced NFAT-responsive reporter activity in these cells, while the P112Q, R895C, and E897K mutant forms of TRPC6 achieved greater levels of NFAT activation (Fig. 2A). To assess which of the four calcium-responsive NFAT genes (NFATc1-4) might be involved, their expression in the human podocyte cell line was assessed by RT-PCR. Transcripts for NFATc1 and c3 were detected in both M1R cells and human podocytes (Fig. 2C); the presence of NFATc3 in podocytes is consistent with publicly available immunohistochemical staining patterns in human kidney samples [www.proteinatlas.org (3)].

Pore mutations impair NFAT activation by FSGS-mutant TRPC6.

Recently, the L-type voltage-dependent calcium channel Ca_v1.2 has been reported to have effects on transcription independent of its channel activity (13). We therefore examined whether the ability of R895C and E897K mutant TRPC6 to activate NFAT-mediated transcription is dependent on channel activity by assessing the ability of a dominant-negative pore mutant TRPC6 to inhibit the effects of the FSGS mutations. Cotransfection of dominant-negative TRPC6 substantially inhibited NFAT activation by R895C, E897K (Fig. 3A), and P112Q TRPC6 (data not shown). Introducing the dominant-negative LFW to AAA pore mutation directly into the R895C TRPC6 largely abolished the ability of the mutant channel to enhance basal or stimulated NFAT activation (Fig. 3C), although overexpression of the R895C dominant-negative channel did induce a small but statistically significant increase in reporter activity. These results suggest that the effects of these FSGS-associated mutants on NFAT activation largely rely on their channel activity. However, we cannot exclude that the pore mutation, in addition to impairing channel function, has additional steric effects that inhibit the ability of mutant TRPC6 to activate NFAT independently of its channel activity.

NFAT activation by FSGS-mutant TRPC6 is dominant over wild-type TRPC6.

Mutations in TRPC6 lead to an autosomal dominant inheritance of FSGS (46, 58). We therefore wanted to assess whether the ability of the R895C and E897K mutants to enhance NFAT activation was dominant in the presence of wild-type TRPC6. Cotransfection of equal amounts of wild-type and mutant TRPC6 expression plasmids in the luciferase assays led to only a slight, statistically insignificant, decrease in NFAT activation compared with transfection of the mutant TRPC6 plasmids alone (Fig. 3A; compare, for instance, R895C/− to R895C/WT). NFAT activation upon cotransfection remained substantially higher compared with cells expressing wild-type TRPC6 alone. These results are consistent with the hypothesis that the mutant TRPC6 channel characteristics, specifically in regard to its ability to activate the calcineurin-NFAT pathway, are dominant over wild-type TRPC6.

Cyclosporine A inhibits TRPC6-mediated NFAT activation without affecting channel activity.

The protein phosphatase calcineurin is responsible for dephosphorylating, and thereby activating, NFAT in response to calcium signaling. As expected, the calcineurin inhibitor cyclosporine A is able to abrogate both TRPC6-mediated and TRPC6-independent NFAT activation (Fig. 4A). Basal and carbachol-induced NFAT activation via the mutant R895C was also substantially reduced in the presence of cyclosporine A.

Calcineurin has been reported to bind to TRPC6 via calmodulin upon M1 acetylcholine receptor activation (29). In addition, FKBP12 binds directly to TRPC6, and high-dose FK506 has been shown to inhibit TRPC6 activity (51). To ascertain whether cyclosporine A may have a direct effect on TRPC6 activity in addition to downstream events in the calcineurin-NFAT pathway, we assessed the effect of cyclosporine A on TRPC6-mediated whole cell currents. Cells were activated by the diacylglycerol analog OAG, because this represents a more direct mechanism for activating TRPC6 than carbachol (20). While high concentrations of FK506 were able to rapidly inhibit OAG-induced TRPC6 currents, similar to previous reports (51), high-dose cyclosporine A did not appreciably affect current amplitudes (Fig. 4, B and C). Similar results were obtained with the R895C and E897K mutants and when cells were stimulated with carbachol instead of OAG (data not shown). Thus, the effect of cyclosporine A on NFAT activation appears to be mediated solely downstream of TRPC6 without an additional effect on the channel itself. In contrast, FK506 can potentially act at two sites in the TRPC6-calcineurin-NFAT pathway, by blocking the binding of FKBP12 to TRPC6 (51) and by inhibiting calcineurin.

