Plasmids and reagents
The human TRPC6 coding sequence, with or without mutations as outlined in the text, and containing an amino-terminal FLAG tag sequence, was cloned into pcDNA4/TO/myc-HIS B (Clontech) using standard PCR-based techniques. Similarly, full-length human TRPC6 carrying an amino-terminal HA tag was amplified by PCR and subcloned into pcDNA3.1. The HA-SNF8 expression plasmid was a gift from C. Bucci . The Matchmaker Two-Hybrid System and S. cerevisiae Y187 pre-transformed with a human kidney cDNA library were purchased from Clontech (Palo Alto, CA). The dual luciferase assay kit and reporter vectors pGL4.30 and pGL4.74 were obtained from Promega. Affinity purified rabbit anti-TRPC6 polyclonal antibody was purchased from Chemicon, anti-FLAG M2 monoclonal antibody and anti-FLAG rabbit polyclonal antibodies were purchased from Sigma, rabbit anti-GFP polyclonal antibody and mouse anti-HA monoclonal antibody were purchased from Abcam Inc, and rabbit anti-HA monoclonal antibody (C29F4) was purchased from Cell Signaling Technologies. Anti-SNF8 rabbit polyclonal antibody was the kind gift of Dr. H. Stenmark . Anti-caveolin-1 mouse monoclonal antibody (clone 2297) was obtained from BD Biosciences.
Yeast two-hybrid screen
cDNA encoding residues 1 through 406 of TRPC6 (wild-type N-terminal domain) was used as bait and cloned in-frame with GAL4 DNA-binding domain in the vector pGBKT7-BD and transformed into yeast strain AH109. The bait strain was mated to Y187 yeast strain pre-transformed with a commercially available human kidney cDNA library cloned into pACT2-AD vector according to the manufacturer’s protocol (Clontech). Based on mating efficiency, 1 × 106 clones were screened. Mated yeast cells were grown on high-stringency selection plates (SD-Leu, Trp, Ade, His) and positive colonies were further verified by growth on high-stringency plates supplemented with X-α-gal as a test for β-galactosidase activity. pACT2-AD plasmids containing library inserts from positive colonies were isolated and transformed into DH5α derived E. coli (New England Biolabs). Plasmids were then isolated from bacteria, sequenced, and analyzed using the Blast-NT alignment algorithm from NCBI.
To confirm the interaction, the full-length SNF8 coding sequence was cloned inframe into the pGAD-T7 plasmid and the resulting plasmid transformed into Y187 strain yeast. The resulting strain was mated with AH109 bait strain harboring pGBKT7 with wild-type TRPC6 N-terminal sequences or the TRPC6 C-terminal domain (corresponding to amino acids 726-931). The resulting diploids were tested for positive interaction by growth on high-stringency plates.
Cell culture and luciferase assays
Cells stably expressing the M1 muscarinic acetylcholine receptor and either wild-type, R895C or E897K TRPC6 under a tetracycline-inducible promoter were generated from T-Rex-293 cells (Invitrogen), as previously described . Stable cell lines were induced to express TRPC6 for 24 hours prior to lysis by adding tetracycline to a 1 μg/ml final concentration in culture medium. Luciferase assays were carried out essentially as previously described  using the dual luciferase reporter assay (DLR; Roche) using a Veritas microplate luminometer (Turner Biosystems). All conditions were tested in triplicate and results normalized to the Renilla luciferase internal control.
SNF8 was knocked down using short hairpin-expressing double-stranded oligonucleotides cloned into pcDNA 6.2-GW/EmGFP-miR plasmids (BLOCK-iT™ Pol II miR RNAi Expression Vector Kit, Invitrogen). A specific shRNA sequence against SNF8 (sense 5′-TGACTTCG[CC]CAATGTCAGTCAA-3′ and antisense 5′-TTGACTGACATCCTGGGCGAA-3′), and a scrabbled control sequence, were obtained from Invitrogen and cloned into the plasmid. Knock-down efficiency was determined by transient transfection of M1R cells with either SNF8 or control plasmid, followed by FACS sorting for GFP expression to isolate the transfected cells after 48 hrs. SNF8 expression in sorted cells was assessed by immunbloting for SNF8.
To assess the effect of SNF8 knock-down on NFAT-mediated transcription, cells were transfected with the indicated shRNA expression vector and luciferase reporter plasmids using Fugene 6. 24 hours after transfection, TRPC6 expression was induced where indicated by the addition of tetracycline (1μg/ml final concentration) to the media. After an additional 24 hours, cells were lysed and processed for dual luciferase reporter assay.
