Interaction of GAT1 with sodium ions: from efficient recruitment to stabilisation of substrate and conformation

  1. Erika Lazzarin
  2. Ralph Gradisch
  3. Sophie MC Skopec
  4. Leticia Alves da Silva
  5. Chiara Sebastianelli-Schoditsch
  6. Dániel Szöllősi
  7. Julian Maier
  8. Sonja Sucic
  9. Marko Roblek
  10. Baruch I Kanner
  11. Harald H Sitte
  12. Thomas Stockner

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    eLife Assessment

    The study elucidates a detailed molecular mechanism of the initial stages of transport in the medically relevant Na+-coupled GABA neurotransmitter transporter GAT1 and thus generates important new insights into this protein family. In particular, it presents convincing evidence for the presence of a "staging binding site" that locally concentrates Na+ ions to increase transport activity, whilst solid evidence for how Na+ binding influences larger scale dynamics.

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Abstract

The human GABA transporter (GAT1) is a membrane transporter that mediates the reuptake of the neurotransmitter GABA from the synaptic cleft into neurons and glial cells. Dysregulation of the transport cycle has been associated with epilepsy and neuropsychiatric disorders, highlighting the crucial role of the transporter in maintaining homeostasis of brain GABA levels. GAT1 is a secondary active transporter that couples the movement of substrate to the simultaneous transport of sodium and chloride ions along their electrochemical gradients. Using MD simulations, we identified a novel sodium recruiting site at the entrance to the outer vestibule, which attracts positively charged ions and increases the local sodium concentration, thereby indirectly increasing sodium affinity. Mutations of negatively charged residues at the recruiting site slowed the binding kinetics, while experimental data revealed a change in sodium dependency of GABA uptake and a reduction of sodium affinity. Simulation showed that sodium displays a higher affinity for the sodium binding site NA2, which plays a role in stabilisation of the outward-open conformation. We directly show that the presence of a sodium ion bound to NA2 increases the stability of the closed inner gate and restrains motions of TM5. We find that sodium is only weakly bound to NA1 in the absence of GABA, while the presence of the substrate strengthens the interaction due to the completed ion coordinating shell, explaining cooperativity between GABA and sodium.

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  1. Version published to 10.7554/elife.93271.3 on eLife
  2. Version published to 10.7554/elife.93271 on eLife
  3. eLife

    eLife Assessment

    The study elucidates a detailed molecular mechanism of the initial stages of transport in the medically relevant Na+-coupled GABA neurotransmitter transporter GAT1 and thus generates important new insights into this protein family. In particular, it presents convincing evidence for the presence of a "staging binding site" that locally concentrates Na+ ions to increase transport activity, whilst solid evidence for how Na+ binding influences larger scale dynamics.

  4. eLife

    Reviewer #1 (Public review):

    The authors have tried to identify the plausible Na+ entry pathway in an important SLC6 member GAT1, using computational approaches to assess residence times of the ions as they enter the vestibule of GAT1. The authors identify a patch of negative residues in TM6a and implicate them for being important to attract the Na+ ions during their movement towards the binding sites Na1 and Na2. Besides this they also suggest that sodium binding at site 1 is flexible and at times can occupy the primary binding site when the substrate is not available. Na2 site as other literature also suggests is demonstrated to be vital for the stability of the outward-open state.

    Studies of ion permeation are challenging given that the states are challenging to trap through structural studies and computational methods are vital for understanding these steps. The authors suggest that two negatively charged residues are vital to attract Na+ ions to the vestibule. Using a combination of simulations and PCA analysis the authors identify the importance of Na+ binding at site 2 that stabilises the outward-open state and the flexibility observed in Na1 site for ion binding which happens alongside substrate in the GABA bound state. The study reconfirms earlier observations in the SLC6 family that Na2 site is critical for conformational transitions and Na1 site is substrate dependent in amino acid transporters.
    One of the challenges in such studies is to conclusively establish the presence of additional Na+ sites or regions of ion-binding with experimental structures as they are nearly impossible to trap. Such studies using simulations therefore become the only resort to understand such phenomena.

    The work is likely to further provide insights into the transport mechanism of GAT1 and lends credence to some structural studies where the sodium at site1 is displaced but the ion remains proximal to the bound substrate.

  5. eLife

    Author response:

    The following is the authors’ response to the previous reviews.

    Public Reviews:

    Reviewer #1 (Public Review):

    Summary:

    The manuscript authored by Stockner and colleagues delves into the molecular simulations of Na+ binding pathway and the ionic interactions at the two known sodium binding sites site 1 and site 2. They further identify a patch of two acidic residues in TM6 that seemingly populate the Na+ ions prior to entry into the vestibule. These results highlight the importance of studying the ion-entry pathways through computational approaches and the authors also validate some of their findings through experimental work. They observe that sodium site 1 binding is stabilized by the presence of the substrate in the s1 site and this is particularly vital as the GABA carboxylate is involved in coordinating the Na+ ion unlike other monoamine transporters and binding of sodium to the Na2 site stabilizes the conformation of the GAT1 by reducing flexibility among the helical bundles involved in alternating access.

