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Molecular bases of human excitatory neurotransmitter transport across the plasma membrane

Final Report Summary - HEAATS (Molecular bases of human excitatory neurotransmitter transport across the plasma membrane)

Glutamate is the main excitatory neurotransmitter in the brain and its extracellular concentration has to be tightly regulated to maintain neurotransmission and prevent cytotoxicity. Key players in this regulatory function are integral membrane proteins of the solute carrier 1 family (SLC1), called Excitatory Amino Acid Transporters (EAATs) or simply Glutamate Transporters.
This ERC-StG project focused on understanding the molecular mechanisms of function and pharmacology of human EAATs. However these transporters, as many other human membrane proteins are biochemically unstable in the detergents solutions required for their purification. Therefore, we obtained stable EAATs mutants that are highly similar to the Wild Type transporters and retain their functional and pharmacological profiles for biophysical characterization.
Following this, we solved the first X-ray crystal structures of a thermostable human EAAT1 in complex with a substrate and thermodynamically coupled ions, as well as with and without an allosteric inhibitor. The structures reveal novel architectural features of the human transporters, including extra- and intra-cellular domains that have a potential role in the transport function, regulation by lipids and post-translational modifications, and aid to understand the impact of mutations associated to human disease. The coordination of substrate and coupled ions also shed light on the transport mechanisms of these proteins.
The binding site of the allosteric inhibitor observed in the structure, as well as the accompanying changes in transport and dynamics of EAAT1 unravel the first allosteric inhibitory mechanism of the SLC1 family of transporters, whereby the allosteric inhibitor blocks important conformational changes of the transport cycle.
The results of this project provide new approaches to obtain thermostable transporters for biophysical analysis, and valuable insights into the molecular mechanisms of function and pharmacology of human SLC1 transporters. This knowledge might help to develop selective allosteric and non-allosteric compounds that modulate the activity of important human membrane proteins to alleviate common neurological disorders and different forms of human cancer.