Fig 2: Representative confocal images showing the expression of (A) GAT-1 or (B) GAT-3 and PV in the SScortex of epileptic stargazers (STG) and non-epileptic littermates (NE). Merged images at low magnification (10×) and medium magnification (40×) are shown. White dotted boxes in the third panel were magnified in the fourth panel. Green boxes in the fourth panel were magnified in the bottom panels of figures (A,B) to show punctate structures.

Fig 3: The GABA reuptake function of the variant GAT-1 transporters was reduced in HEK 293 T cells and mouse astrocytes.(A). HEK293T cells under passage 20 were transfected with wildtype (wt) or the variant GAT-1YFP cDNAs (1 µg per a 35 mm dish) for 48 h before 3H radioactive GABA uptake assay. GAT-1-specific inhibitors Cl-966 (50 µM) and NNC-711 (30 µM) were added during flux time for each experiment. (B). Diagram adapted from the Allen Brain Atlas, demonstrating the areas that were dissected for astrocyte culture. The astrocytes from the cortex, cerebellum, and thalamus of wildtype C57BL/6 J mouse pups at postnatal day 0–3 were prepared and the live cells at passage 2 were imaged before experiment of GABA uptake assay. (C). The graph showing relative GABA uptake assay in astrocytes from cortex, cerebellum and thalamus. The GABA uptake from cerebellum (cb) and thalamus (thal) was normalized to the GABA uptake in the cortex (cor). Mouse cortical (D) or thalamic (E) astrocytes at passage 2 were transfected with wildtype (wt) or the variant GAT-1YFP cDNAs (1 µg per a 35 mm dish) for 48 h before 3H radioactive GABA uptake assay. In C, GABA uptake in astrocytes from the cerebellum and thalamus was normalized to that from the cortex (=100%) each time. In D, GABA uptake in astrocytes expressing the variant or wildtype cDNAs treated with GAT-1 inhibitors Cl-966 or NNC-711 was normalized to the astrocytes expressing the wildtype cDNAs without GAT-1 inhibition. “Un” stands for the astrocytes transfected with the wildtype GAT-1YFP without treatment of GAT-1 inhibitors. (966 stands for Cl-966 while 711 for NNC-711). In E, GABA uptake in astrocytes expressing the variant or wildtype cDNAs treated with GAT-3 inhibitor (SNAP5114 (30 µM)) was normalized to that in astrocytes expressing the wildtype cDNAs without GAT-3 inhibition. GAT-3 inhibitor was applied 1 h before and during uptake assay. In C, D and E, N = 4–5 transfections from 4 batches of cultured astrocytes, ***P < 0.001 vs astrocytes expressing the wildtype GAT-1YFP. In A, §§§P < 0.001 vs G362R. In C, *P < 0.05 vs cortex; §P < 0.05 vs thalamus. In D, §P < 0.05 vs G362R. In E, §P < 0.05 vs G362R without treatment; d P < 0.05 vs G362R with SNAP5114 treatment. In C, D and E, N = 4–5 experiments, cells were prepared from 3 different litters of mouse pups. One-way analysis of variance (ANOVA) and Newman-Keuls test was used to determine significance compared to the wt condition and between mutations. Values were expressed as mean ± S.E.M.

Fig 4: The mutant GAT-1 had reduced total protein expression but increased localization inside the endoplasmic reticulum in astrocytes.A. Mouse cortical astrocytes under passage 2 were transfected with wildtype GAT-1YFP or the variant GAT-1 (E16X)YFP, GAT-1 (G362R)YFP, GAT-1 (L460R)YFP and GAT-1 (W495X)YFP with the pECFP-ER marker (ERCFP) at a 1:1 ratio (1 µg:1 µg cDNAs) for 48 h. Live cells were examined under a confocal microscopy with excitation at 458 nm for CFP and 514 nm for YFP. All images were single confocal sections averaged from 8 times to reduce noise, except when otherwise specified. “Co” stands for overlay, “ER” stands for endoplasmic reticulum marker (ERCFP) marker, and “GAT” stands for GAT-1YFP. (B). The GAT-1YFP fluorescence overlapping with ERCFP fluorescence was quantified by Metamorph with colocalization percentage. (C). Total fluorescence intensity (raw values) over the area was measured for each whole field and the raw mean values were plotted. In B and C, ***p < 0.001 mutant vs. wt, §§ P < 0.01, §§§ < 0.001 vs G362R. N = 7–8 representative fields from 4 different transfections. One-way analysis of variance (ANOVA) and Newman-Keuls test was used to determine significance compared to the wt condition and between mutations. Values were expressed as mean ± S.E.M.

Fig 5: Impaired GABA uptake of the mutant GAT-1(P361T) transporters. (A) HEK293T cells were transfected with wildtype GAT-1YFP (wt), or the mutant GAT-1(P361T)YFP cDNAs (1 µg/35mm2) for 48 h. The GABA uptake assay was carried out with 3H radioactive labeling in HEK 293 T cells. GABA flux was measured after 30 min transport at room temperature. The influx of GABA, expressed in pmol/µg protein/min, was averaged from duplicates for each condition and for each transfection. The average counting was taken as n = 1. The untransfected condition was taken as baseline flux, which was subtracted from both the wild-type and the mutant conditions. The pmol/µg protein/min in the mutant was then normalized to the wildtype from each experiment, which was arbitrarily set as 100%. (**p < 0.01 vs. wt, n = 4–5 different transfections)