    Strengths:

    The study displays results that are generally consistent with available information from experiments on SLC6 transporters particularly GAT1 and puts forth the importance of this added patch of residues in the extracellular vestibule that could be of importance to the ion permeation in SLC6 transporters. This is a nicely performed study and could be improved if the authors could comment on and fix the following queries.

    We thank the reviewer for the overall positive assessment of our work.

    Comments on revised version:

    The authors have satisfactorily addressed my comments and this has significantly improved the clarity of the manuscript.

    The only point that I would like to inquire about is the role of EL4 in modulating Na+ entry.

    In the simulations do the authors see no role of EL4 in controlling Na+ entry. It is particularly intriguing as some studies in the recent past displayed charged mutations in EL4 of dDAT, SERT and GAT1 as being detrimental for substrate entry/uptake. It would therefore be nice to add a small discussion if there is any role for EL4 in Na+ entry.

    In this study we focused on sodium binding to the sodium binding site NA1 and NA2 and discovered the role of negatively charged residues at the beginning of TM6 contribution to sodium binding. Our data shows less than average interactions of sodium ions with EL4. In particular, we do also not observe any prominent role for D355, which is the only negatively charged residues in EL4a. We associate this effect to the presence of four positively charged residues (R69,Y76, K350, R351) surrounded D355 and an electrostatic repulsion by a local positive field, which is also visible in Figure 1k. Following the suggestion of the reviewer, we added a short statement to the last paragraph of the discussion.

    Reviewer #2 (Public Review):

    Summary

    Starting from an AlphaFold2 model of the outward-facing conformation of the GAT1 transporter, the authors primarily use state-of-the-art MD simulations to dissect the role of the two Na+ ions that are known to be co-transported with the substrate, GABA (and a cotransported Cl- ion). The simulations indicated that Na+ binding to OF GAT depends on the electrostatic environment. The authors identify an extracellular recruiting site including residues D281 and E283 which they hypothesized to increase transport by locally increasing the available Na+ concentration and thus increasing binding of Na+ to the canonical binding sites NA1 and NA2. The charge-neutralizing double mutant D281AE283A showed decreased binding in simulations. The authors performed GABA uptake experiments and whole-cell patch clamp experiments that taken together validated the hypothesis that the Na+ staging site is important for transport due to its role in pulling in Na+.

    Detailed analysis of the MD simulations indicated that Na+ binding to NA2 has multiple structural effects: The binding site becomes more compact (reminiscent of induced fit binding) and there is some evidence that it stabilizes the outward-facing conformation.

    Binding to NA1 appears to require the presence of the substrate, GABA, whose carboxylate moiety participates in Na+ binding; thus the simulations predict cooperativity between binding of GABA and Na+ binding to NA1.

    Strengths

    - MD simulations were used to propose a hypothesis (the existence of the staging Na+ site) and then tested with a mutant in simulations AND in experiments. This is an excellent use of simulations in combination with experiments.

    - A large number of repeat MD simulations are generally able to provide a consistent picture of Na+ binding. Simulations are performed according to current best practices and different analyses illuminate the details of the molecular process from different angles.

    - The role of GABA in cooperatively stabilizing Na+ binding to the NA1 site looks convincing and intriguing.

    We thank the reviewer for the overall positive assessment of our work.

    Weaknesses

    - Assessing the effects of Na+ binding on the large scale motions of the transporter is more speculative because the PCA does not clearly cover all of the conformational space and the use of an AlphaFold2 model may have introduced structural inconsistencies. For example, it is not clear if movements of the inner gate are due to a AF2 model that's not well packed or really a feature of the open outward conformation.

    We do not think that the results of the manuscript and in particular the large scale motions are speculative or dependent too much on the limitations of PCA. We only use PCA for Figure 6a-d,6g,h. Motions of SLC6 transporters (and of any other transporter) are much more complex than a single 2D PCA plot could every capture. We therefore used PCA here only to identify the two motions with the largest amplitude, show in Figure 6a-d, 6g,h.

    Given that all the ~13000 degrees of freedom of GAT1 contribute to conformational differences, a dimensionally reduction method like PCA can be very helpful for extracting dominant motions. Structure comparison showed that motions observed in PC1 captured a large portion of the motions of occlusion (Figure 6c,d) when compared to the full transition observed in the unfiltered trajectories (See Figure 6e,f). PCA therefore helps to extract this main motions.

