FIGURE 6.

GABA_AR internalization in COS-7 cells is increased by GABA. A, COS-7 cells were transiently co-transfected with α1-, β2-, and Myc-tagged γ2 subunits. Internalized labeling (red) was obtained after incubating live cells with polyclonal antibody directed at the extracellular Myc tag at 37 °C followed by stripping of antibodies remaining on the surface. Intracellular staining (green) of the same cells was obtained after permeabilization and labeling with a monoclonal antibody directed at the same tag (white frames detailed in lower panels). Scale bar, 10 μm. B, quantification of the area of intracellular punctate labeling of internalized receptors formed by γ2 subunit in cells incubated in culture medium (Ctrl) with GABA, gabazine (GBZ), or picrotoxin (PTX). ***, p < 0.001. C, COS-7 cells transfected as in A were labeled with biotin and returned to 37 °C in control medium, GABA, gabazine, or picrotoxin, as indicated. Biotin remaining on the surface after the incubation at 37 °C was removed by cleaving. Total and internalized (biotinylated) γ2 subunits were detected with an anti-Myc antibody. GAPDH staining shows that intracellular proteins were not biotinylated. D, means of internalized/total expression ratio normalized to total γ2 from experiments described in C. **, p < 0.005; *, p < 0.01. E, COS-7 cells transfected as in A and C and labeled with biotin. Representative Western blot shows Myc-tagged γ2 in total or surface-biotinylated extracts. F, means of surface/total expression ratio normalized to total γ2 from experiments described in E show that surface expression was unchanged by treatment with GABA. G, quantitative analysis of α1β2γ2 GABA_ARs surface expression in COS-7 cells transiently co-transfected with α1-, β2-, and Myc-tagged γ2 subunits and incubated with monensin with or without muscimol for 30 min before surface and total immunofluorescence labeling. A value of 1 for cells incubated without muscimol was used. **, p < 0.005. Error bars, S.E.