Measurements of KCa3.1 K+ currents by whole‐cell patch‐clamp recordings

Whole‐cell patch‐clamp experiments were performed as reported previously (Matsuba et al. 2014). A pipette solution was used, with the following composition (in mmol/L): 110 K‐aspartate, 30 KCl, 1 MgCl₂, 10 HEPES, 10 EGTA, and 2 Na₂ATP (pH 7.2 by KOH). Ca²⁺ concentrations (pCa 6.5) were determined using the WINMAXC program (Stanford University, Stanford, CA). Membrane currents were digitized using an analog‐digital converter (PCI6229, National Instruments, Tokyo, Japan), and data acquisition and analysis were performed using WinWCP4.65, developed by Dr. John Dempster (University of Strathclyde, UK). The liquid junction potential between the pipette and bath solutions (−10 mV) was corrected. Cells were held at −80 mV and currents were evoked by pipette solutions containing Ca²⁺ buffered at 300 nmol/L. Outward K⁺ currents were determined by stepping the cell from −80 to +40 mV for 100 msec, and then to −40 mV to measure the tail current (see Fig. S3A). A standard HEPES‐buffered bathing solution was used, with the following composition (in mmol/L): 137 NaCl, 5.9 KCl, 2.2 CaCl₂, 1.2 MgCl₂, 14 glucose, and 10 HEPES (pH 7.4 by NaOH). All experiments were performed at 25 ± 1°C.