PT - JOURNAL ARTICLE AU - Yang Dong AU - Jiali Wang AU - Rachel-Ann Garibsingh AU - Keino Hutchinson AU - Yueyue Shi AU - Gilad Eisenberg AU - Xiaozhen Yu AU - Avner Schlessinger AU - Christof Grewer TI - Conserved allosteric inhibition mechanism in SLC1 transporters AID - 10.1101/2022.09.21.508810 DP - 2022 Jan 01 TA - bioRxiv PG - 2022.09.21.508810 4099 - http://biorxiv.org/content/early/2022/09/22/2022.09.21.508810.short 4100 - http://biorxiv.org/content/early/2022/09/22/2022.09.21.508810.full AB - Excitatory Amino Acid Transporter 1 (EAAT1) is a plasma-membrane glutamate transporter belonging to the SLC1 family of solute carriers. It plays a key role in neurotransmitter transport and contributes to the regulation of the extracellular glutamate concentration in the mammalian brain. The structure of EAAT1 was determined using cryo-EM, in complex with UCPH-101, a highly potent and non-competitive inhibitor of EAAT1. Alanine Serine Cysteine Transporter 2 (ASCT2) is a neutral amino acid transporter, which regulates pools of amino acids such as glutamine, serine and alanine between intracellular and extracellular compartments in a Na+ dependent manner. ASCT2 also belongs to the SLC1 family and shares 58% sequence similarity with EAAT1. However, allosteric modulation of ASCT2 via non-competitive inhibitors is unknown. Here we explore the UCPH-101 inhibitory mechanisms of EAAT1 and ASCT2 by using rapid kinetic experiments. Our results show that UCPH-101 slows substrate translocation rather than substrate or Na+ binding, confirming a non-competitive inhibitory mechanism, but only partially inhibits wild-type ASCT2 with relatively low affinity. Guided by computational modeling using ligand docking and molecular dynamics (MD) simulations, we selected two residues involved in UCPH-101/EAAT1 interaction, which were mutated in ASCT2 (F136Y, I237M, F136Y/I237M) in the corresponding positions. We show that in the F136Y/I237M double mutant transporter, 100% of the inhibitory effect of UCPH-101 on anion current could be restored, and the apparent affinity was increased (Ki = 9.3 μM), much closer to the EAAT1 value of 0.6 μM. Finally, we identify a novel non-competitive ASCT2 inhibitor, identified through virtual screening and experimental testing against the allosteric site, further supporting its localization. Together, these data indicate that the mechanism of allosteric modulation is conserved between EAAT1 and ASCT2. Due to the difference in binding site residues between ASCT2 and EAAT1, these results raise the possibility that more potent, and potentially selective inhibitors can be designed that target the ASCT2 allosteric binding site.Competing Interest StatementThe authors have declared no competing interest.