Bapta am

HAECs were plated on a Y-shaped slide (ibidi #80126). After reaching confluence, cells were exposed to flow (20 dynes cm⁻²) for 48 h using red perfusion set (ibidi #10962) containing 13 mL of conditioned MCDB-131 with 10% FBS. After 48 h, the cells were treated with 20 µM BAPTA-AM (Life Technologies #B6769) for 20 min. BAPTA-AM was reconstituted in Pluronic F-127 (Invitrogen #P-3000MP) and diluted in MCDB-131 (VEC Technologies #MCDB-131 WOFBS) with 10% FBS (Fisher Scientific, #MT 21-023-CV). After a 20-min incubation with BAPTA-AM, slides were reconnected to perfusion sets and exposed to flow (20 dynes cm⁻²) for 1 h in the presence of 50 µM DAPT or equivalent volume of DMSO. After 1 h, the slides were removed and fixed with 2% PFA and subsequently imaged for confocal microscopy.

Human embryonic kidney line 293 (HEK293T) cells from ATCC authenticated via STR profiling by Eurofins Genomics (ISO 17025) and confirmed negative for mycoplasma via PCR (EZ-PCR Mycoplasma detection kit, Generon, 20-700-20) were cultured to 80% confluence before passaging and used for experimentation between P10 and P40. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM Gibco) containing penicillin (100 U/mL; Sigma), streptomycin (100 µg/mL; Sigma), and 10% fetal calf serum (Sigma). HEK293 cells were cultured in 24 well plates for 24 hr prior to transfection and transfected with 300 ng of each plasmid using FuGene transfection reagent (Promega), according to the manufacturer’s instructions. Plasmids: hKCNQ4-eYFPc (AF105202) or hKCNQ4CCC/AAA -eYFPc a triple cysteine to alanine mutation in the S2–S3 linker (C156A, C157A, and C158A; Gamper et al., 2006 (link)) were co-transfected with WT CaM or lobe mutants. For BAPTA-AM experiments, cells were incubated with 10 µM cell-permeable BAPTA-AM (B1205 ThermoFisher) for 30 min prior to recordings. Cells were also bathed in 10 µM BAPTA-AM for the duration of the recording.

The cells (1 × 10⁵/well) were seeded on flat‐bottom black 96‐well culture plates. After 24 h, they were incubated in serum‐free Dulbecco’s modified Eagle’s medium for 4 h, washed twice with regular buffer [10 mm HEPES (pH 7.4), 140 mm NaCl, 10 mm glucose, 5 mm KCl, 1 mm CaCl₂, and 1 mm MgCl₂], and treated with 5 μm calcium crimson in regular buffer for 30 min at 25 °C. For calcium chelation, the cells were incubated with 10 μm BAPTA‐AM (Thermo Fisher Scientific, Waltham, MA, USA) for 15 min in regular buffer at 37 °C and 5% CO₂, and then, the BAPTA‐AM loading solution was removed and the cells were treated with calcium crimson as described above. After buffer replacement with calcium‐free buffer [10 mm HEPES (pH 7.4), 140 mm NaCl, 10 mm glucose, 5 mm KCl, 1 mm CaCl₂, 1 mm MgCl₂, 1 mm EDTA, and 1 mm EGTA], the cells were stimulated with 5 µm caffeine. The level of intracellular calcium was determined with the 590/615‐nm ratio (emission at 590‐nm excitation/emission at 615‐nm excitation) using a multimode microplate reader (BioTek Instruments).