Mechanism of basal activation of NFAT by TRPC6 mutants.

The constitutive activation of NFAT-mediated transcription by three of the TRPC6 mutants was intriguing. We postulated that this could be due to constitutive low-level leak through the channels. To address this issue further, we performed whole cell voltage-clamping experiments in HEK-293 cells expressing either wild-type or mutant R895C and E897K TRPC6 (Fig. 5A). Basal currents were not significantly different in cells expressing R895C or E897K mutant TRPC6 compared with wild-type TRPC6, suggesting that constitutive low-level channel leak is unlikely to explain the basal activation of NFAT by these mutants. Upon subsequent stimulation of cells with either OAG or carbachol, cells expressing the R895C or E897K mutants showed substantially higher current amplitudes than cells expressing wild-type TRPC6 (data not shown), as had been previously reported (46).

We also examined Fura-2 fluorescence under unstimulated conditions as a surrogate for basal intracellular calcium levels in both transiently transfected cells (Fig. 5B) and cells stably expressing TRPC6 under a tetracycline-inducible promoter (Fig. 5C). Cells overexpressing wild-type TRPC6 had slightly lower median basal 340/380 fluorescence ratios than control-transfected cells. Median F₃₄₀/F₃₈₀ ratios of cells expressing the R895C or E897K mutants were not statistically significantly different (Fig. 5B), or only slightly lower (Fig. 5C) than ratios of control cells. However, the distribution of the ratios in these cells does shows a significant tail at higher ratios, suggesting that at any given time, a subpopulation of cells expressing the mutant channels have elevated intracellular calcium levels.

We wished to identify what signaling pathways might be important for the basal activation of the calcineurin-NFAT pathway by mutant TRPC6. Because several signaling pathways have been implicated in the regulation of TRPC6, including phospholipase C (PLC) (20), Src-family tyrosine kinases (1, 19, 25), PI3K (33, 54), and CaMK II (49), we tested the ability of inhibitors of these enzymes to block mutant TRPC6-mediated NFAT-activation. Cells overexpressing TRPC6 R895C were exposed to various inhibitors for 6 h followed by the assessment of NFAT-mediated transcription (Fig. 6A). To attempt to identify pathways required specifically for activation of the calcineurin-NFAT pathway by mutant TRPC6, and not for TRPC6-independent NFAT activation downstream of the M1 acetylcholine receptor, we also examined the effect of the inhibitors on M1R cells not overexpressing TRPC6, but stimulated with carbachol.

Cyclosporine A effectively inhibited both carbachol- and TRPC6 R895C-mediated NFAT activation (Figs. 4A and 6A). In contrast, PP2 (a Src-family kinase inhibitor), LY-294002 (a PI3K inhibitor), and KN-93 (a CaMK II inhibitor) all partially inhibited NFAT activation by TRPC6 R895C, but not by carbachol (Fig. 6A). The tyrosine kinase inhibitor Genistein enhanced NFAT-mediated transcription by both carbachol and mutant TRPC6 (Fig. 6A), as well as basal NFAT-mediated transcription in unstimulated M1R cells (data not shown). Surprisingly, the PLC inhibitor U-73122 did not significantly inhibit NFAT activation.

There have been conflicting reports regarding the role of Src-family kinases, specifically Fyn, in the activation of TRPC6 (1, 19, 25). In light of the ability of PP2 to partially inhibit NFAT activation by R895C TRPC6, the ability of the R895C mutant to activate NFAT-mediated transcription was also examined in SYF cells, which are deficient in Src, Yes, and Fyn (30). The R895C mutant was able to activate NFAT-mediated transcription in these cells under serum starvation, although to a lesser extent than in M1R cells, while wild-type TRPC6 had no effect (Fig. 6B). P112Q and E897K also activated NFAT mediated transcription (data not shown). The addition of PP2 was able to partially inhibit NFAT activation in R895C-expressing SYF cells, while it had no effect in the absence of TRPC6, suggesting that the ability of PP2 to affect mutant TRPC6 mediated activation of the calcineurin-NFAT pathway is independent of its inhibition of Src, Yes, or Fyn.

Differential glycosylation of TRPC6 mutants.

