Design and production of stably MCU knockdown HeLa cells and their corresponding control cells
HeLa MCU-KD and HeLa control cells have been produced upon request and supplied by TeBu-bio® (Tebu-bio SAS, Le Perray-en-Yvelines Cedex, France). HeLa cells with stable MCU knockdown and the respective scrambled control cells were produced by applying the SilenciX® technology (Tebu-bio, www.tebu-bio.com, Le Perray-en-Yvelines, France) using the following 5′-3′shRNA sequence against MCU: GGTGCAATTTATCTTTATA.
Cell culture and isolation of mitochondria
All cells were grown on DMEM containing 10 % FCS, 50 U/ml penicillin, and 50 μg/ml streptomycin. Mitochondria were freshly isolated as previously described . Mitochondria were prepared from HeLa cells by differential centrifugation. Cells were trypsinized, harvested, and washed with PBS. The cell pellet was suspended in a 200-mM sucrose buffer containing 10 mM Tris-MOPS, 1 mM EGTA and protease inhibitor (1:50, P8340 Sigma, Vienna, Austria) (pH adjusted to 7.4 with TRIS), and homogenized with a glass–Teflon potter (40–50 strokes). Nuclear remnants and cell debris were centrifuged down at 900 g for 10 min. The supernatant was centrifuged at 3,000g for 20 min. The mitochondrial pellet was washed and centrifuged down at 7,000g for 15 min. All fractions were kept on ice until further utilization.
Preparation of mitoplasts
Isolation and preparation of mitoplasts (mitochondria devoid of outer membrane) from HeLa cells was performed as recently described . Briefly, mitoplast formation was achieved by incubation of isolated mitochondria in hypotonic solution (5 mM HEPES, 5 mM sucrose, 1 mM EGTA, pH adjusted to 7.4 with KOH) for 8 min. Then, hypertonic solution (750 mM KCl, 80 mM HEPES, 1 mM EGTA, pH adjusted to 7.4 with KOH) was added to restore isotonicity.
Mitoplast patch-clamp recordings
Single channel measurements were performed in the mitoplast-attached configuration as previously described [2, 9]. In brief, patch pipettes were pulled from glass capillaries using a Narishige puller (Narishige Co., Ltd., Tokyo, Japan), fire-polished, and had a resistance of 8–12 MΩ. Mitoplasts were bathed in the solution containing (in millimolars): 145 KCl, 1 EGTA, HEPES, and pH adjusted to 7.2 with KOH. For single channel recordings, the pipette solution contained 105 mM CaCl2 and 10 mM HEPES, 10 μM cyclosporin A (Tocris Bioscience, Bristol, UK) and 10 μM 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP 37157, Ascent Scientific Ltd., Bristol, UK) to prevent opening of the permeability transition pore, and the activity of the mitochondrial Na+/Ca2+ exchanger (NCXmito), respectively. pH was adjusted to 7.2 with Ca(OH)2. Single channel currents were recorded at a fixed holding potential indicated in the respective figures. For whole-mitoplast recordings, pipette solution contained (in millimolars): 120 Cs methanesulfonate, 30 CsCl, 1 EGTA, 110 sucrose, 2 gluconic acid, and pH by TEAOH to 7.2. For obtaining whole-mitoplast configuration, voltage steps of 300–600 mV and 20–50 ms duration were applied. Voltage ramps of 1 s duration from −160 to +50 mV were delivered every 5 or 10 s from the holding potential 0 mV. Currents were recorded using a patch-clamp amplifier (EPC7, List Electronics, Darmstadt, Germany). Data collection was performed using Clampex software of pClamp (V9.0, Molecular Devices, Sunnyvale, CA, USA). Signals obtained were low pass filtered at 1 kHz using an eight-pole Bessel filter (Frequency Devices) and digitized with a sample rate of 10 kHz using a Digidata 1200A A/D converter (Molecular Devices, Sunnyvale, CA, USA). All measurements were performed at room temperature. For recording cationic currents via whole mitoplasts, bath solution contained (in millimolars): 150 TRIS HCl, 1 EGTA, 1 EDTA, 10 HEPES with pH 7.2. For INa recording, NaCl was substituted for TRIS HCl. Ca2+-containing bath solution for ICa recording contained (in millimolars): 140 TRIS HCl, 3 CaCl2, 10 HEPES, and pH 7.2
Single cell Ca2+ imaging and data acquisition
Imaging mitochondrial targeted cameleon 4mtD3cpv was performed on a digital wide field imaging system, the Till iMIC (Till Photonics Graefelfing, Germany) using a 40× objective (alpha Plan Fluar 40×, Zeiss, Göttingen, Germany). For illumination of the cameleon, an ultrafast switching monochromator, the Polychrome V (Till Photonics), was used for excitation light at 430 nm. Emission light was collected at 480 and 535 nm using a single beam splitter design (Dichrotome, Till Photonics). Images were recorded with a charged-coupled device camera (AVT Stringray F145B, Allied Vision Technologies, Stadtroda, Germany). For the data acquisition and the control of the digital fluorescence microscope, the live acquisition software version 188.8.131.52 (Till Photonics) was used. Experiments were performed at the same day than the isolation, purification, and electrophysiological measurements of the respective mitoplasts.