Immunoprecipitation and Immunoblotting
Transfected cells were rinsed once in phosphate buffered saline and lysed in modified RIPA lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate supplemented with Complete protease inhibitor cocktail (Roche)). After clearing lysates by centrifugation at 14,000 RPM for 15 minutes at 4ºC, supernatants were incubated with 30 μl of FLAG M2 agarose slurry (Sigma-Aldrich), and incubated with constant agitation at 4ºC for 2-3 hours. Immunoprecipitated complexes were washed three times with lysis buffer and eluted off of the beads by boiling in SDS-sample loading buffer.
Cell lysates and immunoprecipitated materials were separated by SDS-PAGE and transferred to PVDF membrane (Bio-Rad). The membrane was blocked with 5% non-fat milk in PBST (PBS with 0.05% Tween-20) for 1 hour at room temperature, followed by overnight incubation in 1:1000 anti-TRPC6, 1:500 anti-HA, 1:500 FLAG M2, or 1:200 anti-SNF8 antibody in 5% non-fat milk PBST. After three washes in PBST, blots were incubated with the appropriate secondary antibody conjugated to HRP (Pierce) in PBST at room temperature, followed by detection with SuperSignal West Pico chemiluminescent substrate (Pierce).
Cells stably expressing wild-type TRPC6 under a tetracycline inducible promoter were grown on collagen I coated glass coverslips and transfected with plasmid encoding for HA-SNF8 using Fugene6 followed by treatment with tetracycline to induce FLAG-TRPC6. 24 hours after inducing TRPC6 expression, cells were washed with PBS, fixed in 2% paraformaldehyde, 4% sucrose in PBS, and permeabilized with 0.3% Triton X-100 in PBS. Nonspecific binding sites were blocked using blocking solution (2% bovine serum albumin, 2% fetal calf serum, 0.2% fish gelatin). Cells were incubated with 1:200 anti-FLAG M2 antibody and 1:1600 anti-HA rabbit monoclonal antibody, followed by Alexa488 conjugated goat anti-mouse and Cy3 conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch), and mounted on slides with ProLong Gold antifade reagent (Invitrogen). Images were taken using a Zeiss LSM 510 confocal microscope.
Patch-clamp electrophysiology (Axopatch 200B amplifier, Axon Instruments, CA) was performed in the whole-cell configuration. Briefly, HEK293T cells (American Type Culture Collection, VA) were plated on glass coverslips at low density and placed in the recording chamber. The patch pipettes with resistances of 3–4 MΩ were pulled from borosilicate glass with a P-97 puller (Sutter Instrument) and filled with a solution containing (in mM): 135 CH3SO3Cs, 10 CsCl, 3 MgATP, 0.2 NaGTP, 0.2 EGTA, 0.13 CaCl2, and 10 HEPES, pH 7.3 with CsOH. The bath solution contained (in mM): 135 CH3SO3Na, 5 CsCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 Glucose, pH 7.4 with NaOH. Carbachol (100 μM) or 1-oleoyl-2-acetyl-sn-glycerol (OAG, 10 μM) was applied to the bath solution. Whole-cell currents were recorded from –100 mV to 100 mV voltage ramps over 150 ms and a holding potential of 0 mV. All data were acquired at room temperature and analyzed using pClamp 10 (Axon Instruments, CA). Statistical analysis was done using one-way ANOVA Fisher’s LSD test, and p values <0.05 were considered significant.
Cells were surface biotinylated on ice using Sulfo-NHS-SS-biotin (Pierce) 48 hours after transfection, essentially as previously described , followed by lysis in modified RIPA buffer. Lysates were incubated with streptavidin beads (Pierce) at 4°C. The streptavidin beads were extensively washed and bound material was eluted and analyzed by SDS-PAGE electrophoresis followed by Western blot analysis.
Lipid raft preparation
M1R cells stably expressing FLAG-tagged TRPC6 were transfected with control or HA-SNF8 expression constructs. 48 hours after transfection, cells were processed using a protocol modified from Alicia et al . Briefly, cells were washed in ice cold PBS, scraped off of plates in PBS and lysed in 1% Triton X-100 in MES buffer (25mM MES, pH 6.5, 150mM NaCl, 2mM EDTA complemented with protease inhibitors) on ice for 20 minutes. Lysates were further homogenized in a glass homogenizer with the loose fitting piston, then mixed with an equal volume of 85% (w/v) sucrose in MES buffer. Lysates were overlayed with 4mls of 40% sucrose in MES buffer followed by 4mls of 5% sucrose in MES buffer. Lysates were spun at 175,000x g for 24 hours in an SW41 Ti rotor. Eight 1.5 ml fractions were obtained by aspiration starting at the top of the gradient. Aliqouts of each fraction were combined with 4x sample loading buffer with β-mercaptoethanol and analyzed by Western blot as above.