GIRK2c and mutant channels were engineered with an extracellular hemagglutinin (HA) epitope inserted between I126 - E127 for immunohistochemical detection with anti-HA antibodies46. HEK-293T cells were transfected with 0.5 μg of channel cDNA and examined 24−48 hr after transfection. Cells were washed with 1X Dulbecco's Phosphate Buffer Saline (DPBS; Invitrogen), fixed with 2% para-formaldehyde (PFA) in 1X DPBS for 10 min and rinsed with 1X DPBS (at 22−25° C). To label surface channels, cells were incubated with blocking buffer (3% BSA in 1X DPBS) for 1 hr and then with anti-HA mouse antibody (1:400 in blocking buffer; Covance) for 2 hr at 22° C. To label cytoplasmic channels, cells were rinsed with 1X DPBS, permeabilized with 0.25% TritonX-100 (Sigma) in blocking buffer for 10 min at 22° C and incubated with blocking buffer for 1 hr. Cells were then incubated with rabbit anti-GIRK2 (1:200 in blocking buffer; Alomone) for 2 hrs. Following rinses in 1X DPBS, cells were incubated with fluorescent secondary antibodies, anti-mouse Alexa-647 and anti-rabbit Alexa-488 (1:300; Invitrogen) for 1 hr in the dark. Cells were rinsed with
