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

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.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

* We thank our reviewer for their thorough evaluation and their constructive comments, which have helped to improve the manuscript. We have enhanced the description of the sodium recruiting site, created new figures, which clearly show structural details and sodium density. We conducted the suggested experiments, created the single GAT1 mutants D281A and E283A at the sodium recruiting site and measured surface expression, GABA uptake and sodium dependence. The data shows that E283A is wild-type like, while D281A is intermediate between wild-type and the D281A-E283A double mutant for all measured properties. A sequence alignment of all human SLC6 transporters corroborates these experimental data. The conservation of E283 is low, while D281 is an aspartate in all GABA transporters and a glutamate in all monoamine transporters. We now also give standard deviations and/or p-values for all experimental data. The SI now shows traces of distances between the sodium ion and the recruiting site for every trajectory in which sodium binds to GAT1. We have improved the visualisation of most structural figures to enhance clarity. For the analysis of the principal components (PC), we created new panels in figure 6 that visualise the motions described by the first two PCs. We have added a paragraph describing potential limitations of the AlphaFold model. The observed long-range effect of sodium binding to GAT1 and destabilisation of the inner gate has, based on our data, a causal effect. Motions of TM5a were among the 2 largest motions identified by PCA, which suggest to reflect relevant motions. To directly quantify the structural dynamics of the inner gate, we measured informative distances at the inner gate of GAT1, as shown in Figure 5i,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 AlphaFold and therefore not reflecting functionally relevant effect: If depending on the model instead of sodium binding, the effect should not be correlated with the presence of sodium in the NA2 binding site. We have observed the same property in SERT, for which we used experimental structures as starting positions (doi: 10.1038/s41467-023-44637-6), suggesting that this could be a generally mechanism. 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 stabilizing the outward-open state.

* https://doi.org/10.5281/zenodo.10686813