Transiently overexpressed TRPC6 was observed to run at two distinct molecular masses of approximately 100 and 130 kDa (Fig. 1C, top, and Fig. 7A, top). To confirm that the higher-molecular form represents the fully glycosylated form, while the lower represents a precursor, as has previously been reported (5, 37), we subjected lysates from cells overexpressing various forms of TRPC6 to treatment with PNGase to remove all forms of N-linked glycosylation. After deglycosylation, only the lower-molecular-weight form of TRPC6 remained (Fig. 7A, bottom). Furthermore, while the higher-molecular-weight form of various mutant forms of TRPC6 had slightly different mobilities on SDS-PAGE, this difference was no longer detected after deglycosylation, indicating that these mutants are differently glycosylated from wild-type TRPC6.

To assess whether the 100-kDa form of TRPC6 represents a precursor form found only intracellularly, or whether this form can reach the cell surface, transiently transfected cells were surface biotinylated, and biotinylated TRPC6 was compared with total TRPC6 (Fig. 7B). Both the 100- and 130-kDa forms were biotinylated, with the relative abundance of the two forms on the cell surface generally mirroring their relative abundance in total lysates.

TRPC6 activity has been reported to be at least partially controlled through regulated transport to the cell surface (8), and a greater percentage of P112Q mutant TRPC6 has been reported to reside on the cell surface compared with wild-type TRPC6 (58). We therefore examined whether the amount of cell surface expression of the TRPC6 mutants correlated with their ability to activate the calcineurin-NFAT pathway by comparing the amount of cell surface expressed TRPC6 to the total amount of TRPC6 by Western blot analysis (Fig. 7, B and C). When the relative intensities of the TRPC6 bands in the cell surface and whole lysate fractions were compared, there was a trend toward a higher proportion of the carboxyl-terminal mutants being present on the cell surface (Fig. 7C). However, there was significant heterogeneity from experiment to experiment, and the differences were not statistically significant.

The relative abundance of the 100- and 130-kDa forms of TRPC6 differed between the wild-type and mutant forms (Fig. 7, A and B). Specifically, the three mutations that constitutively activate NFAT, as well as the K874* truncation mutant, are predominantly present as the lower form, while a larger fraction of wild-type TRPC6 and the other mutations exist as the 130-kDa species. Interestingly, introducing the dominant-negative pore mutation into the R895C mutation shifted the protein predominantly into the more heavily glycosylated form (Fig. 7A).

Differences in N-linked glycosylation between TRPC3 and C6 have been shown to be important for the different basal channel characteristics of these proteins, with glycosylation deficient TRPC6 displaying increased constitutive channel activity (10). Is it possible that the differences in the relative abundances of the 100- and 130-kDa forms of TRPC6 could explain the ability of some TRPC6 mutants to constitutively activate the calcineurin-NFAT pathway? Alternatively, the presence of the unglycosylated form of TRPC6 could represent an overwhelming of the protein folding and trafficking machinery by transient transfection. We therefore generated stable M1R cell lines in which wild-type, R895C, or E897K TRPC6 is stably expressed under a tetracycline-inducible promoter. Induction of TRPC6 expression in these cell lines leads to the predominant expression of the fully glycosylated, mature form of wild-type and mutant protein (Fig. 8A), suggesting that the lower-molecular-weight form of TRPC6 accumulates at least in part due to a limited ability of the cell to properly glycosylate the channel in the setting of overexpression by transient transfection. Furthermore, surface biotinylation demonstrated that in these cell lines, only the mature 130-kDa form was present on the cell surface (Fig. 8B). To confirm that a predominance of the 100-kDa form of the mutant proteins is not required for the constitutive activation of NFAT-mediated transcription, NFAT-responsive luciferase reporter assays were repeated in these stable cell lines (Fig. 8C). Interestingly, induction of wild-type TRPC6 expression did not lead to any change in basal transcription from the NFAT-responsive reporter plasmid. Induction of either the R895C or E897K mutants led to significant activation of NFAT-mediated transcription, though the relative induction was less than was seen with transient transfection. Although we cannot completely exclude a contribution of improper glycosylation as a mechanism for NFAT activation by transiently overexpressed TRPC6, our results making use of the inducible stable cell lines demonstrate that abnormal glycosylation is not the only mechanism involved in constitutive activation of NFAT by mutant TRPC6.

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