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Site-directed mutagenesis and DNA transfection of HEK293 cells. Point mutations, N4104K, R4496C, and N4895D, in the mouse RyR2 (1) were made by the PCR-mediated overlap extension method (2). All mutations were confirmed by DNA sequencing. HEK293 cells grown on 100-mm tissue culture dishes were transfected with WT or mutant RyR cDNA by using Ca²⁺ phosphate precipitation, as described (3).

Generation of stable inducible HEK293 cell lines expressing WT RyR2 [RyR2(wt)] and Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) mutants. Stable inducible HEK293 cell lines expressing RyR2(wt) and mutants were generated using the Flp-In T-REx Core Kit from Invitrogen. Briefly, the full-length cDNA encoding the RyR2(wt) or mutant channel was subcloned into the inducible expression vector, pcDNA5/FRT/TO. Flp-In T-Rex-293 cells were cotransfected with the inducible expression vector, pcDNA5/FRT/TO containing the RyR2(wt) or mutant cDNA and the pOG44 vector encoding the Flp recombinase in 1:5 ratios using the Ca²⁺ phosphate precipitation method. Transfected cells were washed with PBS (137 mM NaCl/8 mM Na₂HPO₄/1.5 mM KH₂PO₄/2.7 mM KCl) 1 day after transfection and allowed to grow for 1 more day in fresh medium. The cells were then washed again with PBS, harvested, and plated onto new dishes. After the cells had attached (» 4 h), the growth medium was replaced with a selective medium containing 200 m g/ml hygromycin (Invitrogen). The selective medium was changed every 3-4 days until the desired number of cells were grown. The hygromycin-resistant cells were pooled, aliquoted, and stored at - 80°C. These positive cells are believed to be isogenic, because the integration of RyR2 cDNA is mediated by the Flp recombinase at a single FRT site. Each HEK293 cell line was tested for RyR2 expression by using immunocytofluorescence staining with an anti-RyR antibody, and the RyR2 protein was detected in all cells examined.

Single-cell Ca²⁺ imaging. Intracellular Ca²⁺ transients in stable inducible HEK293 cells expressing the RyR2(wt) or CPVT mutant channels were measured by using single-cell Ca²⁺ imaging and the fluorescence Ca²⁺ indicator dye fura-2 acetoxymethyl ester (fura-2-AM), as described, with some modifications (4). Cells grown on glass coverslips for 17-20 h after induction by 1 m g/ml tetracycline (Sigma) were loaded with 5 m M fura-2-AM in Krebs- Ringer- Hepes (KRH) buffer (125 mM NaCl/5 mM KCl/1.2 mM KH₂PO₄/6 mM glucose/1.2 mM MgCl₂/25 mM Hepes, pH 7.4) plus 0.02% pluronic F-127 (Molecular Probes) and 0.1 mg/ml BSA for 20 min at room temperature. The coverslips were then mounted in a perfusion chamber (Warner Instruments, Hamden, CT) on a Zeiss Axiovert 135 microscope. Cells were perfused continuously with KRH buffer and various concentrations of CaCl₂ (0-1 mM) at room temperature. Fura-2 fluorescence was captured every 4 sec through a Fluor ´ 20 objective and a Chroma filter set using the ImageMaster System and the DeltaRAM rapid wavelength-switching illuminator (Photon Technology International, Lawrenceville, NJ).

Preparation of cell lysate from HEK293 cells. Cell lysates were prepared from HEK293 cells as described (3). Briefly, HEK293 cells grown on 100-mm tissue culture plates 24-26 h after transfection were washed three times with PBS plus 2.5 mM EDTA and harvested in the same solution by centrifugation for 8 min at 700 × g in an IEC Centra-CL2 centrifuge. The cells were then washed with PBS without EDTA and centrifuged again at 700 × g for 8 min. The PBS-washed cells were solubilized in a lysis buffer containing 25 mM Tris/50 mM Hepes (pH 7.4), 137 mM NaCl, 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 0.5% soybean phosphatidylcholine, 2.5 mM DTT, and a protease inhibitor mix (1 mM benzamidine/2 m g/ml leupeptin/2 m g /ml pepstatin A/2 m g/ml aprotinin/0.5 mM PMSF). The mixture was incubated on ice for 1 h. Cell lysate was obtained by centrifugation at 16,000 × g in a microcentrifuge at 4°C for 30 min twice to remove the unsolubilized materials.

