PMC

γ2L(Q351X)^pHluorin subunit protein had minimal expression on the surface of rat hippocampal neurons. A–C, Rat hippocampal neurons were transfected with α1β2γ2L^pHluorin (wt) or α1β2γ2L(Q351X)^pHluorin (mut) subunits for 6 d and were imaged as puncta on the surface of neurons. A, Representative images of wt γ2L^pHluorin or mut γ2L(Q351X)^pHluorin subunits are presented. B, C, Enlarged views of the boxed areas for wt (B) and mut (C) receptors in A. In A–C, wt stands for wild-type γ2L^pHluorin subunits, mut stands for mutant γ2L(Q351X)^pHluorin subunits, CO stands for colocalized images, T stands for transmitted images, and F stands for fluorescent images.

With heterozygous expression, surface γ2S subunit levels were reduced and γ2S subunits were retained in the ER. A, HEK 293-T cells were cotransfected with α1 and β2 subunits and γ2S^YFP (1:1:1 cDNA ratio, wt), γ2S^YFP (1:1:0.5 cDNA ratio, hem), γ2S^YFP/γ2S(Q351X)^YFP (1:1:0.5:0.5 cDNA ratio, het), or γ2S(Q351X)^YFP (1:1:1 cDNA ratio, hom) subunits, and surface proteins were biotinylated, isolated, separated by SDS-PAGE, and probed with anti-GFP antibody. Surface proteins were normalized to the wild-type γ2S^YFP subunit protein. Values are mean ± SEM (n = 7) (***p < 0.001 vs wt). B, COS-7 cells were cotransfected with α1 and β2 subunits and γ2S^YFP (1:1:1 cDNA ratio, wt), γ2S^YFP/γ2S(Q351X)^YFP (1:1:0.5:0.5 cDNA ratio, het), or γ2S(Q351X)^YFP (1:1:1 cDNA ratio, hom) subunits, and the images were obtained 48 h later under confocal microscopy. Mem stands for membrane, R for γ2S^YFP subunit, ER for endoplasmic reticulum, and CO for colocalized images.

Expression of heterozygous γ2S/γ2S(Q351X) subunits and α1 and β2 subunits resulted in reduced peak current amplitudes compared with expression of wild-type or hemizygous control α1, β2, and γ2S subunits. A, GABA_A receptor currents were obtained from HEK 293-T cells cotransfected with wild-type α1 and β2 subunits and γ2S (1:1:1 cDNA ratio; wt, black trace), hemizygous control γ2S (1:1:0.5 cDNA ratio; hem, gray trace), or heterozygous γ2S/γ2S(Q351X) (1:1:0.5:0.5 cDNA ratio; het, green trace) subunits with application of 1 mm GABA applied for 6 s. Peak wt, hem, and het currents were marked with a black arrow, a gray arrow or a green arrow, respectively. B, The amplitudes of GABA_A receptor currents from A were plotted. Values were mean ± SD (n = 15–16 cells from 6 different transfections) (***p < 0.001 vs wt, ^† p < 0.05 vs hem).

Glycosylation arrest caused the reduction of mature wild-type γ2S subunit protein. A, HEK 293-T cells were cotransfected with α1 and β2 subunits and wild-type γ2S^YFP (1:1:1 cDNA ratio, wt), heterozygous γ2S^YFP/γ2S(Q351X)^YFP (1:1:0.5:0.5 cDNA ratio, het), or homozygous γ2S(Q351X)^YFP (1:1:1 cDNA ratio, hom) subunits. Total proteins were analyzed by SDS-PAGE and probed with anti-GFP antibody. B, C, The total cell lysates with wild-type γ2S^YFP (B) or mutant γ2S(Q351X)^YFP (C) subunit transfection were undigested (U) or digested with PNGase F (F) or Endo-H (H). D, Total cell lysates of hemizygous control α1β2γ2S^YFP/pcDNA or α1β2γ2S^HA/pcDNA (1:1:0.5:0.5 cDNA ratio) subunits and heterozygous α1β2γ2S^YFP/γ2S(Q351X) or α1β2γ2S^HA/γ2S(Q351X) subunits (1:1:0.5:0.5 cDNA ratio) were transfected into HEK 293-T cells and undigested (U) or digested with Endo-H (H) or PNGase F (F). E, The protein bands insensitive to Endo-H were quantified and presented as a fraction of their total undigested bands (*p < 0.05 vs wt; ^§§§ p < 0.001 vs hem; n = 5). Values are mean ± SEM.

