TRPC6 activated NFAT-mediated transcription in cultured podocytes. A: dual luciferase assays were performed on cultured human podocytes transfected with luciferase reporter constructs and the indicated TRPC6 expression plasmid. Results are means ± SE. *P < 0.01 vs. control; †P < 0.01 vs. WT. B: Western blot analysis of podocyte lysate using FLAG M2 antibody to detect overexpressed TRPC6. C: the presence of NFATc1-4 transcripts in M1R cells and cultured human podocytes (Podo) was assessed by RT-PCR. GAPDH was used as a control. Reactions in which the reverse transcriptase was omitted failed to amplify any fragments (data not shown).

Effect of calcineurin inhibitor cyclosporine A (CsA) on TRPC6-mediated NFAT activation. A: M1R cells were transfected with luciferase reporter constructs and the indicated TRPC6 expression plasmids. After overnight serum starvation, cells were treated without (−) or with 100 μM carbachol (CCh) in the absence or presence of 10 μM cyclosporine A. Luciferase activity was assessed using the dual luciferase reporter assay. Results are shown as means ± SE. *P < 0.05 vs. no CsA; **P < 0.001 vs. no CsA. B: whole cell membrane currents were recorded from 293T cells transiently transfected with the WT TRPC6 channel. A 400-ms voltage ramp from −100 mV to 100 mV was applied every 4 s, and the current measured at 80 mV (open circles) and at −80 mV (filled circles) during each ramp is plotted as a function of time. In the constant presence of 1-oleoyl-2-acetyl-sn-glycerol (OAG), 30 μM FK506 and 10 μM cyclosporine A were perfused during the time indicated by the horizontal bars. C: current-voltage relationships recorded during voltage ramps at the times indicated in B (a and b).

Pharmacologic inhibition of basal NFAT activation by TRPC6 R895C. A: M1R cells were transfected with the NFAT luciferase reporter construct either alone (control) or with TRPC6 R895C expression plasmid (R895C). Cells were serum starved overnight followed by a 6-h incubation in serum-free media with vehicle alone, 10 μM cyclosporine A, 10 μM U-73122, 50 μM Genistein, 4 μM PP2, 50 μM LY-294002, or 2 μM KN-93. Control cells were also stimulated with 100 μM carbachol during the 6-h incubation. NFAT-response element-driven luciferase activity (means ± SE) is shown as a percentage of the activity in the corresponding vehicle-treated cells. *P < 0.05 vs. vehicle-treated cells; **P < 0.05 vs. vehicle-treated cells. B: Western blot of TRPC6 R895C-transfected M1R cells. C: SYF murine embryonic fibroblasts were transfected with NFAT-responsive luciferase plasmid and either control plasmid or the indicated FLAG-TRPC6 expression plasmid. The dual luciferase assay was performed after cells were treated with either vehicle or 4 μM PP2 for 6 h. Results are means ± SE. *P < 0.001 vs. control, †P < 0.05 vs. R895C vehicle treated.

Inducible expression of R895C and E897K TRPC6 activates basal NFAT-mediated transcription. A: M1R cells stably transfected with constructs allowing for tetracycline-inducible expression of WT, R895C, or E897K TRPC6 were cultured in the absence or presence of 5 μg/ml tetracycline (Tet) for 24 h before analysis of TRPC6 expression by Western blot. Parental refers to the original M1R cell line. B: surface-expressed (S) TRPC6 was compared with TRPC6 in total lysate (T) from cell lines expressing the indicated form of FLAG-tagged TRPC6 under the tetracycline-inducible promoter. C: dual luciferase assays were performed on the indicated cell lines transfected with an NFAT-responsive luciferase construct after cells were incubated in the absence or presence of 5 μg/ml tetracycline for 24 h. Results are means ± SE. *P < 0.001 vs. same cell line without tetracycline and vs. parental cell line with tetracycline.

