FIGURE 2.

γ2(R43Q) internalization is clathrin- and dynamin-dependent. A, COS-7 cells were transiently co-transfected with α1-, β3-, and Myc-tagged γ2 subunits (1:1:1 ratio), either wild-type (γ2) or bearing the R43Q (γ2(R43Q)) substitution, as indicated at the top. Surface labeling was obtained after immunostaining living cells with a polyclonal antibody directed at the extracellular Myc tag and Alexa Fluor-568-conjugated secondary antibodies (surface), and intracellular staining of the same cells was revealed using a monoclonal antibody directed at the same tag and FITC-conjugated secondary antibodies (intra). Merged images show intracellular retention of the γ2(R43Q) subunit (control), whereas surface labeling was detected when cells were incubated with dynasore. Scale bar, 10 μm. B, the ratio of surface/total expression was measured in COS-7 cells expressing α1β3γ2 GABA_ARs, after incubation with dynasore (n = 13–22). C, cells were transiently co-transfected with α1-, β2-, or β2LL/AA and Myc-tagged γ2 subunit (1:1:1 ratio), either wild-type (γ2) or bearing the R43Q (γ2(R43Q)) substitution, as indicated at the top. Surface and intracellular labeling were obtained as above. Merged images show intracellular retention of the γ2(R43Q) subunit when cells were co-transfected with β2, whereas surface labeling was detected when cells were co-transfected with β2LL/AA. Scale bar, 10 μm. D, quantitative analysis of α1β2γ2 and α1β2(LL/AA)γ2 GABA_ARs surface expression in transfected COS-7 cells, (n = 48–65). ***, p < 0.001. Error bars, S.E.