    For completeness, we show a series of structures from the unfiltered trajectories in figure 6e,f. In the overlay, the motion of occlusion is more difficult to observe, because convoluted with all other degrees of freedom. In figure 6e,f, the structures are aligned with the maximum likelihood method theseus, while the coloring is based on the amplitudes measured by PCA to visualize the regions moving relative to each other with largest amplitude. All other structural measures, including the opening of the inner gate (Figure 6i-k), are direct measures of the raw trajectories.

    With respect to the question of the instability of the inner gate, we made similar observations for hSERT (please see DOI: 10.1038/s41467-023-44637-6) using the experimentally determined structure as starting point. We find a weakening of the inner gate for sodium free SERT and at intermediate or full occlusion of sodium- and serotonin-bound SERT. These previous data on SERT corroborate our finding and indicates that the effect could be a general feature of the SLC6 transporter family.

    Unfortunately no outward-open structure of GAT1 was available for this study. AlphaFold2 models have limitations and we are well aware of these limitations, but AlphaFold2 can also make high quality models including small adjustment of backbone positions, if the sequence identity is high, as in the current project (43% sequence identity for the transmembrane region). For GAT1 (as described in the manuscript) we initially tested hSERT based model created with MODELLER. MODELLER uses as premises the assumption that the protein backbone does not change or only very little between the template protein and the target protein. These MODELLER created models did not perform well, because of a slight shift in the position of the backbone, which is a consequence of consistently smaller side chains in the bundle domain-scaffold domain interface of GAT1 as compared to SERT.

    In the simulations described in the manuscript (using the AlphaFold created model) we observed that the overall structural and dynamic parameters and in particular also observation at the inner gate are very similar to the results described in our papers on sodium binding to SERT using experimental SERT structures. The differences of Na1 binding are explained in the manuscript and are contingent to the residue difference of D98 in SERT and the corresponding residue G65 in GAT1. This makes us confident about the quality of the obtained data. Please see DOI: 10.3390/cells11020255; DOI: 10.3389/fncel.2021.673782.

    - Quantitative analyses are difficult with the existing data; for example, the tICA "free energy" landscape is probably not converged because unbinding events haven't been observed.

    The tICA analysis is a Marco State Model approach, which relies on the convergence of transitions between a large number of microstates. A limited number of trajectories showing full sodium unbinding are not obligatory for converged dataset, but the transitions between the microstates must to be converged. For the transitions within the S1 we have many transitions and very good convergence for transition probabilities within the S1. We limit interpretation of free energy data and discussion on this part of the free energy surface. The supporting information (Figure S5) reports on the quality of the tICA analysis. Flat lines with a time lag larger than 40 ns is consistent with a converged model based on the data of the trajectories used for the analysis, and consistently, also the Chapman-Kolmogorov tests show minimal difference between estimates and predictions.

    We see about 40 binding event from the extracellular side to the S1, which seems insufficient for a converged quantification for sodium transiting from the extracellular side to the S1. We state this limitation of the dataset in the results section of the manuscript.

  6. Version published to 10.1101/2023.10.10.561652v3 on bioRxiv
  7. Version published to 10.7554/elife.93271.2 on eLife
  8. eLife

    Author response:

    The following is the authors’ response to the original reviews.

    eLife assessment

    The study elucidates a detailed molecular mechanism of the initial stages of transport in a medically relevant GABA neurotransmitter transporter GAT1 and thus generates useful new insights for this protein family. In particular, it presents convincing evidence for the presence of a "staging binding site" that locally concentrates Na+ ions to increase transport activity, whilst solid evidence for how Na+ binding affects the larger scale dynamics.

    Public Reviews:

    Reviewer #1 (Public Review):

    Summary:

    The manuscript authored by Stockner and colleagues delves into the molecular simulations of Na+ binding pathway and the ionic interactions at the two known sodium binding sites site 1 and site 2. They further identify a patch of two acidic residues in TM6 that seemingly populate the Na+ ions prior to entry into the vestibule. These results highlight the importance of studying the ion-entry pathways through computational approaches and the authors also validate some of their findings through experimental work. They observe that sodium site 1 binding is stabilized by the presence of the substrate in the S1 site and this is particularly vital as the GABA carboxylate is involved in coordinating the Na+ ion unlike other monoamine transporters and binding of sodium to the Na2 site stabilizes the conformation of the GAT1 by reducing flexibility among the helical bundles involved in alternating access.

    Strengths:

    The study displays results that are generally consistent with available information from experiments on SLC6 transporters particularly GAT1 and puts forth the importance of this added patch of residues in the extracellular vestibule that could be of importance to the ion permeation in SLC6 transporters. This is a nicely performed study and could be improved if the authors could comment on and fix the following queries.

    We thank our reviewer for the overall positive evaluation.

