Animal care and maintenance
Postnatal Day 30 (P30) Sprague-Dawley rats were used for biochemistry experiments and were housed on a 12 h:12 h light:dark cycle in the Florida State University (FSU) vivarium. All procedures were performed in accordance with the FSU Animal Care and Use Committee and NIH-approved guidelines.
Solutions used for protein sample or tissue preparation, including phosphate buffered saline (PBS), lysis buffer (LB), wash buffer (WB), and protease and phosphatase inhibitor solution (PPI), were made as described in . Tissue extract buffer (TEB) was prepared as in . Cell-surface biotinylation solutions, including biotinylation lysis buffer and biotinylation quench buffer, were made as described by . Immunoblot stripping buffers, including tris stripping buffer (TSB) and sodium citrate stripping buffer (SCSB), were also made as described in Colley et. al. (2007). Electrophysiology solutions were prepared as in  and were as follows (in mM): intracellular pipette 150 KCl, 10 HEPES pH 7.2, 2 MgCl2, 10 glucose; extracellular bath 140 NaCl2, 10 HEPES pH 7.4, 4 KCl, 4 CaCl2, 1 MgCl2, and 10 glucose. All chemicals were obtained from either Sigma Chemical Company (St. Louis, MO, USA) or Fisher Scientific (Suwanne, GA, USA).
Plasmids and antibodies
All encoded cDNAs were downstream from a cytomegalovirus (CMV) promoter. TRPC2 clone 14, with an N-terminal myc-epitope (EQKLISEEDL), was prepared in the pcDNA3 vector and was a kind gift from Dr. L Birnbaumer (National Institute of Environmental Health Sciences) . RTP1 and REEP1 were in the pCI vector and were kind gifts of Dr. H. Matsunami (Duke University) . pCDM8 was a kind gift from Dr. Brian Seed (Harvard University) . DNA encoding human CD8 was amplified from pCDM8 and subcloned into the pcDNA3 vector (Carlsbad, CA, Invitrogen) between the BamH1 and EcoR1 restriction sites. cDNA encoding Kv1.3 was subcloned into the pcDNA3 vector (Carlsbad, CA, Invitrogen) at the unique HindIII restriction site within the multiple cloning region . Kv1.3 was also epitope-tagged via insertion of the myc sequence on the extracellular face of the channel between the S1 and S2 transmembrane domains .
T1NH, T2NH, and T3NH are anti-peptide polyclonal antibodies specific for the type-1, -2 and -3 IP3R isoforms, respectively, and were raised against the following amino terminal sequences (amino acid position in parentheses): T1NH = CLATGHYLAAEVDPDQEVDPDQ-DASR (308-326), T2NH = CPDYRDAQNEGKTVRDGKTVRDGELP (320-338) and T3NH = CENPSYKGDVSDPGDVSDPKAAGPGA (319-337). These antibodies were a generous gift of Dr Gregory Mignery (Loyola University Chicago, Stritch College of Medicine, Maywood, IL, USA) . An antiserum detecting TRPC2 as raised in guinea pig and directed against the N-terminal cytosolic domain CSSDASGAGPGGPLRNVE was a generous gift of Dr. Richard Axel (Columbia University, New York, NY, USA) . Anti-peptide polyclonal antibodies detecting the different forms of Homer were made by immunizing rabbits with the synthetic C-terminal peptides of Homer 1b/c (IFELTELRDNLAKLLECS), 2a/b (GKIDDLHDFRRGLSKLGTDN), or 3 (RLFELSELRE-GLARLAEAA) conjugated to thyroglobulin with glutaraldehyde . A fourth, rabbit polyclonal antiserum recognizing all Homer 1 (1a, 1b, and 1c) isoforms was generated against the full-length GST-Homer 1a fusion protein . These antibodies were a generous gift of Dr. Paul Worley (Johns Hopkins University, Baltimore, MD, USA). A polyclonal antibody recognizing all Homer proteins (Pan-Homer; 1a, 1b, 1c, 2a, 2b, 3) was made by immunizing rats with recombinant Homer 1a (AB5875, Chemicon/Millipore, St. Louis, MO, USA). The mouse monoclonal antiserum for the c-myc epitope was from Roche (Indianapolis, IN, USA). Rabbit polyclonal antiserum specific to RTP1 was a kind gift of Dr. Hiro Matsunami (Duke University, Durham, NC, USA) . When necessary to validate equal protein loading, a rabbit polyclonal antibody detecting cellular actin was used. The immunogen for this antibody was SGPSIVHRKCF attached to a Multiple Antigen Peptide (MAP) backbone (Sigma Chemical).
