Patch clamp experiment

Isolated human atrial myocytes were kept in a perfusion chamber for 10~15 min and then perfused with extracellular solution (in mmol/l: 140 N-methyl glucamine, 4 KCl, 1 MgCl₂, 5 glucose, 10 Hepes, pH 7.4 with HCl) at 1 ml/min. Under an inverted microscope (Olympus, Japan), cardiomyocytes with clear cross-striation were chosen for use in the electrophysiological experiment. Borosilicate glass electrodes (outer diameter 1 mm) were pulled using a micropipette puller (model P-97, Sutter Instrument, USA). The microelectrode had tip resistances of about 2~4 MΩ when filled with the pipette solution (in mmol/l: 144 potassium gluconate, 1.15 MgCl_2, 5 EGTA, 10 HEPES, pH 7.4 with KOH). SK current was recorded using a whole-cell configuration. In the presence of 5 mM EGTA, the MaxChelator program (http://maxchelator.stanford.edu) was used to calculate the amount of CaCl₂ that needed to be added to pipette solution to control the free intracellular Ca²⁺ level. SK current was measured using an EPC-10 amplifier (HEKA Instruments, Germany) and Patchmaster software (HEKA, Germany). When a giga-ohm seal was obtained, the cell membrane was ruptured using negative pressure to establish the whole-cell configuration. The series resistance was compensated up to 70~80%. The sampling frequency was 10 KHz. The SK channel current was identified as apamin-sensitive current (I_KAS). I_KAS was elicited by depolarizing step pulses from −130 mV to +60 mV for 300 ms with a holding potential of −60 mV. Data were acquired, stored, analyzed using Patchmaster and Pulse (HEKA Instruments), and plotted using Origin 8.0 software (OriginLab Corporation, USA).