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

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.

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.