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

Author:

Lazzarin Erika1ORCID,Gradisch Ralph12ORCID,Skopec Sophie M.C.1,Alves da Silva Leticia1ORCID,Sebastianelli-Schoditsch Chiara1,Szöllősi Dániel13ORCID,Maier Julian1ORCID,Sucic Sonja1ORCID,Roblek Marko1ORCID,Kanner Baruch I.4,Sitte Harald H.156ORCID,Stockner Thomas1ORCID

Affiliation:

1. Centre for Physiology and Pharmacology, Institute of Pharmacology, Medical University of Vienna

2. Institute of Pharmacology and Toxicology, University of Zurich

3. Department of Theoretical and Computational Biophysics, Max Planck Institute for Multidisciplinary Sciences

4. Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, Hebrew University, Hadassah Medical School

5. Hourani Center for Applied Scientific Research, Al-Ahliyya Amman University

6. Center for Addiction Research and Science-AddRess, Medical University Vienna

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.

Publisher

eLife Sciences Publications, Ltd

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