FIGURE 2.

Surface expression and radioligand binding of GlyR α3 subunit variants. A, HEK 293 cells were transfected with the indicated subunits, and crude membrane preparations were analyzed using the pan-α antibody mAb-4a and detected with a cy5-coupled goat anti-mouse antibody. The subunits are: 1, α3L^Y367F; 2, α3L^S370A; 3, α3L^Y367F/S370A; 4, α3L^T358A; 5, α3L^{T358A/Y367F/S370A}; 6, α3L^Δ6; 7, α1(wt); 8, α3L^T358A/Y367F; 9, α3L^T358A/S370A; 10, α3L; 11, α3K; 12, water transfected cells; and 13, α3L⁺⁶. The arrows indicate the 48-kDa band, and the arrowheads indicate 45- and 36-kDa bands. B, quantitation of receptor expression via dot blot receptor assay (means ± S.D.). The entire assay was performed three times using duplicate determinations for each data point to give n = 6 measurements/subunit. C, comparison of surface expression (upper panel) and intracellular protein (lower panel) of GlyRα3 variants (α3K, α3L, α3L^Δ6, α3L⁺⁶, and α3L^T358A/S370A) differing within the splice cassette of α3L. Untransfected COS7 cells (lane UT) as well as a mock transfection using a green fluorescent protein (lane GFP) plasmid were used as negative controls. GlyRα1 wild type served as positive control. The arrows point to the specific GlyR protein band at 48 kDa. Double bands in the surface fraction are most likely due to varying degrees of glycosylation. D, radioligand binding assays. Displacement of [³H]strychnine (K_D = ∼9.6 nm) by cold strychnine was measured. For each tested subunit, specific binding was normalized to the maximum binding (at zero concentration of cold strychnine) and plotted. Unspecific binding was determined at the maximum concentration of cold strychnine. All of the determinations were carried out in triplicate, see Table 1 for constants. The data are shown for α3L (■, solid line), α3K (○, dashed line), α3L^T358A/S370A (◇, dotted line), and α3L^Δ6 (▲, dashed and dotted line). The specific counts measured for each subunit were: α3L (dpm_max = 11118 ± 890, dpm_nonspec = 613 ± 45), α3K (dpm_max = 20711 ± 910, dpm_nonspec = 1524 ± 230), α3L^T358A/S370A (dpm_max = 18626 ± 674, dpm_nonspec = 1224 ± 68), and α3L^Δ6 (dpm_max = 22714 ± 1427, dpm_nonspec = 1546 ± 105).