Internalization of GABAA Receptor Subunits

Cell-surface receptors were labelled in living cultured neurons or in α1β2-HEK293 cells transiently transfected with the myc-γ2 cDNA, as described previously 19 (link). Briefly, coverslips were incubated with ice-cold Buffer A (mM: 150 NaCl; 3 KCl; 2 MgCl₂; 10 HEPES, pH 7.4; 5 Glucose), containing 0.35 M sucrose for 5 minutes, followed by incubation with mouse anti-β_2/3 antibody (MAB341-MerckMillipore) for 30 minutes at 4°C in Buffer A containing 0.35 M sucrose, 1 mM EGTA and 1% BSA. Cells were further incubated at 37°C in Buffer A containing 1 mM CaCl₂ and 5 µg/ml leupeptin, in the absence or presence of diazepam (1 µM, Tocris), for 1 hour to allow internalization of the labelled β_2/3 subunit-containing GABA_ARs. Cells were fixed with 4% paraformaldehyde/4% sucrose/PBS (PFA/PBS) and processed for immunolabelling with anti-mouse Alexa-555 antibodies overnight at 4°C. Cells were subsequently permeabilized and labelled with anti-mouse Alexa-488 antibodies (Supplementary Table S1) for 1 hour at room temperature. Samples were imaged using a Zeiss LSM 710 confocal microscope as above.