Tissue homogenization and Western blotting
Tissue extracts were prepared as in . Briefly, P30 rats were killed by CO2 inhalation, decapitated, and VNOs were rapidly dissected and placed in ice cold TEB with PPI. Samples on ice were sonicated (Sonic Dismembrator, Model 60, Fisher Scientific) twice for eight seconds on setting number five. Non-soluble matter was removed by ultracentrifugation (Beckman Coulter, Fullerton, CA, USA) at 37,000 × g for 30 min at 4°C. The supernatant was aliquoted and stored at -80°C until use. The resultant pellet was solubilized in 2% sodium dodecyl sulfate (SDS), aliquoted, and stored at -80°C in case the initial screening of the lysates did not yield membrane-associated fractions.
Immunoprecipitations were performed as in . In brief, P30 rat VNOs were homogenized with a size 20 Kontes glass tissue-homogenizer (Kontes Glass, Vineland, NJ, USA) on ice in LB with PPI. Lysis was continued on a Roto-Torque (Model 7637, Cole-Parmer Instruments, Vernon Hills, IL, USA) for 30 min at 4°C. Lysates were clarified by centrifugation at 15,000 × g (Eppendorf, Model 5415C, Westbury, NY, USA) for 10 min at 4°C and then precleared for 1 hour (hr) with 3 mg/ml protein A sepharose (GE Healthcare, Uppsalla). This was followed by another centrifugation step to remove the protein A sepharose. Proteins of interest (Homer 1b/c, Homer 2, Homer 3, TRPC2, IP3R3, RTP1) were immunoprecipitated from the clarified lysates by overnight incubation on a Roto-Torque at 4°C with 5 μg/ml of appropriate antiserum. Samples were then incubated for 3 hr with protein A sepharose and centrifuged as above. Immunoprecipitates were washed four times with 5 volumes of WB. Lysates and washed immunoprecipitates were diluted in SDS gel loading buffer containing  1 mM Na3VO4 and stored at -20°C until use.
Purified VNO tissue extracts or immunoprecipitated proteins were separated on 6 to 15% acrylamide gels by SDS-PAGE and electro-transferred to nitrocellulose membranes. Equal protein loading (30 μg) was controlled by Bradford protein assay (BioRad, Hercules, CA, USA) and confirmed by 0.1% Fast Green staining and α-β-actin labeling. The nitrocellulose membrane was blocked with 5% non-fat milk for 60 min, incubated overnight at 4°C in primary antisera against TRPC2 (1:2000), IP3R3 (1:2000), RTP1 (1:1000), Homer 1b/c (1:1000), Homer 2a/b (1:2000), Homer 3 (1:2000), Homer 1 isoforms (Worley, 1:1000), all Homer isoforms (Chemicon, 1:1000), or RTP1 (1:1000). Membranes were then incubated with horseradish peroxidase-conjugated species-specific secondary antibody (donkey anti-rabbit, Amersham Biosciences or rabbit anti-guinea pig, Sigma) for 90 min at room temperature (rt). Labelled protein was detected with enhanced chemiluminescence (ECL; Amersham Biosciences, Piscataway, NJ, USA) using Classic Blue autoradiography film BX (MidSci, St. Louis, MO, USA). To ensure equal loading, nitrocellulose membranes were stripped by incubating blots in eight ten-minute washes of TSB, followed by eight ten-minute washes of SCSB, then re-probed using α-β-actin (1:1000). Autoradiographs were scanned using a Hewlett-Packard Photosmart Scanner (Model 106-816; Hewlett-Packard, San Diego, CA, USA) and quantified by line scanning densitometry using Quantiscan Software (Biosoft, Cambridge, UK).