Mutant mRNA and truncated protein were produced from the γ2(Q351X) subunit intron 8 minigene in HEK 293-T cells. A, The γ2 subunit amino acid Q351 is located in the TM3–TM4 loop of the protein. B, The point mutation Q351X is located in the last (ninth) exon of the GABRG2 gene. Wild-type and mutant γ2 and γ2^YFP subunit minigenes were constructed by including the entire intron 8 between exon 8 and 9 in γ2 and γ2^YFP subunit cDNA constructs. Primers in flanking exons 7 and 9 were used to determine whether the γ2 subunit minigene was correctly spliced. C, HEK 293-T cells were transfected with γ2 or γ2(Q351X) subunit minigenes, and each construct displayed a band at 334 bp, suggesting correct splicing in the reverse-transcribed cDNA products from mRNAs of the γ2 subunit minigene. D, HEK 293-T cells were cotransfected with α1 and β2 subunit cDNAs and wild-type γ2^YFP (1:1:1 ratio), heterozygous γ2^YFP/γ2(Q351X)^YFP (1:1:0.5:0.5 ratio), and homozygous γ2(Q351X)^YFP (1:1:1 ratio) subunit minigenes. Total cell lysates were directly analyzed by SDS-PAGE.

The truncated γ2S(Q351X) subunit impaired trafficking of α1β2 receptors to the cell surface due to its oligomerization with wild-type subunits. A, COS-7 cells were transfected with α1^CFPβ2 (left), α1^CFPβ2γ2S (middle), or α1^CFPβ2γ2S(Q351X) subunits, and the confocal images were obtained 48 h later. B, HEK 293-T cells were transfected with empty pcDNA vector (mock) or with α1β2 (1:1 cDNA ratio), α1β2γ2S (1:1:1 cDNA ratio, wt), α1β2γ2S (1:1:0.5 cDNA ratio, hem), α1β2γ2S/γ2S(Q351X) (1:1:0.5:0.5 cDNA ratio, het), or α1β2γ2S(Q351X) (1:1:1 cDNA ratio) subunits, and surface proteins were prepared as described in the methods and visualized with anti-human α1 subunit antibody. C, Surface protein IDVs were normalized to α1β2 receptors (n = 4–8) (*p < 0.05, ***p < 0.001 vs wt; ^†† p < 0.01 vs hem; ^§§§ p < 0.001 vs α1β2). Values were mean ± SEM. D, HEK 293-T cells were transfected with α1β2 (1:1 cDNA ratio) or α1β2γ2S(Q351X) (1:1:1, 1:1:2.5, 1:1:5, 1:1:10 cDNA ratio) subunits, and the peak current amplitudes were plotted (***p < 0.0001 vs α1β2) [n = 19 for α1β2, n = 62 for α1β2γ2S(Q351X) (1:1:1), n = 16 for α1β2γ2S(Q351X) (1:1:2.5), n = 5 for α1β2γ2S(Q351X) (1:1:5 and 1:1:10)]. Values were mean ± SD. In A–D, the total amounts of cDNAs were normalized by the empty vector pcDNA. E, Total lysates from HEK 293-T cells expressing α1β2γ2S^FLAG or α1β2γ2S(Q351X)^FLAG (1:1:1 cDNA ratio) subunits were purified with agarose-immobilized anti-FLAG M2 antibody, and the conjugated subunits were liberated with FLAG-peptide and probed with anti-α1 (E, left) or anti-β2 (E, right). F, Total lysates from HEK 293-T cells expressing α1β2γ2S^HA/γ2S(Q351X)^FLAG (het1) or α1β2γ2S^FLAG/γ2S(Q351X)^HA (het 2) (1:1:0.5:0.5) were purified with FLAG M2 antibody as described in E and visualized with anti-HA antibody.

