HEK293 cells (ATCC; Manassas, VA) were cultured at 37 °C/5%CO2 according to supplier recommendations. For electrophysiological studies, cells were plated on 8-mm glass coverslips (15,000 cells/well), and transfected using calcium phosphate with a mixture of pcDNA3-based expression plasmids harboring coding sequences for EGFP (10 ng/well), GABABR1 (25 ng/well), GABABR2 (25 ng/well), GIRK1 (25 ng/well), and either GIRK2a or GIRK2c (25 ng/well); experiments were conducted 16–24 h later. For immunoblotting experiments, cells were plated on 6-well plates (80,000 cells/well) and transfected using Lipofectamine 2000 reagent (Thermo Fisher Scientific; Waltham, MA); 2 μg of GIRK1, GIRK2a, GIRK2c or GIRK3 expression plasmids were used per well, and cells were harvested 48 h after transfection.
