Syncropatch 384pe

Automated patch-clamp on the SyncroPatch 384PE (Nanion Technologies, Munich, Germany) was used to measure Gárdos activity, with a modified protocol previously described [14 (link)]. Briefly, after three washes with PBS and upon cell catch, external recording solution was added to the wells followed by 10 µM NS3623 (a powerful chloride channel inhibitor [15 (link)]), 10 µM NS309 (Alomone labs, Jerusalem, Israel), 5 µM TRAM-34 (a specific Gárdos channel inhibitor, Tocris, Bristol, UK), and 30 µM GdCl₃ (a generic non selective cation channel inhibitor, Sigma-Aldrich, St. Louis, MO, USA). Internal solution (in mM): 10 KCl, 110 KF, 10 NaCl, 10 EGTA, and 10 HEPES/KOH (pH 7.2). External solution (in mM): 140 NaCl, 4 KCl, 2 CaCl₂, 1 MgCl₂, 5 D-Glucose monohydrate, and 10 HEPES/NaOH (pH 7.4) (Nanion Technologies, Munich, Germany). All compounds (NS3623, NS309, TRAM-34, and GdCl₃) were diluted in an external solution. Currents were measured at room temperature, applying −100 to +80 mV ramp voltage protocol for 300 ms every 10 s at a holding potential of −30 mV. The cell response was measured in pA at +80 mV. Each cell underwent strict quality control (QC) filters, and only NS309 and TRAM-34 responder cells were used in the analysis (Supplementary Table S1).

Second pharmacology screening was performed at Eurofins Pharma Discovery Services in a panel of 190 in vitro radioligand binding and enzyme assays covering a diverse set of enzymes, receptors, ion channels, and transporters. The cardiovascular panel (hERG, Nav1.5, Kv4.3 and IKs) were performed on the SyncroPatch 384PE (Nanion Technologies) high throughput patch clamp platform (Chinese hamster ovary K1 cell line) at room temperature in a 6 point cumulative assay.