Patch-clamp recordings: Voltage step current

For conventional whole-cell experiments, the pipette solution contained 10 mM HEPES, 110 mM K-aspartate, 1 mM MgCl₂, 30 mM KCl, 10 mM NaCl, 5 mM EGTA and 2.5 mM CaCl₂, with pH adjusted to 7.2 with KOH. Free Ca²⁺ was 200 nM, calculated using the online Ca-Mg-ATP-EGTA calculator V1.0 using constants from NIST database #46 V8 (http://maxchelator.stanford.edu/CaMgATPEGTA-NIST.htm). Outward currents were measured during 250-ms voltage steps from -40 mV to +50 mV in 10-mV increments, from a holding potential of -50 mV, in the presence or absence of the selective BK channel inhibitor, paxilline (1 μM). BK current amplitude was estimated from difference currents (±paxilline) calculated using Clampex 10.4. All recordings were performed at room temperature (~22°C). Corrections for liquid junction potentials were applied using the amplifier prior to recordings. For perforated-patch protocols with voltage-stepped current measurements, the pipette and bath solutions were the same as outlined above for the measurement of STOCs with nifedipine and ryanodine in the bath solution.