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. 2017 Aug 17;6:e27443. doi: 10.7554/eLife.27443

Figure 3. γ2 is required for assembly of the native GARLHed complex.

(A) Membranes from cRNA-injected oocytes (0.18 ng ea) were solubilized in Triton X-100 and analyzed by BN-PAGE. α1 and β2 with or without HA-tagged γ2 migrated at 480 kDa. Addition of an anti-γ2 antibody induced an upward shift of GABAARs from α1/β2/HAγ2-injected oocytes but had no effect on GABAARs from α1/β2-injected oocytes, indicating nearly complete incorporation of HAγ2 into GABAAR pentamers. The images are representative of two independent experiments. (B) GABAAR complexes in cerebellum were examined by antibody shift assay. In cerebellum, anti-δ antibody caused most β2/3 signal at 480 kDa to shift up, but did not affect β2/3 signal at 720 kDa. In contrast, anti-γ2 antibody shifted up β2/3 signal at 720 kDa. When anti-δ and anti-γ2 antibodies were combined, both 480 and 720 kDa bands shifted up almost completely. The images are representative of two independent experiments. (C) Primary cultured cerebellar granule cells were prepared from conditional γ2 knockout mice with or without a transgene encoding tamoxifen-inducible Cre recombinase (CreERT), and treated with 4-hydroxytamoxifen (4-OHT) from DIV1.5 to DIV3. At DIV9, cell membranes were solubilized in MNG and examined by BN-PAGE. In neurons expressing CreERT, γ2 was eliminated, and α1 and β2 at 720 kDa collapsed to 480 kDa. The images are representative of three independent experiments. The arrow and arrowhead indicate the GARLHed and GARLHless GABAAR, respectively, and antibody-bound complexes are indicated.

Figure 3.

Figure 3—figure supplement 1. A schematic diagram of an antibody shift assay for distinct GABAAR complexes on BN-PAGE.

Figure 3—figure supplement 1.

GABAARs comprised of α6, β2/3 and δ form a complex at 480 kDa, and the addition of an anti-δ antibody to the complex shifts its mobility on BN-PAGE. On the other hand, GABAARs comprised of α1, β2/3 and γ2 form complexes with GARLH and NL2 at 720 kDa, and the addition of an anti-γ2 antibody to the complex shifts its mobility. When both anti δ and γ2 antibodies are added, mobility of both the complexes at 480 and 720 kDa are shifted.