    Weaknesses:

    (1) How conserved are the residue pair of D281-E283 in other SLC6 transporters. The authors commented on the presence of these residues in SERT but it would be nice to know how widespread these residues are in other SLC6 transporters like NET, GlyT, and DAT.

    We have created a sequence alignment of the entire human SLC6 family (Supplementary Figure 1) and found that E283 is polar or charged in all SLC6 transporters. D281 shows a higher level of conservation across the family compared to E283. D281 is negatively charged in approximately 50% of the SLC6 family members, an aspartate in all GABA transporters and a glutamate in all monoamine transporters.

    (2) Further, one would like to see the effect of individual mutations D281A and E283A on transport, surface expression, and EC50 of Na+ to gauge the effect on transport.

    We have carried out experiments to investigate the effects of the individual mutations. The results revealed intermediate effects between WT and the double mutant (D281A-E283A) and showed that the effects mostly align with the degree of conservation, as a neutralisation of D281 by alanine has a stronger effect than the E283A mutant. Both single mutants had minimal effects on the sodium dependence of uptake, D281A had a stronger effect on expression, Km and Vmax as compared to E283. Only D281A reduced surface expression, while E283A expresses to a similar level as wild type GAT1.

    (3) A clear figure of the S1 site where Na+ tends to stay prior to Na1 site interactions needs to be provided with a clear figure. Further, it is not entirely clear how access to S1 is altered if the transporter is in an outwardoccluded conformation if F294 is blocking solvent access. Please comment.

    We have modified the structural images in Figure 1, 5, 6 and 7 to improve their comprehensibility. We have also added a comment on the role of F294 as part of the outer hydrophobic gate to the discussion. In short, F294 does not occlude the passage to the S1 as long as GAT1 is outward open, and we find that GAT1 is outward open in all sodium binding simulations.

    (4) The p-value of the EC50 differences between GAT1WT and GAT1double mutant need to be mentioned. The difference in sodium dependence EC50 seems less than twofold, and it would be useful to mention how critical the role of the recruitment site is. Since the transport is not affected the site could play a transient role in attracting ions.

    We have added p-values or standard deviation to our data.

    (5) It would be very nice to know how K+ ions are attracted by this recruitment site. This could further act as a control simulation to test the preference for Na+ ions among SLC6 members.

    We think that attraction of potassium to the recruitment site is not of relevance, as the residues are at the extracellular side and exposed to bulk, where the concentration of sodium is high (typically 130-150 mM), while the concentration of potassium is very small (3-5 mM). Exploring sodium binding by simulations for all SLC6 members could be interesting, but clearly outside the scope of this manuscript.

    (6) Some of the important figures are not very clear. For instance, there should be a zoomed-in view of the recruitment site. The current one in Fig. 1b and 1c could be made clearer. Similarly as mentioned earlier the Na residence at the S1 site away from the Na1 and Na2 sites needs to be shown with greater clarity by putting side chain information in Fig. 6d.

    We have modified the structural images in Figure 1, 5, 6 and 7 to improve their comprehensibility.

    (7) The structural features that comprise the two principal components PC1 and PC2 should be described in greater detail.

    We have modified Figure 6 and added images that show the motions along PC1 and PC2. In addition, these are now better explained in the text.

    Reviewer #2 (Public Review):

    Summary:

    Starting from an AlphaFold2 model of the outward-facing conformation of the GAT1 transporter, the authors primarily use state-of-the-art MD simulations to dissect the role of the two Na+ ions that are known to be cotransported with the substrate, GABA (and a co-transported Cl- ion). The simulations indicated that Na+ binding to OF GAT depends on the electrostatic environment. The authors identify an extracellular recruiting site including residues D281 and E283 which they hypothesized to increase transport by locally increasing the available Na+ concentration and thus increasing binding of Na+ to the canonical binding sites NA1 and NA2. The charge-neutralizing double mutant D281A-E283A showed decreased binding in simulations. The authors performed GABA uptake experiments and whole-cell patch clamp experiments that taken together validated the hypothesis that the Na+ staging site is important for transport due to its role in pulling in Na+.

    Detailed analysis of the MD simulations indicated that Na+ binding to NA2 has multiple structural effects: The binding site becomes more compact (reminiscent of induced fit binding) and there is some evidence that it stabilizes the outward-facing conformation.

    Binding to NA1 appears to require the presence of the substrate, GABA, whose carboxylate moiety participates in Na+ binding; thus the simulations predict cooperativity between binding of GABA and Na+ binding to NA1.

    Strengths:

    - MD simulations were used to propose a hypothesis (the existence of the staging Na+ site) and then tested with a mutant in simulations AND in experiments. This is an excellent use of simulations in combination with experiments.

    - A large number of repeat MD simulations are generally able to provide a consistent picture of Na+ binding. Simulations are performed according to current best practices and different analyses illuminate the details of the molecular process from different angles.

