Fig 2: Modeling of the mutant GAT-1 protein with machine learning tools. a-b. Tertiary structures of both the wildtype (a) and P361T mutant (b) GAT-1 protein are predicted by I-TASSER and DynaMut. The proline at residue 361 is mutated to threonine, both highlighted in light green, alongside with the surrounding residues. The interatomic interactions were predicted by DynaMut, where halogen bonds are depicted in blue and hydrogen bonds are colored in red. The P361T mutation results in the loss of two hydrogen bonds, those between residues 361 and 365 (yellow arrow with red border) and between 361 and 364 (yellow arrow with blue border). This supports the result in Table 1 that this mutation destabilized the global conformation of the GAT-1 protein. c. Machine learning tools predicted ??G (Kcal/mol) of the mutant GAT-1 protein. Bars in the positive direction are predicted as stabilizing while bars in the negative direction are predicted as destabilizing

Fig 3: Gamma-aminobutyric acid transporters 1 and 3 are expressed in the nTS. (A) Quantification of mRNA transcript of GAT-1 and GAT-3 in the nTS. GAT-3 mRNA transcript was greater than in GAT-1 (N = 6, *p = 0.05 paired t-test). (B) Immunoblot example GAT-1 and 3 in the nTS [left, GAT-1 (20 and 40 µg) and right, GAT-3 (20 and 40 µg)]. Note the GAT-1 band at ~67 and the GAT-3 band at ~70 kDa. Tubulin was used as a control. (C) Quantification of the GAT-1/tubulin ratio (left) and GAT-3/tubulin ratio (right). GAT-3 is found in higher quantities in the nTS than GAT-1. N = 5, *p = 0.05 paired t-test. (D) IHC of GAT-1 and GAT-3 in nTS. TS, solitary tract; DmnX, dorsal motor nucleus of the vagus, AP, area postrema. Scale, 200 µm.

Fig 4: Enrichment of GAT-1 and GAT-3 expression in the DLS versus NAcC.a, c Representative immunofluorescence signals for GAT-1 (cyan, a) and GAT-3 (green, c) using confocal microscopy in coronal sections across the rostral-caudal limits containing striatum prepared from an individual C57BL/6 J mouse with heatmaps for striatal GAT intensity. Boxes indicate representative locations for GAT intensity measurements in the dorsolateral striatum (DLS) and nucleus accumbens core (NAcC). Scale bars: 1 mm. Note enriched GAT-3 in the medial NAc shell contiguous with the medial septal nucleus and enriched GAT-3 expression in the claustrum. b, d Left, Mean GAT-1 (b) and GAT-3 (d) intensity in DLS and NAcC normalized to total striatum and averaged across rostral-caudal sites for each hemisphere (n = 12 hemispheres/6 mice for each GAT-1 and GAT-3). Right, Representative single plane images of GAT-1 (b) and GAT-3 (d) immunofluorescence from DLS and NAcC; imaging parameters were kept constant across regions. Scale bars: 50 µm. Mann–Whitney U tests (b, d). **p < 0.01. Error bars are ±SEM. Source data are provided as a Source Data file.

Fig 5: The mature and functional GAT-1 protein was reduced in the variant GAT-1.HEK293T (A, B) or mouse cortical astrocytes at passage 2 (C, D, E) were transfected with the wildtype or the variant GAT-1YFP cDNAs (3 µg per 60 mm dish or 9 µg per 100 mm dish) for 48 h before harvest. Equal protein amounts were loaded for SDS-PAGE. The membrane was immunoblotted with anti-rabbit GAT-1. In A, protein lysates from HEK293T cells expressing the wildtype and the variant GAT-1 cDNAs were either undigested (U) or digested with Endo-H (H) followed by SDS-PAGE fractionation. Bands 1, 2, 3, 4 and 5 represent the main bands detected in lysates without Endo-H treatment, while band 3 was shifted down to a lower molecular mass than band 4 after Endo-H digestion. To better show both mature and immature bands of GAT-1YFP, a slightly overexposed blot is presented, but note that this has no effect on the quantification data in Image Studio. (B). Graph showing normalized total protein IDVs in HEK293T cells. The variant GAT-1 protein integrated density values (IDVs) were normalized to the wildtype untreated (U) or Endo-H treated (H). In C, Total lysates from mouse cortical astrocytes transfected with wildtype or the variant GAT-1YFP cDNAs (9 µg per a 100 mm dish) for 48 h before harvest were subject to SDS-PAGE. The red arrow pointed bands were quantified. The band run at 67 KDa is likely the endogenous GAT-1 from astrocytes. In D, the protein samples of the wildtype and the GAT-1(E16×) were run separately due to the small protein mass of GAT-1(E16X). The loaded protein amount of the GAT-1E(16X) was tripled for better visualization. The membrane was immunoblotted by a rabbit anti-GAT-1 in A and C and by a mouse anti-GFP in D because GAT-1(E16X) is undetectable by anti-GAT-1 antibody. (E). The total protein IDVs in the mouse cortical astrocytes were plotted. In A and D, CHO stands for Chinese hamster ovary cells. CHO cells were used for loading control because of the low level of endogenous GAT-1 expression (N = 4–5 transfections from 4 to 5 batches of cultured astrocytes, ***P < 0.001 vs astrocytes expressing the wildtype GAT-1YFP; §§§ < 0.001 vs G362R. One-way analysis of variance (ANOVA) and Newman-Keuls test was used to determine significance compared to the wt condition and between variants. Values were expressed as mean ± S.E.M).