GST-FKBP12.6 pull-down and immunoblotting analyses. GST-FKBP12.6 pull-down assay and immunoblotting analysis were carried out as described with some modifications (5). HEK293 cells expressing the RyR2(wt) or CPVT mutants were grown for 17-18 h after induction by 1 m g/ml tetracycline. The same amount of cell lysate (5 mg) prepared from each HEK293 cell line was incubated with glutathione-Sepharose (15 m l) that was prebound to 60 m g GST-FKBP12.6 at 4°C for 17-19 h. The glutathione-Sepharose precipitates were washed with ice-cold lysis buffer containing the protease inhibitor mix three times, each time for 10 min. The proteins bound to the Sepharose beads were solubilized by the addition of 20 m l of 2´ Laemmli’s sample buffer (6) plus 5% 2-mercaptoethanol and boiled for 5 min. An equal portion of the solubilized proteins from different samples was then separated by 6% SDS/PAGE. The SDS/PAGE-resolved proteins were transferred to nitrocellulose membranes at 45 V for 18-20 h at 4°C in the presence of 0.01% SDS, according to the method of Towbin et al. (7). The nitrocellulose membranes containing transferred proteins were blocked for 30 min with PBS containing 0.5% Tween-20 and 5% skim milk powder. The blocked membrane was incubated with the anti-RyR(34c) antibody and washed three times, each time for 15 min, with PBS containing 0.5% Tween-20. The membrane was then incubated with the secondary anti-mouse IgG (H&L) antibodies conjugated with alkaline phosphatase for 30-40 min. After washing three times, each time for 15 min, the bound antibodies were visualized by the alkaline phosphatase-mediated color reaction using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as the substrates.

[³H]Ryanodine binding. Equilibrium [³H]ryanodine (NEN Life Science) binding to cell lysate was performed as described, with some modifications (3). Briefly, a binding mixture (300 m l) containing 30 m l of cell lysate (3-5 mg/ml), 25 mM Tris/50 mM Hepes (pH 7.4), 5 nM [³H]ryanodine, the protease inhibitor mix, and various concentrations of CaCl₂, and KCl as indicated, was incubated at 37°C for 2-3 h. The binding mix was diluted with 5 ml of ice-cold washing buffer containing 25 mM Tris (pH 8.0), and 250 mM KCl, and immediately filtered through Whatman GF/B filters presoaked with 1% polyethylenimine. The filters were washed four times with 5 ml of ice-cold washing buffer and the radioactivity associated with the filters was determined by liquid scintillation counting. Nonspecific binding was determined by measuring [³H]ryanodine binding in the presence of 50 m M unlabeled ryanodine. All binding assays were done in duplicate. Statistical comparisons were made using the unpaired Student’s t test.

Single channel recordings in planar lipid bilayers. Recombinant WT and mutant RyR2 proteins were partially purified from cell lysate by sucrose density gradient centrifugation. Heart phosphatidylethanolamine and brain phosphatidylserine (Avanti Polar Lipids), dissolved in chloroform, were combined in a 1:1 ratio (wt/wt), dried under nitrogen gas, and suspended in 30 m l of n-decane at a concentration of 12 mg lipid per ml. Bilayers were formed across a 250-m m hole in a Delrin partition separating two chambers. The trans chamber (800 m l) was connected to the head stage input of an Axopatch 200A amplifier (Axon Instruments, Austin, TX). The cis chamber (1.2 ml) was held at virtual ground. A symmetrical solution containing 250 mM KCl and 25 mM Hepes (pH 7.4), was used for all recordings, unless indicated otherwise. A 4-m l aliquot (» 1 m g of protein) of the sucrose density gradient-purified recombinant WT or mutant RyR2 proteins was added to the cis chamber. Spontaneous channel activity was always tested for sensitivity to EGTA and Ca²⁺. The chamber to which the addition of EGTA inhibited the activity of the incorporated channel presumably corresponds to the cytoplasmic side of the Ca²⁺ release channel. The direction of single channel currents measured was always from the luminal to the cytoplasmic side of the channel, unless mentioned otherwise. Recordings were filtered at 2,500 Hz. Data analyses were carried out using the pclamp 8.1 software (Axon Instruments). Free Ca²⁺ concentrations were calculated using the computer program of Fabiato and Fabiato (8).

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