    - The role of GABA in cooperatively stabilizing Na+ binding to the NA1 site looks convincing and intriguing.

    We thank the review for the very supportive assessment.

    Weaknesses:

    - Assessing the effects of Na+ binding on the large-scale motions of the transporter is more speculative because the PCA does not clearly cover all of the conformational space and the use of an AlphaFold2 model may have introduced structural inconsistencies. For example, it is not clear if movements of the inner gate are due to an AF2 model that's not well packed or really a feature of the open outward conformation.

    The long range effect of sodium binding to GAT1 and destabilisation of the inner gate has, based on our data, a causal effect. PCA separates conformational motions into degrees of freedom and sorts them according to the largest motions. Motions of TM5a were among the 2 largest motions, which suggests that these are relevant motions. To directly quantify their behaviour, we measured informative distances at the inner gate of GAT1, as shown in Figure 6i,j,k and separated data according to the presence of sodium in NA2.

    For the following reasons we exclude that the results are a consequence of structural inconsistencies introduced by AlphaFold2 and therefore not reflecting functionally relevant effects:

    (1) If depending on the model instead of sodium binding, the effects should not be correlated with the presence of sodium in the NA2 binding site.

    (2) We carried out new simulations starting from the occluded GAT1 structure (Figure 6j,k). The data shows that in the occluded state the distance across the inner vestibule and the length of TM5a differ, consistent with our interpretation of the data. As sodium binding fixes GAT1 outwardfacing, as it also occurs in other SLC6 family members (Szöllősi and Stockner, 2022), the distances of the outward-open GAT1 are at the short extreme of the scale, distances of the inward-open state of the cryo-EM structure(s) are at the other extreme, while the occluded conformation of GAT1 shows intermediate values.

    (3) We have observed the same property in SERT, for which we used experimental structures as starting structure (Gradisch et al., 2024), suggesting that this could be a generally mechanism.

    (4) All available structures from the entire SLC6 family are consistent with structural effects of TM5a in response to bundle domain motions and therefore to binding of sodium to NA2 as it stabilized the outward-open state as well as transition to the inward facing conformation.

    - Quantitative analyses are difficult with the existing data; for example, the tICA "free energy" landscape is probably not converged because unbinding events haven't been observed.

    Simulations can always be too short and therefore not fully describe the complete underlying conformational ensemble. We added a statement in the discussion indicating this shortcoming. With respect to the tICA analysis in our manuscript, the tICA approach does, by design, not need long simulations that capture the full binding and unbinding in multiple instances to construct a correct free energy landscape. Instead, the tICA method builds on Markov chain dependencies and relies only on the convergence of transitions between hundreds of conformational microstates and the fluxes between them. The free energy profile derived for the S1, including NA1, TMP and NA2 and up to the salt bridge of the outer gate is well converged and we observed many transitions. In contrast, the entry from the recruitment side to the S1 has most likely a too low density of microstate and a too small number of transition to be considered converged with respect to quantifying the free energy of binding from bulk. We now explain this shortcoming.

    Recommendations for the authors:

    Reviewer #1 (Recommendations for The Authors):

    Authors should furnish p-values in the figure legends for experimental results.

    We have added the p-values to text and figure legends.

    Reviewer #2 (Recommendations For The Authors):

    - Deposit simulation data in a public repository (input files, trajectories (possibly subsampled)).

    We deposited the data to Zenodo and provided the DOI: 10.5281/zenodo.10686813 to the data. As we were unable to upload the trajectories to zenodo, we deposited the starting and the end structures of the simulations.

    - Please include a short discussion of the reliability of using an AF2 model instead of experimental structures. What is expected to be correct/which parts of the structure are potentially incorrect? What makes you think that the AF2 model is a good model of the OF conformation of GAT1?

    Unfortunately, an outward-facing structure of GAT1 is not available. We have initially worked with an outward-open homology model of GAT1 based on SERT (build with MODELLER), but the structural differences between SERT and GAT1 are sufficiently large that these models did not behave well in simulations and too frequently could not maintain a sealed inner gate, also forming a channel. In contrast to the SERT-based GAT1 model, the AlphaFold2 model of GAT1 behaved as expected and consistent with the behaviour of SERT in simulations and with general knowledge of protein dynamics from literature. Based on structural analysis of our simulations and on the comparison to SERT we could not identify a region of GAT1 which would be potentially behave incorrect or unexpectedly. We added a statement to the discussion on this potential limitation of the use of homology models.

    - Fig 1a: Na+ densities are not very clear (both due to small size and the transparency). I have a hard time seeing where bulk, 2*bulk regions are --- are you showing "onion shells" of density? Perhaps investigate presenting as cuts through the full density?

    I like the labelling in terms of absolute density and multiples of bulk.