Focal segmental glomerulosclerosis (FSGS)-associated canonical transient receptor potential channel TRPC6 mutants P112Q, R895C, and E897K enhance basal and G protein-coupled receptor-activated nuclear factor of activated T cell (NFAT)-dependent transcription. A: M1R cells were transiently transfected with an NFAT-response element-driven luciferase reporter plasmid and either control plasmid (control), FLAG-tagged TRPC6 [wild type (WT)], or FLAG-tagged TRPC6 with an FSGS-associated mutation or a dominant-negative pore mutation (DN). Cells were serum starved (open bars) or treated with 100 μM carbachol (shaded bars) for 6 h. Firefly luciferase activity was normalized to Renilla luciferase activity driven by the thymidine kinase promoter. Results are expressed as a fold increase over control. Values are means ± SE. *P < 0.01 vs. control; †P < 0.001 vs. wild type; **P < 0.001 vs. carbachol-treated control. B: detail of basal NFAT-responsive reporter activity from graph in A. C: expression of the TRPC6 mutants in cells used for the luciferase assay in A was assessed by Western blot using an anti-TRPC6 polyclonal antibody. D: M1R cells were transiently transfected with NFATc3-green fluorescent protein (GFP) plasmid and the indicated TRPC6 expression construct. GFP localization was scored as exclusively cytoplasmic (C), cytoplasmic more than nuclear (C>N), nuclear more than cytoplasmic (N>C), or exclusively nuclear (N). At least 100 cells were scored for each transfection condition. E: representative examples of cells showing distinct NFATc3-GFP localization. A control cell transfected with NFATc3-GFP alone shows exclusively cytoplasmic localization, as seen in the lack of colocalization of GFP and 4′,6-diamidino-2-phenylindole (DAPI) signals (top). Two cells transfected with TRPC6 R895C and NFATc3-GFP are shown (bottom). They demonstrate cytoplasmic more than nuclear (arrowhead) and nuclear more than cytoplasmic (arrow) localization of NFATc3-GFP.

TRPC6 mutant R895C and E897K activation of NFAT-mediated transcription is dominant over WT TRPC6 but is inhibited by dominant-negative TRPC6. A: M1R cells were transfected with NFAT-responsive luciferase reporter plasmid and the indicated TRPC6 expression plasmids. Equal total amounts of expression plasmids were used, such that WT/WT indicates 0.25 μg of WT TRPC6 expression plasmid, while WT/− indicates 0.125 μg of WT TRPC6 expression plasmid and 0.125 μg of control plasmid. DN refers to a dominant-negative pore mutant of TRPC6. Cells were serum starved overnight followed by 6 h of incubation in fresh serum-free media. Data are shown as means ± SE. *P < 0.05 vs. R895C/DN; †P < 0.05 vs. E897K/DN. B: expression of TRPC6 in cells used for the luciferase assay in A was assessed by Western blot using an anti-TRPC6 polyclonal antibody; actin was used as a loading control. C: M1R cells were transfected with an NFAT-responsive luciferase reporter and either control plasmid, or the indicated TRPC6 expression plasmid. TRPC6 R895C DN harbors both the R895C mutation and the LWF to AAA pore mutant. Inset: TRPC6 expression assessed by Western blot. **P < 0.001 vs. control and R895C DN; *P < 0.05 vs. control.

Basal channel currents and Fura-2 fluorescence. A: average normalized basal TRPC6 currents measured at 80 mV (gray bars) and −80 mV (black bars) from cells expressing the WT TRPC6 channel or the mutant channels R895C or E897K (pA/pFz ± SE). An unpaired Student's t-test did not reveal statistically significant differences between WT and the two mutant channels. B: histogram of ratio of Fura-2 fluorescence upon excitation at 340 and 380 nm under unstimulated conditions of M1R cells transfected with control plasmid (−), or the indicated TRPC6 expression plasmid. Bar indicates median ratio. ***P < 0.001 vs. all other groups, by Kruskal-Wallis and Dunn's multiple-comparison tests. C: Fura-2 fluorescence ratio of unstimulated M1R cells stably expressing either WT or the indicated TRPC6 mutants under a tetracycline-inducible promoter, or the parental M1R cell line. Bar indicates median ratio of population. ***P < 0.001 vs. parental; †††P < 0.001 vs. WT by Dunn's multiple-comparison test.

TRPC6 mutants exhibit different glycosylation patterns and are transported to the cell surface. A: lysates from M1R cells transiently transfected with the indicated FLAG-tagged TRPC6 constructs were treated without (mock) or with peptide N-glycosidase F (PNGase F), followed by detection of TRPC6 by Western blot analysis. B: M1R cells transiently transfected with the indicated FLAG-tagged TRPC6 constructs were subject to surface biotinylation. Western blot analysis compares total TRPC6 (T; 5% of the total lysate) to surface-expressed TRPC6 (S; streptavidin-agarose bound fraction). C: relative surface expression of WT and mutant TRPC6. The relative intensities of total to surface expressed protein were normalized to that of WT TRPC6; results are means ± SE of four experiments. P > 0.05 for all mutants vs. WT.