RNA extraction and reverse-transcriptase PCR
P30 rats were killed by CO2 inhalation, decapitated, and VNOs were rapidly dissected from the surrounding tissues and placed on dry ice. RNA was extracted using the SV Total RNA Isolation System as per the manufacturer's protocol (Promega, Madison, WI, USA). RNA purity and concentration was determined by UV spectroscopy (NanoDrop-1000, Wilmington, DE, USA). cDNA was reverse-transcribed using the ImPromII kit as per manufacturer's protocol (Promega). Gene-specific primers have been previously reported  and were as follows: RTP1_forward, AAGCGTGACCACAGATGAGTG; RTP1_reverse, GAGCAGAAGTTCCAGCCTGAG; REEP1_forward, CAATGAATTCCCACCATGGTGTCATGGATCATCTCCAGGC; REEP1_reverse, GACTAGCGGCCGCCTAGGCGGTGCCTGAGCTGCTAG. PCR products were resolved using 1.0% agarose gel electrophoresis and visualized via UV excitation of the incorporated ethidium bromide.
Human embryonic kidney cell (HEK293) maintenance and transfection
HEK 293 cells were maintained in minimum essential medium (MEM), 2% penicillin/streptomycin, and 10% FBS (Gibco BRL). Before transfection, cells were grown to confluence (~7 days), dissociated with trypsin, and re-plated to low density onto Corning substrate-coated plasticware on poly-d-lysine-treated glass coverslips as previously described . HEK293 cells were transfected for 4-5 hrs at 37°C with plasmid cDNA and LipofectamineTM transfection reagent (Invitrogen, Carlsbad, CA, USA) in OptiMEM serum-reduced media (Gibco BRL). Either 1.5 μg of plasmid DNA and 7.5 μl Lipofectamine were applied to 30-50% confluent glass coverslips in 35 mm dishes or 3.0 μg of plasmid DNA and 15 μl Lipofectamine were applied to 80-95% confluent 60 mm dishes, for immunocytochemical and biochemical experiments, respectively. pcDNA3 vector was used to normalize total DNA concentration in co-transfection conditions, as previously .
Thirty-six hrs post-transfection, HEK293 cells were washed in PBS and fixed in ice-cold St. Marie's fixative (1.0% acetic acid in 95% ethanol). Cells were washed three times in PBS and incubated for 30 min at rt in PBST-block (0.1% Triton x-100 in PBS and 1% Bovine Serum Albumin). Cells were immunolabeled with primary antisera diluted in PBST-block for 90 min at rt or overnight at 4°C with α-c-myc. Cells were then washed three times in PBS. The secondary antisera were applied at rt for 1.5 hr in PBS using fluorescein isothiocyanate-conjugated goat anti-mouse antiserum (1:200). Following three washes in PBS, cells were counter-stained with a five-minute incubation in diamidino-phenyindole (DAPI) in PBS (1:5000). The cells were mounted on glass slides with Vectashield (Vector Laboratories, Burlingame, CA, USA) to prevent photobleaching.
Tissue sections (~16 μm) were prepared from P30 rats that had been fixed-perfused (4% paraformaldehyde) and sucrose cryoprotected as previously described . Sections were air-dried on the bench for 60 minutes, re-hydrated with PBS, and then incubated at 80°C in 10 mM sodium citrate for 30 minutes for antigen retrieval . The sections were cooled and non-specific binding was blocked by a 60-minute incubation in blocking solution (5% normal goat serum/2.5% BSA/0.3% triton in PBS). During the block, the primary antibodies were treated with L-glutathione to cap reactive SH-functional groups. On ice, an L-glutathione/Tris-EDTA solution was diluted in the blocking solution containing the primary antiserum to a final concentration of 30 mM L-glutathione Tris-EDTA/1.25% normal goat serum/0.625% BSA/0.075% triton . After the block, sections were rinsed with Tris-EDTA - 0.3% triton and incubated with antiserum overnight in a darkened, humidified chamber. Antiserum for RTP1 was used at a final dilution of 1:200 and antisera for TRPC2 was used at 1:400. Sections to be stained using the ABC method were treated according to the manufacturer's protocol (Vector, Burlingham, CA, USA) and the precipitate was visualized with the chromagen AEC (Sigma). Immunofluorescence was detected with either donkey anti-rabbit Texas Red (Amersham Biosciences) or rabbit anti-guinea pig FITC (Sigma).