    We have created new images to improve the visualisation of data. The data are shown as onion shells (isosurface), with the shells at the indicated densities. This is now clearly stated. Transparency is needed, otherwise e.g. the inner onion shells would not be visible. The cut-through is intuitive, but we could not find a useful plain, as the densities are too extensively distributed in 3D and not on a single plain.

    - Fig 1h-k: would be clearer if "recruitment site" (TMP?) was indicated in the figure.

    We have created a new image for the recruiting site (Figure 1b,c) and temporary site (Figure 1g) and indicated these two sites as appropriate.

    - Show time series of Na+ binding with a suitable order parameter (z or distances to NA1 and NA2?) to show how ions bind spontaneously. Mark the different sites. Mark pre- and post-binding parts of trajectories.

    We have added time series for every simulation that shows sodium binding to the NA1 or NA2 to the supplementary information Figure 2a,b,c. These quantify the distances to the recruiting site, the temporary site and the respective sodium binding site.

    - PCA - how much of the total variance was captured by PC1 and PC2?

    The variance captured by the PCs are shown as eigenvalues in supplementary information Figure 4. PC1 captures about 19% of the variance, PC2 8%.

    - "We found that the inner hydrophobic gate is dynamic in the absence of Na2" -- is this instability due to the AF2 model or likely realistic? E.g. was similar behaviour ever observed in simulations of the occluded state?

    In simulations of the occluded state we do not see such instabilities as observed in the outward-open state in the absence of sodium (Figure 6). As these larger scale fluctuations are not randomly distributed across all simulations starting from the AlphaFold2 models, but confined to the systems without sodium, it is unlikely an effect of the AlphaFold2 model.

    Please note, we have seen comparable behaviour in simulations of SERT starting from experimental structures (Gradisch et al., 2024), therefore suggesting a more general mechanism.

    - Cooperativity between GABA-binding and Na+ binding to NA1: How would this lead to an experimentally measurable signature, i.e., which experiments could validate this interesting prediction?

    Direct detection of cooperativity is difficult to separate from other effects in experiments, as sodium binding and transport involves NA1 and NA2, NA2 has a higher affinity according to our data, while mutations will not only affect cooperativity, but will also have other effects.

    Conformational changes can also complicate experimental detection, as NA2 stabilises the outward-open conformation, while NA1+GABA binding triggers the transition to the inward-open state. To quantify cooperativity, it would be important to isolate the cooperative from all other effects, which is a challenge. Support for cooperativity has been found by (Zhou, Zomot and Kanner, 2006; Meinild and Forster, 2012) using this route. In the first paper the authors make use of lithium that only binds to the NA2, even though lithium is not only a mere NA2 selective ligand and otherwise identical to sodium. By comparing two GABA concentrates the authors showed that the sodium dependence of GABA transport is left shifted at higher GABA concentrations, which is not the case in the absence of lithium. This data is indirect, but consistent with cooperativity between GABA and NA1-bound sodium, as GABA transport mainly reflects binding of sodium to NA1. Similar approaches could be further explored, for example by varying the GABA concentration instead of sodium. Other options could be to create an outward-facing and conformationally locked GAT1 and to measure the cooperativity of sodium and GABA binding using for example the scintillation proximity assay. Most likely the assay would also need a way to be NA2 binding independent. We are not aware of such a GABA transporter system.

    - There are some instances of [SI Figure] or [citation needed] that should be cleaned up.

    We have corrected these instances.

    References

    Gradisch, R. et al. (2024) ‘Ligand coupling mechanism of the human serotonin transporter differentiates substrates from inhibitors’, Nature Communications, 15(1), p. 417. Available at: https://doi.org/10.1038/s41467-023-44637-6.

    Meinild, A.-K. and Forster, I.C. (2012) ‘Using lithium to probe sequential cation interactions with GAT1’, American Journal of Physiology. Cell Physiology, 302(11), pp. C1661-1675. Available at: https://doi.org/10.1152/ajpcell.00446.2011.

    Szöllősi, D. and Stockner, T. (2022) ‘Sodium Binding Stabilizes the Outward-Open State of SERT by Limiting Bundle Domain Motions’, Cells, 11(2), p. 255. Available at: https://doi.org/10.3390/cells11020255.

    Zhou, Y., Zomot, E. and Kanner, B.I. (2006) ‘Identification of a lithium interaction site in the gamma-aminobutyric acid (GABA) transporter GAT-1’, The Journal of Biological Chemistry, 281(31), pp. 22092–22099. Available at: https://doi.org/10.1074/jbc.M602319200.

