Expression Vector Construction and HEK-293 Cell Transfection

For each mutation, the appropriate nucleotide changes were engineered by site-directed mutagenesis into a construct containing the most common splice variant of SCN5A (hH1c1, GenBank {"type":"entrez-nucleotide","attrs":{"text":"AC137587","term_id":"27413169","term_text":"AC137587"}}AC137587) (27) to encode an altered Na⁺ channel α-subunit, Na_V1.5, using the QuikChange II XL site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA) according to the manufacturer’s instructions. The integrity of the constructs and the presence of the desired mutation were verified by DNA sequencing. The primers used for mutagenesis are listed in Table 1. Wild-type (WT) (27) or mutant constructs were cotransfected with pEGFP-C1 (Clontech, Palo Alto, CA) into HEK-293 cells (American Type Culture Collection, Manassas, VA) using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA), as described previously (8, 37). After overnight culture, transfected cells positive for green fluorescent protein (GFP) were selected for whole cell voltage clamp.

Primers used for mutagenesis