Macroscopic Membrane Current Measurement

Macroscopic membrane current was recorded 24–72 hours after injection as described previously.^{31 (link)} Oocytes were perfused at room temperature in Barth’s solution containing (in mM) 88 NaCl, 1 KCl, 2.4 NaHCO₃, 0.3 NaNO₃, 0.71 CaCl₂, 0.82 MgCl₂, and 15 HEPES for pH 8.5 or 15 MES for pH 5.5 at a rate of 2 mL/min. The tested compounds were dissolved in DMSO and applied (100 μM) at pH 5.5 when the inward current reaches maximum. The compounds were applied for 2 min, and residual membrane current was compared with the membrane current before the application of compounds. The compounds were typically applied for 2 min, and residual membrane current was compared with the membrane current before the application of compounds. Membrane currents were recorded at −20 mV and analyzed with pCLAMP 10.0 software package (Axon Instruments, Sunnyvale, CA).