  9. eLife

    eLife assessment

    The study elucidates a detailed molecular mechanism of the initial stages of transport in the medically relevant Na+-coupled GABA neurotransmitter transporter GAT1 and thus generates useful new insights into this protein family. In particular, it presents convincing evidence for the presence of a "staging binding site" that locally concentrates Na+ ions to increase transport activity, whilst solid evidence for how Na+ binding influences larger scale dynamics.

  10. eLife

    Reviewer #1 (Public Review):

    Summary:

    The manuscript authored by Stockner and colleagues delves into the molecular simulations of Na+ binding pathway and the ionic interactions at the two known sodium binding sites site 1 and site 2. They further identify a patch of two acidic residues in TM6 that seemingly populate the Na+ ions prior to entry into the vestibule. These results highlight the importance of studying the ion-entry pathways through computational approaches and the authors also validate some of their findings through experimental work. They observe that sodium site 1 binding is stabilized by the presence of the substrate in the s1 site and this is particularly vital as the GABA carboxylate is involved in coordinating the Na+ ion unlike other monoamine transporters and binding of sodium to the Na2 site stabilizes the conformation of the GAT1 by reducing flexibility among the helical bundles involved in alternating access.

    Strengths:

    The study displays results that are generally consistent with available information from experiments on SLC6 transporters particularly GAT1 and puts forth the importance of this added patch of residues in the extracellular vestibule that could be of importance to the ion permeation in SLC6 transporters. This is a nicely performed study and could be improved if the authors could comment on and fix the following queries.

    Comments on revised version:

    The authors have satisfactorily addressed my comments and this has significantly improved the clarity of the manuscript.

    The only point that I would like to inquire about is the role of EL4 in modulating Na+ entry. In the simulations do the authors see no role of EL4 in controlling Na+ entry. It is particularly intriguing as some studies in the recent past displayed charged mutations in EL4 of dDAT, SERT and GAT1 as being detrimental for substrate entry/uptake. It would therefore be nice to add a small discussion if there is any role for EL4 in Na+ entry.

  11. eLife

    Reviewer #2 (Public Review):

    Summary

    Starting from an AlphaFold2 model of the outward-facing conformation of the GAT1 transporter, the authors primarily use state-of-the-art MD simulations to dissect the role of the two Na+ ions that are known to be co-transported with the substrate, GABA (and a co-transported Cl- ion). The simulations indicated that Na+ binding to OF GAT depends on the electrostatic environment. The authors identify an extracellular recruiting site including residues D281 and E283 which they hypothesized to increase transport by locally increasing the available Na+ concentration and thus increasing binding of Na+ to the canonical binding sites NA1 and NA2. The charge-neutralizing double mutant D281A-E283A showed decreased binding in simulations. The authors performed GABA uptake experiments and whole-cell patch clamp experiments that taken together validated the hypothesis that the Na+ staging site is important for transport due to its role in pulling in Na+.

    Detailed analysis of the MD simulations indicated that Na+ binding to NA2 has multiple structural effects: The binding site becomes more compact (reminiscent of induced fit binding) and there is some evidence that it stabilizes the outward-facing conformation.

    Binding to NA1 appears to require the presence of the substrate, GABA, whose carboxylate moiety participates in Na+ binding; thus the simulations predict cooperativity between binding of GABA and Na+ binding to NA1.

    Strengths

    - MD simulations were used to propose a hypothesis (the existence of the staging Na+ site) and then tested with a mutant in simulations AND in experiments. This is an excellent use of simulations in combination with experiments.

    - A large number of repeat MD simulations are generally able to provide a consistent picture of Na+ binding. Simulations are performed according to current best practices and different analyses illuminate the details of the molecular process from different angles.

    - The role of GABA in cooperatively stabilizing Na+ binding to the NA1 site looks convincing and intriguing.

    Weaknesses

    - Assessing the effects of Na+ binding on the large scale motions of the transporter is more speculative because the PCA does not clearly cover all of the conformational space and the use of an AlphaFold2 model may have introduced structural inconsistencies. For example, it is not clear if movements of the inner gate are due to a AF2 model that's not well packed or really a feature of the open outward conformation.

    - Quantitative analyses are difficult with the existing data; for example, the tICA "free energy" landscape is probably not converged because unbinding events haven't been observed.

  12. Version published to 10.1101/2023.10.10.561652v2 on bioRxiv
  13. Version published to 10.7554/elife.93271.1 on eLife
  14. eLife

    eLife assessment

    The study elucidates a detailed molecular mechanism of the initial stages of transport in a medically relevant GABA neurotransmitter transporter GAT1 and thus generates useful new insights for this protein family. In particular, it presents convincing evidence for the presence of a "staging binding site" that locally concentrates Na+ ions to increase transport activity, whilst solid evidence for how Na+ binding affects the larger scale dynamics.

