Fig 2: Substrate-activated Cl– conductance.(A) Current-voltage (IV) relationships for hEAAT1 (black diamond), P290R (red diamond), M128R (gray empty diamond), A329T (gray circle), and V393I (gray diamond) were plotted, where reversal potentials (Erev) were measured (B). The exact numbers of cells (n) used are indicated in parentheses. Currents were elicited by 100 µM L-aspartate in 10 mM Cl– buffer containing 100 mM Na+ (gluconate was used as Cl– substituent to maintain equal osmolarity), except M128R. Erev was not determined for M128R due to no detectable L-aspartate–activated current, even when 1 mM L-aspartate was applied in the presence of 100 mM NaCl. One-way ANOVA tests (Brown-Forsythe) were performed F(5, 13.12) = 50.6, ***P < 0.001 and ****P < 0.0001. (C) Positions of residues that, when mutated to the residues indicated, have previously been demonstrated to increase (P98G and P392V) or reduce (S103V and K114L) Cl– conductance in hEAAT1 are highlighted in the structure of glutamate transporter homolog GltPh in the Cl– conducting state (Cl– pathway indicated as transparent blue density; PDB 6WYK). Positions of EA6-related mutations are shown on the same structure rotated 90°. Transmembrane domains are colored as per Figure 1C. For clarity, hEAAT1 numbering is used for annotation.

Fig 3: Spinal astrocytes proteins are altered in a mouse model of late-onset SMA before the first loss of motor neurons. (A) Immunostaining of astrocyte-specific proteins such as GFAP (green), Kir4.1 (green), and EAAT1 (green) in spinal cord slices of SMA and control mice at P28. Nucleic DNA was stained with Dapi (blue). (B) The relative GFAP level in SMA mice spinal cord ventral horns was increased compared to control mice (***, p < 0.001). (C,D) The relative level of Kir4.1 and EAAT1 was reduced in SMA mice, compared to control animals (***, p < 0.001). VH = ventral horn. n = 6 animals (see bars). Three slices per spinal cord were investigated. Each data point reflects the mean of those three spinal cord slices. Scale bar: 20 µm.

Fig 4: Stoichiometry of transport and locations of EA6 mutations in the hEAAT1 structure.(A) The transport of each substrate molecule (glutamate or aspartate) is coupled to the cotransport of 3 sodium ions (Na+) and 1 proton (H+), and the countertransport of a potassium ion (K+); the transport process also activates a thermodynamically uncoupled chloride channel. (B and C) Locations of EA6-related mutations in the structure of hEAAT1 (PDB: 5LLU) (B) and on a topology schematic (C) are shown. Cartoon representation of transmembrane domains (TMDs) 1 and 2 from the scaffold domain are omitted for clarity, while the whole scaffold domain is shown in transparent white surface representation. TMDs in B are coloured as per C. Figure was made using ChimeraX (84).

Fig 5: EA6-related mutations M128R, A329T, and V393I fail to rescue Drosophila crawling.(A) dEAAT1-null animals were rescued by WT hEAAT1 or 1 of 5 EA6-related mutations (M128R, C186S, T318A, A329T, or V393I), and representative trajectories of crawling paths of 9 L1 larvae for each genotype are shown, as captured by infrared tracking for 180 seconds. Color heatmap indicates the average speed over a moving bin of 0.5 seconds. Scale bar: 5 mm. (B–D) Quantification (mean ± SEM) of crawling parameters achieved by larvae over 60 seconds of continuous tracking, including mean speed (B), total path length (C), and the beeline distance between the origin at t = 0 and the termination point (D). The exact numbers of animals (n) used for panels B–D are hEAAT1 (40, 74, 75), M128R (50, 53, 53), C186S (53, 51, 50), T318A (78, 79, 78), A329T (76, 84, 84), and V393I (52, 56, 55). One-way ANOVA tests (Brown-Forsythe) were performed for mean speed F(5, 130) = 18.32, *P < 0.05, ****P < 0.0001; for total path length F(5, 197.6) = 27.16, ****P < 0.0001; and for beeline distance F(5, 199.5) = 24.04, ****P < 0.0001. (E and F) Astrocyte-specific expression (with alrm-Gal4) of hEAAT1 and EA6-related mutations. Representative high-power images of infiltrative astrocyte processes within the ventral nerve cord of a dissected larva for each genotype, labeled by immunohistochemistry for hEAAT1 (E) or the plasma membrane-associated GABA transporter (GAT, F). Each panel represents a single optical confocal section from the middle of the dorsal-ventral axis of the neuropil. Scale bars: 10 µm. Like controls where dEaat1-null larvae are rescued with hEAAT1, all the EA6-related mutations of hEAAT1 except M128R were well expressed and addressed to astrocyte processes within CNS neuropil. Anti-GAT staining (F) reveals that astrocytes rescued with M128R infiltrate the neuropil normally. At least 5 animals were dissected, immunostained, and examined for each genotype.