Conventional light microscopy was performed on an Axiovert S-100 (Zeiss, Thornwood, NY, USA) equipped with epifluoresence, an AxioCam camera (#412-312, Zeiss), and Axiovision data capture software (version 3.1, Zeiss). Images were captured with a pixel resolution of 1300 × 1030. Laser confocal microscopy was performed on an Axioplan 2 microscope attached to an LSM510 two-confocal system (Zeiss). FITC was excited at 488 and DAPI at 700 nm with argon/2 and titanium/sapphire lasers, respectively. Images were captured at 1024 × 1024 pixels resolution in LSM file format, and then were converted to a 16-bit TIFF file format using LsMB software (Zeiss). The TIFF file was opened in NIH ImageJ http://rsb.info.nih.gov/ij/ and a uniform rectangle of 200 pixels × 20 pixels was applied across the center of the cell (Z-axis) to obtain the plot density profile of the pixels underneath, as previously described .
Transfection efficiency analysis
Three to nine fields of view for each transfection condition (TRPC2 + pcDNA3, TRPC2 + RTP1, TRPC2 + REEP1, and Kv1.3 + pcDNA3) were captured under a fluorescent and either a brightfield or a DAPI emission. Each field of view was approximately 90,000 μm2. Transfection efficiency was calculated for each field of view as the ratio of the number of fluorescent cells divided by the total number of cells. Cell counts were performed using NIH ImageJ software.
Cell-surface biotinylation was performed as described . Briefly, either 24- or 48-hr post-transfection, HEK293 cells were washed with ice-cold PBS and then incubated with 1.0 mg/ml biotin (Pierce, Rockford, Ill, USA) in PBS for 30 min at 4°C. Following a PBS rinse, cells were incubated for 30 min at 4°C in quench buffer, rinsed again, and then lysed for 30 min at 4°C in lysis buffer. Lysed cells were scraped, triturated, and centrifuged at 12,000 rpm for 10 min at 4°C. The supernatants were collected and aliquots were set aside for confluency controls. Protein concentration was calculated using a Bradford assay (BioRad). Equal amounts of protein were brought up to 1 ml with PBS (pH 8.0) and incubated overnight at 4°C with 40 μl streptavidin-conjugated agarose-beads (Pierce). The beads were pelleted with a centrifugation of 12,000 rpm for 10 min at 4°C, washed, and then stored at -20°C until further use.
Hoffman modulation contrast optics was used to visualize cells at 40× magnification (Axiovert 135, Carl Zeiss). Thirty-six hrs post-transfection, HEK293 cells were rinsed with bath solution and incubated with anti-hCD8 beads (Dyna-Beads, Invitrogen) to mark transfected cells (either hCD8+/TRPC2+/RTP1- or cCD8+/TRPC2+/RTP1+) . Co-expression with CD8 allows visualization of cells taking up the cDNA encoding the channel or receptor of interest by marking transfected cells with a red polypropylene-antibody-linked bead. We  and others  have demonstrated that single cells take up multiple constructs equivalently and that the density of the beads is proportional to the expression of channel of interest. Cells were rinsed two times before beginning a recording session to remove any unbound beads. Cells were rinsed two more times before beginning a recording session. Electrophysiological records were analyzed using software from Microcal Origin (Northampton, MA) and Quattro Pro (Borland International, Scotts Valley, CA).
Patch electrodes were fabricated from Jencons glass (Jencons Limited, Bridgeville, PA) with pipette resistances between 9 and 14 MΩ. Macroscopic currents were recorded in the whole-cell configuration using an Axopatch-200B amplifier (MDS Analytical Technologies/Axon Instruments, Sunnyvale, CA), filtered at 5 kHz, digitized at 5 kHz, and stored for later analysis. All voltage signals were generated and data were acquired with the use of an Axon Digidata 1200 board with pClamp v9.2 software (Axon Instruments). Cells were held routinely at a holding potential (Vh) of -60 mV, stepped to 80 mV (Vc), held for 40 milliseconds (ms), and then changed to a ramp protocol by falling to -80 mV over 100 ms (-1.6 mV/ms). The total pulse duration was 140 ms and the inter pulse interval was 60 seconds. Several sweeps were taken over the 3-6 minutes after establishing the whole-cell configuration to establish a stable baseline. ATP stimulation was achieved with bath application of 0.5 ml of 500 μM for a final bath concentration of ~166 mM ATP. Peak inward current amplitude was measured at -80 mV from a subsequent sweep.
Numerical data were statistically analyzed using Prizm software (version 4, GraphPad, San Diego, CA, USA). One-way Analysis of Variance (ANOVA) with either a Student-Newman-Keuls (snk) or a Dunnett's post-hoc test was performed with statistical significance determined at the 95% confidence interval.