FIGURE 6.

Interactions of the α1(AD) subunit with wild type GABA_AR subunits. We transfected HEK293T cells with 2 μg of α1(AD)^h subunit and 0.125:0.250:0.250 μg ratios of either α1^HAβ2γ2 (A), α1^rβ2^HAγ2 (B), or α1^rβ2γ2^HA subunits to form receptors in which either the α1, β2, or γ2 subunit is tagged with the HA epitope. In addition, to determine whether the α1(AD)^h subunit interacted nonspecifically with the TGF^HA protein, we transfected cells with 2 μg of α1(AD)^h subunit, a 0.125:0.250:0.250 μg ratio of α1^rβ2γ2 subunits, and 0.250 μg of TGF^HA (D). We permeabilized the cells and stained them with either the anti-α1^h-647 antibody (FRET acceptor), the anti^HA-555 antibody (FRET donor), or both anti-α1^h-647 and anti^HA-555 antibodies. A–D are flow cytometry histograms of FRET fluorescence. The gray line plots the histogram for cells stained with only the α1^h-647 acceptor antibody, and the black line plots the histogram for cells stained with both HA-555 donor and α1^h-647 acceptor antibodies. The arrows point to the regions of the histograms where one would find specific FRET fluorescence. We quantified the specific FRET fluorescence and plotted it in E (n ≥ 5). Samples transfected with α1β2^HAγ2 and α1β2γ2^HA receptors possessed substantial FRET fluorescence that significantly differed compared with samples transfected with TGF^HA protein. This difference in FRET fluorescence did not result from reduced HA fluorescence from the TGF^HA protein because TGF^HA possessed substantially more HA florescence than HA-tagged GABA_AR subunits (F). AU, arbitrary units.