2.2. Cell culture and transfection

HEK 293 cells (European Collection of Cell Cultures, Porton Down, UK) transfected with Kv4.3 or a stable HEK 293 cell line expressing wild-type (WT) hERG (Kv11.1) channels (kindly provided by Professor Craig January) [37] were used. Additional K⁺ channel β–subunits were also transfected into the cells (as described below). Cells were maintained at 37 °C in a humidity controlled incubator with 5% CO₂ atmosphere and cultured in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, Life technologies division, Paisley, UK) supplemented with 10% (v/v) heat inactivated fetal bovine serum (Thermo Fisher Scientific, Life Technologies division, Paisley, UK), 1% (v/v) non-essential amino acids (Thermo Fisher Scientific, Life Technologies division, Paisley, UK) and 50 μg/mL Gentamicin (Merck KGaA, Darmstadt, Germany). In the case of hERG-expressing HEK 293 cells, the medium was further supplemented with 400 μg/ml G418 selection antibiotic (Thermo Fisher Scientific, Life technologies division, Paisley, UK). Prior to transfection, cells were plated for 48 h onto 12-well plates using a non-enzymatic agent (Merck KGaA, Darmstadt, Germany) before transfection. Transfection reactions were prepared using OPTI-MEM® I (Thermo Fisher Scientific, Life Technologies division, Paisley, UK). For Kv4.3 experiments 0.3 μg of DNA was transfected along with either 1 μg of a GFP construct (control) or KChIP2.1/2.2. In experiments where a KChIP2 isoform and human DPP6 were co-transfected, 0.8 μg of each DNA was transfected and compared to a “control” condition, in which 1.6 μg of GFP alone was transfected. For hERG experiments, a matching concentration of GFP DNA was used, in order to have an equivalent DNA concentration across all conditions. Lipofectamine™ 2000 transfection agent (Thermo Fisher Scientific, Life Technologies division, Paisley, UK) was used at a 2:1 ratio with DNA. The culture medium was replaced 5 h after transfection. After 24 h, cells were collected and re-plated onto 13 mm round glass coverslips, pre-treated for 4 h with 200 μg/mL Poly-D-Lysine (Merck KGaA, Darmstadt, Germany). Patch-Clamp recordings started 3 h after plating.