cDNAs for mouse GIRK2c46 (GIRK2c is referred to as ‘GIRK2’ in this study for clarity), rat GIRK4[Krapivinsky, 1998 #126], mouse IRK124, human m2R46, and bovine m-Phos26 were subcloned into pcDNA3.1 vector (Invitrogen). GIRK4* contains a S143T mutation in the pore-helix27. Point mutations were introduced by Quickchange site directed mutagenesis kit (Stratagene). GIRK2-PIP2 mutant was created by overlap-PCR method47. Briefly, a region of GIRK2-D228-L234 was replaced with the homologous region of IRK1-N216-L222; this region contains seven amino acids in the βC-βD region implicated previously in PIP2 binding28,29 (we refer to this mutant as GIRK2-PIP2). All mutations were confirmed by DNA sequencing. HEK-293T cells were cultured in DMEM supplemented with 10% fetal bovine serum, and 1X Glutamax (Invitrogen) in a humidified 37° C incubator with 5% CO2. Cells were plated in 12 well dish, and transiently transfected with DNA using Lipofectamine 2000 (Invitrogen). Cells were replated to 12-mm glass coverslips coated with poly-D-Lysine (20 μg/ml) 12−24 hr after transfection.
