2.7. Electrophysiological data acquisition and analysis

HEK cells were transfected with 0.5 μg of KCNQ1 plasmid and 0.5 μg of KCNE1 plasmid, and 0.1 μg EGFP was co-transfected as a transfection marker. Conventional whole-cell patch-clamp was conducted to study the function of KCNQ1/KCNE1 channels 48–56 h after transfection.

For recording in HEK cells, the extracellular recording solution contained (in mM): 145 NaCl, 5.4 KCl, 1.8 CaCl₂, 1 MgCl₂, 10 HEPES, 10 D-Glucose. pH value was adjusted to 7.4 with NaOH, and the osmolality between 320 and 330 mOsm with D-Glucose. The pipette solution contains (in mM): 130 K Aspartate, 11 EGTA, 1 MgCl₂, 1 CaCl₂, 10 HEPES, and 5 K₂ATP. pH was adjusted to 7.2 with KOH. For recording in cardiomyocytes, the extracellular recording solution contained (in mM): 132 NaCl, 4 KCl, 1 CaCl₂, 1.2 MgCl₂, 10 HEPES, 5 D-Glucose. pH value was adjusted to 7.4 with NaOH. The pipette solution contained (in mM): 125 K Aspartate, 20 KCl, 10 EGTA, 1 MgCl₂, 5 HEPES, and 5 K₂ATP. pH was adjusted to 7.2 with KOH.

IKs current was recorded from HEK cells expressing KCNQ1/KCNE1, or cardiomyocytes expressing KCNQ1-GFP/KCNE1, which were voltage-clamped from — 80 mV or — 50 mV to +100 mV in 10 mV interval for 3 s, respectively, before clamped at –20 mV for tail current to measure channel activation. Data were acquired using Axopatch 200B (Axon Instruments) equipped with an A/D converter Digidata 1322A (Axon Instruments) and recorded and stored in a pc computer using Clampex 10 software. Data were analyzed in Clampfit software. Peak tail currents were fit into a Boltzmann equation, G(v) = Gmax/[1 + exp(–(V – V_1/2)/k)] for conductance Gmax, which reflects channel density on plasma membrane, and midpoint voltage V_1/2, where channels are half-maximally activated. More details have been previously described [23].