2.1 | Molecular biology
PCR-based mutagenesis was used to insert mutations into the hKv1.5 (KCNA5 ) cDNA. The Kv1.5 cDNA is based on the database entry NM_002234 but differs by two residues (K418R and K565E). Compared to the previous database entry M60451 it includes two additional amino acids in the N-terminus, leading to a shift of the numbering of +2. The PCR products were fully sequenced (ABI 3100, Applied Biosystems, California, USA). Kv1.5 cDNA was linearized with NheI and complementary cRNA was produced with the mMESSAGE mMACHINE T7-Kit (AM1344 – Ambion, Texas, USA) and its quality was checked by UV spectroscopy (NanoDrop 2000 – Thermo Fisher Scientific, Texas, USA) and gel electrophoresis.