Experimental buffers for Ca2+ measurements
Ca2+ measurements in HeLa cells were performed by stimulating cells in Ca2+-containing environment. Cells were superfused by a Ca2+-containing buffer, which was composed of (in millimolars): 138 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 d-glucose and 10 HEPES, and pH adjusted to 7.4 with NaOH. Stimulation was performed using 100 μM of the IP3-generating agonist histamine.
HeLa cells that were washed with ice-cold PBS or isolated mitochondria were lysed with RIPA buffer containing protease inhibitor cocktail (Sigma-Aldrich, Vienna, Austria). The protein concentration was measured using the BCA protein assay (Thermo Fisher Scientific Inc., Vienna, Austria). Forty micrograms of protein were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was incubated with the primary antibody at 4 °C overnight and the primary antigen–antibody complex was detected by incubating the blot with a horseradish peroxidase-conjugated secondary antibody at room temperature for 2 h. The membrane was further developed with the ECL Plus Western blotting detection system (GE Healthcare, Vienna, Austria). To control the equal amount of protein loading of whole cell lysates and isolated mitochondria, MCU expression (sc-246071; Santa Cruz, Vienna, Austria) were densitometrically normalized to β-actin (sc-47778; Santa Cruz) and VDAC (sc-32063 and sc-32059; Santa Cruz), respectively.
RNA was isolated from HeLa cells using a Total RNA isolation kit (PEQLAB Biotechnologie GmbH, Erlangen, Germany), and it was reverse transcribed using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA). The analysis of the expression of the target genes was performed by conventional polymerase chain reaction (PCR) using GoTaq Green master mix (Promega, Madison, WI, USA) and real-time PCR using QuantiFast SYBR Green RT-PCR kit (Qiagen, Hilden, Germany) on LightCycler 480 (Roche Diagnostics, Vienna, Austria). RNA polymerase II (RPOL2) was used as a housekeeping control [12, 16, 24]. Primers for RPOL2 and MCU were obtained from Invitrogen (Vienna, Austria) and their sequences (5′–3′) were as follows: RPOL2: CATTGACTTGCGTTTCCACC, RPOL2 rev: ACATTTTGTGCAGAGTTGGC, MCU: TTCCTGGCAGAATTTGGGAG, and MCU rev: AGAGATAGGCTTGAGTGTGAAC.
The occurrence probability was calculated as a fraction of patches displayed specific channel activity relative either to the total number of patches studied or the number of active patches displayed any type of the channel activity. Single channel analysis was performed using Clampfit 9.2 (Molecular Devices, Sunnyvale, CA, USA). Data are expressed as means with standard error. Statistical comparisons were conducted with a two-tailed unpaired t test. Values of p < 0.05 (*) were taken as statistically significant. Statistical analysis was performed by Graph Pad Software version 5.01 (La Jolla, CA, USA).