Whole-Cell Voltage-Clamp Recordings

Recordings were performed at room temperature (22°C–25°C) using HEK293 (human embryonic kidney 293) cells stably transfected with Nav1.5 cDNA. For Nav1.5 recordings, the extracellular solution (mM) included 137 NaCl, 10 HEPES, 4 KCl, 1 MgCl2, 1 CaCl2, 10 dextrose, and the intracellular solution included 120 aspartic acid, 120 CsOH, 10 CsCl, 10 HEPES, 10 EGTA, 5 MgATP, 0.4 TrisGTP. The voltage protocol (1 s duration) was repeated at 0.1 Hz. Briefly, cells were repolarized from −95 to −120 mV for 200 ms, then depolarized from −120 to −15 mV for 40 ms, and further depolarized to +40 mV for 200 ms, followed by a voltage ramp down phase for 100 ms from +40 to −95 mV. Nav1.5 late current was induced by 20 nmol/L anemone toxin in the extracellular solution, which allowed for the study of late sodium current contribution from Nav1.5 that is separate from fast sodium current, as previously described.⁵⁸ Nav1.5 fast current was measured in the absence of anemone toxin. For positive control, 30 µM tetrodotoxin (sodium channel blocker) was directly applied to recordings and resulted in 98.3±0.7% Nav1.5 current block (n=3). To ensure baseline recording was stable enough for drug application, cells were presented with this voltage protocol in control solution until Nav1.5 current amplitudes for 12 consecutively recorded current traces (2-minute duration) exhibit <10% difference. Effects were monitored for 3 minutes before and after application of MEHP (n=4 per dose). To quantify drug potency against Nav1.5 channels, the steady state Nav1.5 current amplitude (averaged value from 5 steady state current traces) in drug solution was divided by the averaged amplitude from the last 5 traces measured in control solution to calculate the fractional block. Then, fractional block was plotted against drug concentration tested, fitted with the Hill Equation to generate an half-maximal inhibitory concentration (IC50) and the Hill coefficient.