35 mm dishes

The cells were grown on 35 mm dishes (Nunc) to a confluency of about 50%. The cells in each dish were transfected with 4 µg of wild-type rCav1.3 or mutant rCav1.3 R930H cDNA in pCMV6b, 1 µg α₂δ₁ (Supplementary File: Figure S1) in pCDNA3.1, 1 µg β2b in pCDNA3.1 and 0.2 µg of pEGFP vector using Jetprime (peqlab, Erlangen, Germany), according to the instructions of the manufacturer. After 48 h, CHO cells were recorded in whole cell configuration at room temperature (22 °C) with an EPC-10 amplifier (HEKA). All recordings were digitized at 10 kHz, low-pass filtered at 2 or 5 kHz, analyzed with PulseFIT software (HEKA) and compensated for 60–70% of the series resistance. The pipettes had a tip resistance of 2.5–4.0 MΩ when filled with a solution containing (in mM): Cs-methane sulfonate 120, CaCl₂ 5, MgCl₂ 2, EGTA 10, MgATP 2 and HEPES 10 (pH 7.4 CsOH), yielding a [Ca²⁺_i] of about 110 nM (calculated with WinMAxc). The cells were bathed in a solution containing (in mM): NMDG 130, CaCl₂ 15, KCl 5 and HEPES 10 (pH 7.4, HCl).

Fluorescence images and bright-field movies were performed with a Zeiss Axio Observer.Z1 as described above, using a Zeiss LD A-Plan 40×/0.05 Ph2 objective and 35-mm dishes (Nunc) without oil immersion. Movies were taken by Axio Vision LE Modul ‘Digital High Speed Recorder’ with an exposure time of 70 ms per picture, resulting in a 15-s movie with 14 frames per second. To determine the action potential frequency, the beats per minute (bpm) were visually analyzed by four different persons, who simultaneously counted the beating rate in order to determine the average bpm. The average bpm was analyzed from four to six independent transfections and dishes with untransfected HL-1 cells.

HL‐1 cells were grown in supplemented Claycomb medium at 37°C in an incubator supplied with 5% CO₂ as previously described (Claycomb et al, 1998). Cells were split in a 1:3 ratio when they were 100% confluent and had the ability to beat. For transfection, HL‐1 cells were seeded on 35‐mm dishes (Nunc) previously coated with gelatin/fibronectin in a 1:2 ratio; 24 h after seeding, cells were transfected with either 1.5 μg of TREK‐1‐EGFP or TREK‐1^I267T‐EGFP cDNA constructs using Lipofectamine 2000 (Invitrogen) following manufacturer instructions. After 24 h, transfection media was exchanged and cells were incubated for 4 h in supplemented Claycomb media. Subsequently, the 100% confluent and beating cells were used in patch clamp and imaging experiments.