Figure 8. Multiple truncated or misfolded GABAA receptor subunits formed SDS-resistant protein complexes, and cysteines 151 and 165 in the N terminus were involved in the formation of the SDS-resistant protein complexes. A, Schematic locations of mutations that were introduced to produce truncated γ2S subunits were indicated by red crosses. B, Total lysates of HEK 293T cells transfected with γ2S(Q351X)FLAG, γ2S(G234X)FLAG, γ2S(N258X)FLAG, γ2S(R284X)FLAG, and γ2S(V321X)FLAG subunits were immunopurified using anti-FLAG antibody and analyzed by SDS-PAGE. C, The ratios of the total IDVs of dimers over monomers for each truncated γ2S subunit in B were plotted. The data were normalized to wild-type γ2SFLAG subunit (wt). The ratio of the total IDVs of dimer in wild type was arbitrarily taken as 1 (n = 6) (C). D, Schematic topology of α1, β2, and γ2S subunits with deletions of a major portion of the intracellular TM3–TM4 loops were marked with red crosses to represent the portion of the intracellular TM3–TM4 loop that was removed. E, Total lysates of HEK 293T cells transfected with α1FLAG, β2FLAG, and γ2SFLAG with loop deletion, γ2SFLAG(wt) and γ2S(Q351X)FLAG subunits, and their partnering subunits were immunopurified using anti-FLAG antibody and analyzed by SDS-PAGE. The loop-deleted α1FLAG subunits were cotransfected with wild-type β2 and γ2S subunits; the loop-deleted β2FLAG subunits were cotransfected with α1 and γ2S subunits; and the wild-type, loop-deleted and mutant γ2S subunits were cotransfected with α1 and β2 subunits. F, The percentages of total IDVs of dimers over monomers for each condition were plotted [n = 4; ***p < 0.001 vs wild type; ††p < 0.01 vs γ2S(Q351X)FLAG; §§p < 0.01 vs β2FLAG loop deletion]. In B and E, the green arrows designated mutant γ2SFLAG subunits that migrated as monomers, and the red arrows designated potential subunit dimers. G, Schematic topology of γ2S(Q351X) subunits with cysteines C151 and C165 in the signature Cys-loop mutated to alanines. H, Total lysates of HEK 293T cells untransfected (U) or transfected with γ2S(Q351X)FLAG subunits without cysteines mutated (con) or with mutant γ2S(Q351X)FLAG subunits containing mutations in C151 (151), C165 (165), or both (151 + 165). Mutant γ2S(Q351X)FLAG subunits without or with mutation of the cysteines were immunopurified using anti-FLAG antibody and analyzed by SDS-PAGE. I, The total IDVs in H for γ2S(Q351X)FLAG subunits without or with mutation of the cysteines were plotted. The data were normalized to the γ2S(Q351X)FLAG subunit without mutant cysteines. The total IDV of γ2S(Q351X)FLAG subunits was arbitrarily taken as 1 [n = 4 from four different transfections; ***p < 0.001 vs γ2S(Q351X)FLAG subunit].