Conserved allosteric inhibition mechanism in SLC1 transporters
Excitatory amino acid transporter 1 (EAAT1) is a glutamate transporter belonging to the SLC1 family of solute carriers. It plays a key role in the regulation of the extracellular glutamate concentration in the mammalian brain. The structure of EAAT1 was determined in complex with UCPH-101, apotent,...
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eLife Sciences Publications Ltd
2023-03-01
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| Series: | eLife |
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| Online Access: | https://elifesciences.org/articles/83464 |
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| author | Yang Dong Jiali Wang Rachel-Ann Garibsingh Keino Hutchinson Yueyue Shi Gilad Eisenberg Xiaozhen Yu Avner Schlessinger Christof Grewer |
| author_facet | Yang Dong Jiali Wang Rachel-Ann Garibsingh Keino Hutchinson Yueyue Shi Gilad Eisenberg Xiaozhen Yu Avner Schlessinger Christof Grewer |
| author_sort | Yang Dong |
| collection | DOAJ |
| description | Excitatory amino acid transporter 1 (EAAT1) is a glutamate transporter belonging to the SLC1 family of solute carriers. It plays a key role in the regulation of the extracellular glutamate concentration in the mammalian brain. The structure of EAAT1 was determined in complex with UCPH-101, apotent, 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 between intracellular and extracellular compartments . 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. Guided by computational modeling using ligand docking and molecular dynamics 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 could be restored, and the apparent affinity was increased (Ki = 4.3 μM), much closer to the EAAT1 value of 0.6 μM. Finally, we identify a novel non-competitive ASCT2 inhibitor, 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 ASCT2 allosteric inhibitors can be designed . |
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| language | English |
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| spelling | doaj.art-73cb33321df14974abd825f88d0a87d32023-03-15T09:37:31ZengeLife Sciences Publications LtdeLife2050-084X2023-03-011210.7554/eLife.83464Conserved allosteric inhibition mechanism in SLC1 transportersYang Dong0Jiali Wang1https://orcid.org/0000-0002-9520-8140Rachel-Ann Garibsingh2Keino Hutchinson3Yueyue Shi4Gilad Eisenberg5Xiaozhen Yu6Avner Schlessinger7https://orcid.org/0000-0003-4007-7814Christof Grewer8https://orcid.org/0000-0002-8342-9878Department of Chemistry, Binghamton University, Binghamton, United StatesDepartment of Chemistry, Binghamton University, Binghamton, United StatesDepartment of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, United StatesDepartment of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, United StatesDepartment of Chemistry, Binghamton University, Binghamton, United StatesDepartment of Chemistry, Binghamton University, Binghamton, United StatesDepartment of Chemistry, Binghamton University, Binghamton, United StatesDepartment of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, United StatesDepartment of Chemistry, Binghamton University, Binghamton, United StatesExcitatory amino acid transporter 1 (EAAT1) is a glutamate transporter belonging to the SLC1 family of solute carriers. It plays a key role in the regulation of the extracellular glutamate concentration in the mammalian brain. The structure of EAAT1 was determined in complex with UCPH-101, apotent, 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 between intracellular and extracellular compartments . 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. Guided by computational modeling using ligand docking and molecular dynamics 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 could be restored, and the apparent affinity was increased (Ki = 4.3 μM), much closer to the EAAT1 value of 0.6 μM. Finally, we identify a novel non-competitive ASCT2 inhibitor, 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 ASCT2 allosteric inhibitors can be designed .https://elifesciences.org/articles/83464SLC1glutamate transporteralanine serine cysteine transporterinhibitiondockingelectrophysiology |
| spellingShingle | Yang Dong Jiali Wang Rachel-Ann Garibsingh Keino Hutchinson Yueyue Shi Gilad Eisenberg Xiaozhen Yu Avner Schlessinger Christof Grewer Conserved allosteric inhibition mechanism in SLC1 transporters eLife SLC1 glutamate transporter alanine serine cysteine transporter inhibition docking electrophysiology |
| title | Conserved allosteric inhibition mechanism in SLC1 transporters |
| title_full | Conserved allosteric inhibition mechanism in SLC1 transporters |
| title_fullStr | Conserved allosteric inhibition mechanism in SLC1 transporters |
| title_full_unstemmed | Conserved allosteric inhibition mechanism in SLC1 transporters |
| title_short | Conserved allosteric inhibition mechanism in SLC1 transporters |
| title_sort | conserved allosteric inhibition mechanism in slc1 transporters |
| topic | SLC1 glutamate transporter alanine serine cysteine transporter inhibition docking electrophysiology |
| url | https://elifesciences.org/articles/83464 |
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