Heterozygous α1β2γ2S(Q351X) receptor current amplitudes were less than half of wild-type peak current amplitudes in hippocampal neurons. A, Rat hippocampal neurons in the absence or presence of 5 μg of siRNA targeting the rat γ2 subunit gene for 8 d were permeabilized and stained with anti-rat γ2 antibody and visualized with FITC. B, The whole-cell lysates from the sister dishes were probed with rabbit anti-rat γ2 antibody by Western blot. C, Whole-cell currents evoked by GABA (2 μm) and GABA (2 μm) plus diazepam (1 μm) were recorded from 16-d-old hippocampal neurons in the absence (Control) or presence of 5 μg of siRNA targeting the rat γ2 subunit gene for 6–8 d (siRNA). D, Wild-type α1β2γ2S^YFP, heterozygous α1β2γ2S^YFP/γ2S(Q351X)^YFP, and homozygous α1β2γ2S(Q351X)^YFP receptor currents from 15- to 16-d-old endogenous γ2 subunit silenced hippocampal neurons were evoked with GABA (1 mm) for 28 s. E, The peak current amplitudes of neurons untreated (con) or treated with siRNA (siRNA) and transfected with human α1β2γ2S^YFP (wt), α1β2 γ2S^YFP/γ2S(Q351X)^YFP (het), and α1β2γ2S(Q351X)^YFP (hom) were plotted (***p < 0.001 vs wt, ^†††hom vs het, data from 13–15 cells from 5 batches of cells). F, G, GABAergic mIPSCs for cells transfected with α1 and β2 subunits and wild-type γ2S^YFP or heterozygous γ2S^YFP/γ2S(Q351X)^YFP subunits were recorded at a holding potential of −60 mV. G, Individual mIPSCs from a 5 min recording period were averaged for wild-type and heterozygous subunit-transfected cells. The amplitude distributions of mIPSCs obtained during a 5 min recording period were displayed in a histogram. The histograms were fitted using Mini Analysis program by Justin Lee (**p < 0.01 het vs wt, n = 6 for each group).

The α1 subunit was subject to glycosylation arrest and enhanced degradation in the presence of the γ2S(Q351X) subunit. A, HEK 293-T cells were cotransfected with α1 and β2 subunits and wild-type γ2S (1:1:1 wt or 1:1:0.5 hem cDNA ratio), heterozygous γ2S/γ2S(Q351X) (1:1:0.5:0.5 hem cDNA ratio), and homozygous γ2S(Q351X) (1:1:1 cDNA ratio) subunits. The cell lysates were either undigested (U) or digested with Endo-H (H) and analyzed by SDS-PAGE. B, Total undigested α1 subunit protein IDVs were quantified, and the data were normalized to GAPDH IDVs (n = 5). C, The fractions of Endo-H-digested α1 subunit proteins were quantified and expressed as percentage maturation [taken as 100% if there was only a single 48.4 kDa present and a percentage of the 48.4 kDa band (A, top lanes in H) over 48.4 plus 46 kDa bands (A, bottom lanes in H) if there were two bands present]. In B and C, *p < 0.05, **p < 0.01, ***p < 0.001 versus wt; ^†† p < 0.01 versus het 0.5; ^§ p < 0.05, ^§§ p < 0.01, ^§§§ p < 0.001 versus hemizygous control (n = 5). D, HEK 293-T cells were cotransfected with α1, β2, and γ2S(Q351X) subunits using cDNA ratios of 1:1:1, 1:1:5, and 1:1:10. The lysates were either undigested (U) or digested with Endo-H (H) and analyzed by 10% SDS-PAGE. E, The fractions of Endo-H-digested α1 subunit protein IDVs were quantified as described in C. In A–E, the total amounts of cDNAs were normalized by the empty vector pcDNA. **p < 0.01, ***p < 0.001 versus α1β2γ2S(Q351X) (1:1:1) (n = 4). F, G, HEK 293-T cells containing ³⁵S methionine-radiolabeled α1^FLAGβ2γ2S (wt) and α1^FLAGβ2γ2S(Q351X) (hom) receptors were lysed, immunopurified with FLAG M2 antibody, and analyzed by SDS-PAGE. After 20 min of labeling, the cells were chased for the indicated times (n = 4). The same amount of total protein (800 μg) from each sample was used for immunopurification (F). The graph plots the percentage radioactivity normalized to α1^FLAG subunit IDV of α1^FLAGβ2γ2S receptors at the given chase time (G) (**p < 0.01 vs α1^FLAGβ2γ2S). H, The total lysates from cells expressing α1β2γ2S and α1β2γ2S(Q351X) receptors with or without treatment by lactacystin (Lac, 10 μm) for 6 h were analyzed by SDS-PAGE and blotted with anti-α1 antibody.