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.