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.