  15. eLife

    Reviewer #1 (Public Review):

    Summary:
    The manuscript authored by Stockner and colleagues delves into the molecular simulations of Na+ binding pathway and the ionic interactions at the two known sodium binding sites site 1 and site 2. They further identify a patch of two acidic residues in TM6 that seemingly populate the Na+ ions prior to entry into the vestibule. These results highlight the importance of studying the ion-entry pathways through computational approaches and the authors also validate some of their findings through experimental work. They observe that sodium site 1 binding is stabilized by the presence of the substrate in the s1 site and this is particularly vital as the GABA carboxylate is involved in coordinating the Na+ ion unlike other monoamine transporters and binding of sodium to the Na2 site stabilizes the conformation of the GAT1 by reducing flexibility among the helical bundles involved in alternating access.

    Strengths:
    The study displays results that are generally consistent with available information from experiments on SLC6 transporters particularly GAT1 and puts forth the importance of this added patch of residues in the extracellular vestibule that could be of importance to the ion permeation in SLC6 transporters. This is a nicely performed study and could be improved if the authors could comment on and fix the following queries.

    Weaknesses:
    1. How conserved are the residue pair of D281-E283 in other SLC6 transporters. The authors commented on the presence of these residues in SERT but it would be nice to know how widespread these residues are in other SLC6 transporters like NET, GlyT, and DAT.

    2. Further, one would like to see the effect of individual mutations D281A and E283A on transport, surface expression, and EC50 of Na+ to gauge the effect on transport.

    3. A clear figure of the S1 site where Na+ tends to stay prior to Na1 site interactions needs to be provided with a clear figure. Further, it is not entirely clear how access to S1 is altered if the transporter is in an outward-occluded conformation if F294 is blocking solvent access. Please comment.

    4. The p-value of the EC50 differences between GAT1WT and GAT1double mutant need to be mentioned. The difference in sodium dependence EC50 seems less than twofold and it would be useful to mention how critical the role of the recruitment site is. Since the transport is not affected the site could play a transient role in attracting ions.

    5. It would be very nice to know how K+ ions are attracted by this recruitment site. This could further act as a control simulation to test the preference for Na+ ions among SLC6 members.

    6. Some of the important figures are not very clear. For instance, there should be a zoomed-in view of the recruitment site. The current one in Fig. 1b and 1c could be made clearer. Similarly as mentioned earlier the Na residence at the S1 site away from the Na1 and Na2 sites needs to be shown with greater clarity by putting side chain information in Fig. 6d.

    7. The structural features that comprise the two principle components PC1 and PC2 should be described in greater detail.

  16. eLife

    Reviewer #2 (Public Review):

    Summary:
    Starting from an AlphaFold2 model of the outward-facing conformation of the GAT1 transporter, the authors primarily use state-of-the-art MD simulations to dissect the role of the two Na+ ions that are known to be co-transported with the substrate, GABA (and a co-transported Cl- ion). The simulations indicated that Na+ binding to OF GAT depends on the electrostatic environment. The authors identify an extracellular recruiting site including residues D281 and E283 which they hypothesized to increase transport by locally increasing the available Na+ concentration and thus increasing binding of Na+ to the canonical binding sites NA1 and NA2. The charge-neutralizing double mutant D281A-E283A showed decreased binding in simulations. The authors performed GABA uptake experiments and whole-cell patch clamp experiments that taken together validated the hypothesis that the Na+ staging site is important for transport due to its role in pulling in Na+.

    Detailed analysis of the MD simulations indicated that Na+ binding to NA2 has multiple structural effects: The binding site becomes more compact (reminiscent of induced fit binding) and there is some evidence that it stabilizes the outward-facing conformation.

    Binding to NA1 appears to require the presence of the substrate, GABA, whose carboxylate moiety participates in Na+ binding; thus the simulations predict cooperativity between binding of GABA and Na+ binding to NA1.

    Strengths:
    - MD simulations were used to propose a hypothesis (the existence of the staging Na+ site) and then tested with a mutant in simulations AND in experiments. This is an excellent use of simulations in combination with experiments.

    - A large number of repeat MD simulations are generally able to provide a consistent picture of Na+ binding. Simulations are performed according to current best practices and different analyses illuminate the details of the molecular process from different angles.

    - The role of GABA in cooperatively stabilizing Na+ binding to the NA1 site looks convincing and intriguing.

    Weaknesses:
    - Assessing the effects of Na+ binding on the large-scale motions of the transporter is more speculative because the PCA does not clearly cover all of the conformational space and the use of an AlphaFold2 model may have introduced structural inconsistencies. For example, it is not clear if movements of the inner gate are due to an AF2 model that's not well packed or really a feature of the open outward conformation.

    - Quantitative analyses are difficult with the existing data; for example, the tICA "free energy" landscape is probably not converged because unbinding events haven't been observed.

  17. Version published to 10.1101/2023.10.10.561652v1 on bioRxiv