2.2. Single-cell electrophysiological recordings
Core human cardiac channels were heterologously expressed in recombinant HEK293 or CHO cell lines and studied using conventional patch clamp methods. Stable cell lines expressing Nav1.5 and Cav1.2/β2/α2/δ1, hERG, Kir2.1, Kv4.3, KCNQ1/E1 were used. All currents were recorded using a MultiClamp 700A amplifier, 1550A digitizer and pCLAMP 10.6 software (Molecular devices, USA). Patch electrodes with resistances of 2-5 MΩ were pulled with a P-97 micropipette puller (Sutter Instruments, USA). The conventional whole-cell recordings were obtained by manual suction for hERG, Kir2.1, Kv4.3 and Nav1.5 studies. Perforated whole-cell recordings of KCNQ1/E1 and Cav1.2 were obtained following perforation with 200 µg/ml Amphotericin included in the pipette solution. After whole-cell mode were obtained, cells were used in voltage clamp mode for the current recordings. The external solution contained (mM):NaCl 137;MgCl2 1;KCl 4;glucose 10; HEPES 10;CaCl2 1.8, pH adjusted to 7.4 with NaOH. The K+-containing pipette solution contained (mM):KCl 40;KF 100;MgCl2 2; EGTA 5;HEPES 10,pH adjusted to 7.2 with KOH. The sodium and calcium current pipette solution contained (mM): CsCl 65;CsF 75;MgCl2 2.5;EGTA 5;HEPES 10, pH adjusted to 7.3 with CsOH.