Sodium binding and GAT1 electrostatics

a) The GAT1 surface is shown in white, with transmembrane helices TM1, TM2, and TM6 highlighted in grey. Averaged density of sodium is shown in close proximity to GAT1 in a side view. The sodium density is represented as isovolume, colour-coded according to the legend and shown in three layers above bulk (double bulk, 10* bulk, bound), as indicated by the legend. The trajectories were fitted to the Cα atoms of the transmembrane domain. The residues D281 and E283 (Recruitment Site) are illustrated as cyan sticks. b) The focus zooms in on the recruitment site in GAT1-WT and c) the D281A-D283A mutant, showing helices of TM1, TM2, TM6, and TM8 along with the recruitment site residues. Sodium density from the wild-type and the double alanine (D281A and E283A) mutant simulations is colour-coded according to the legend. d) Quantification of sodium binding events calculated as cumulative sodium ions entering the vestibule. e) Zoom to the S1 showing Ion density and surrounding residues of NA1, f) NA2 and g) the temporary binding sites. h) Separation of ion position observed for sodium ions binding directly to NA1 (green), i) directly to NA2 in purple or j) first residing in the temporary binding site (yellow) before reaching NA1 or NA2. All residues of the respective binding sites are represented as sticks and coloured accordingly. k) Top view and side view of the electrostatic potential of GAT1 wild-type mapped onto its surface. l) Top and side view of the electrostatic potential of the double alanine mutant (D281A and E283A).

Effect of D281A-E283A mutation

a) Representative confocal microscopy images of GAT1 wild-type, single mutants (D281A, E283A) and double alanine mutant (D281A-E283A) stably expressed in HEK293 cells. Transporters were N-terminally tagged with YFP., The cell membrane was stained by trypan blue. Composite images display the superimposition of tagged GAT1 and trypan blue stained membranes. The inserts in the lower left corner show a zoom-in on the cell membrane. b) Mean fluorescence of cytosolic and membrane resident YFP-tagged GAT1 of n=23 (wild-type) and n=20 (D281A,E283A,and the double mutant) randomly analysed confocal images per condition. c) Concentration-dependent [3H]GABA uptake in HEK293 cells stably expressing GAT1 wild-type, single mutants and D281A-E283A. d) Insert is depicting the quantification of the Km (µM) and the Vmax (pmol/106 cells/3 min) of the GABA uptake. Data is displayed as mean ± SD. Concentration dependent uptake was measured in triplicates in n=7 (wild-type) and n=6 (D281A, E283A, and D281A-E283A) biologically independent experiments. Single data points for Km and Vmax are the averages for individual experiments. Confidence intervals (95%) for curve fittings are plotted within ± SD and delimited by black dashed lines. After conducting statistics with one-way ANOVA, we performed Dunnett’s post hoc multiple comparison vs. wild-type. Statistical significance was denoted as follows: *=P < 0.05, **=P < 0.01, ***P < 0.001, ****P < 0.0001.

Impact of the charge neutralisation on Na+ affinity

a) Sodium-dependent [3H]GABA uptake curves normalised to maximal uptake. b) Sodium EC50 values for GAT1 wild-type, D281A, E283A, and the double mutant D281A-E283A observed in panel a. Data is shown as mean ± SD from n=5 biological independent replicas of [3H]GABA uptake experiments measured in triplicates. Single EC50 data points are the averages for individual triplicates. Confidence intervals (95%) for curve fittings are plotted within ± SD and delimited by black dashed lines. Statistical analysis was performed with one-way ANOVA and Dunnett’s post hoc multiple comparison vs. wild-type. Statistical significance was denoted as follows: *=P < 0.05, **=P < 0.01, ***P < 0.001, ****P < 0.0001.

Electrophysiological characterisation of the transport cycle

a) Representative traces of whole-cell patch clamp recordings showing GAT1 wild-type mediated currents in response to different GABA concentrations. b) Concentration dependent average steady state currents of wild-type GAT1. c) Representative traces of whole-cell patch clamp experiments showing concentration-dependent GAT1 D281A-E283A mediated currents (data points are shown as running average (n=11) traces). d) Average steady state currents of GAT1 double mutant (D281A-E283A) plotted against the used GABA concentration. e) Comparison of concentration dependent average steady state currents of GAT1 wild-type and double mutant, normalised to the respective currents observed at 300 μM GABA. Data are represented as the mean ± SD from n=11 recordings of independent cells per condition. 95% confidence intervals of curve fittings are plotted within ± SD with dashed lines.

Short-range effect of sodium binding

a) GAT1 structure represented as a cartoon, highlighting the ion binding sites b) Visualisation of the sodium binding sites NA1 (green) and c) NA2 (purple). The dashed lines indicate the distances used for determining the compactness NA1 and NA2. d) Compactness of NA1 and e) NA2 before (in grey) and after sodium binding (coloured according to panels a and b) The vertical dashed lines indicate the measure in the cryo-EM structure (PDB ID: 7Y7W), where GAT is pictured in a holo inward-occluded conformation. f) Reference orientation of GAT1 as used for panel g. g) Top view projection of sodium ion positional density after binding to NA1 and NA2. The canonical binding positions of Na1 and Na2 are indicated by stars. h) Free energy landscape of sodium binding, where coordinates are projected along IC1 and IC2, respectively.

Global structural effects of sodium binding to NA2

Scatter plot of principal component 1 (PC1) against PC2 of all simulations that showed NA2 binding. Trajectories are divided into a) before and b) after Na2 binding. The pink triangle corresponds to the outward-open AlphaFold model of GAT1. All trajectories are projected to the same PC1 and PC2 axis. (c,d) Direction of motions along PC1 mapped as vectors on the TM helices of GAT1 g,h) Motions of GAT1 along PC2. (e,f) Using the same orientation as in panel c, superpositioning of 1 ns separated frames with the maximum likelihood method. Structures are coloured according to the degree of correlated motions along PC1 for frames e) before Na2 binding and f) after Na2 binding. g) Distribution of the distances Y241-F51 and Y241-F227 visualised in panel h, where values before Na2 binding are shown in grey, and after Na2 binding are coloured according to the legend. The vertical dashed lines indicate the distance measured in the cryo-EM structures of the GAT1 holo occluded and apo inward-open conformations (PDB ID: 7Y7W) (Zhu et al., 2023) and in the AlphaFold outward-open model h) The distances shown in panels g are indicated in orange between TM1a (F51) and TM5a (Y241), and in yellow between TM4 (F227) and TM5b (Y241)

The substrate GABA completes the sodium binding site NA1

a-c) Comparison of the sodium binding site NA1 in a) the cryo-EM holo inward-occluded GAT1 (PDB ID: 7y7w (Zhu et al., 2023), b) AlphaFold holo outward-open GAT1, c) and SERT. The group donating the sodium-coordinating carboxylate moiety is GABA in GAT1 (a, b) and D98 in SERT c). d) Zoom onto S1 showing as isovolume densities the regions in which sodium ions Na1 and Na2 are found in the four 0.5 μs long simulations in the absence of GABA. e) Time course and averaged histogram of the compactness of NA1. The vertical dashed line highlights the measured NA1 compactness in the holo inward-occluded GAT1 cryo-EM structure (PDB ID: 7Y7W (Zhu et al., 2023) f,g) Same data as shown in panels d, and e, but in the presence of GABA.