Conserved allosteric inhibition mechanism in SLC1 transporters
- Department of Chemistry, Binghamton University, United States
- Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, United States
Figures
Figure 1 with 1 supplement
Structure of the excitatory amino acid transporter 1 (EAAT1) UCPH-101-bound state.
(A) UCPH-101 structure and substituents used for constraints in docking calculations. (B) EAAT1 structure (PDB id: 5MJU) in complex with the competitive inhibitor TFB-TBOA …
Figure 1—figure supplement 1
EAAT1/ASCT2/ASCT1 sequence alignment.
Sequence alignment was performed with Bioedit software, total alignment length: 571 amino acid, 220 identical residues, 109 similar residues. EAAT1 and ASCT2 show 38.53% identity and 57.62% …
Figure 2
Alanine serine cysteine transporter 2 (ASCT2) structures and predicted allosteric binding site.
(A) ASCT2 structure (PDB id 7BCS) with the competitive inhibitor L-cis-PBE (cyan sticks). The trimerization and transport domains are highlighted in dark green and dark orange, respectively. (B) …
Figure 3 with 1 supplement
UCPH-101 is a high-affinity, non-competitive inhibitor of excitatory amino acid transporter 1 (EAAT1) anion current.
(A) Typical whole-cell current recordings in the absence (black) and presence of 2 μM (red) and 100 μM (blue) UCPH-101. [Glutamate] was 100 μM. Experiments were performed using 140 mM sodium …
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Figure 3—source data 1
The source data contains original current traces for Figure 3A, and the original data for panels (B) and (C).
- https://cdn.elifesciences.org/articles/83464/elife-83464-fig3-data1-v3.xlsx
Figure 3—figure supplement 1
Excitatory amino acid transporter 1 (EAAT1) anion current is inhibited by UCPH-101 independent of voltage.
Voltage dependence of EAAT1 anion currents under forward transport conditions, activated using 500 μM extracellular Glu (n=13) (A) or 500 μM Glu in the presence of 50 μM UCPH-101 (n=14) (B). The …
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Figure 3—figure supplement 1—source data 1
The source data contains the current traces for Figure 3—figure supplement 1A and B, and the original data for panel (C).
- https://cdn.elifesciences.org/articles/83464/elife-83464-fig3-figsupp1-data1-v3.xlsx
Figure 4 with 1 supplement
UCPH-101 has only minor effect on recovery kinetics of excitatory amino acid transporter 1 (EAAT1) current after glutamate removal.
(A) Anion currents in response to two pulses of rapid glutamate application (1 mM), with varying inter-pulse interval (pulse protocol shown at the top) under forward transport conditions. The …
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Figure 4—source data 1
Current traces for Figure 3A,B,C,E,F and G, and the original data for panels (D) and (H).
- https://cdn.elifesciences.org/articles/83464/elife-83464-fig4-data1-v3.xlsx
Figure 4—figure supplement 1
UCPH-101 does not eliminate, but reduces capacitive charge movement caused by Na+ binding to the apo-form of excitatory amino acid transporter 1 (EAAT1).
Voltage jumps (protocol shown in top panel) were used to disturb the Na+ binding equilibrium in the apo-form of EAAT1 (absence of glutamate). (A) and (B) show the transient currents caused by Na+ …
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Figure 4—figure supplement 1—source data 1
Original current traces for Figure 4—figure supplement 1A and B, and the original data for panel (C).
- https://cdn.elifesciences.org/articles/83464/elife-83464-fig4-figsupp1-data1-v3.xlsx
Figure 5 with 1 supplement
EAAT1, ASCT2WT, and ASCT2F136Y/I237M residues contribute to UCPH-101 stability in the binding site.
(A) Original state of the EAAT1-UCPH-101 complex from structure 5LLM (Canul-Tec et al., 2017). Y127 and M271 (EAAT1 sequence number) contribute to the EAAT1-UCPH-101 interaction. The binding state …
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Figure 5—source data 1
Trajectory and RMSD data for panels (D-F).
- https://cdn.elifesciences.org/articles/83464/elife-83464-fig5-data1-v3.xlsx
Figure 5—figure supplement 1
Molecular dynamics (MD) simulations to 500 ns for ASCT2WT and ASCT2F136Y/I237M.
Conditions were as described in the legend of Figure 5.
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Figure 5—figure supplement 1—source data 1
Trajectory and RMSD data.
- https://cdn.elifesciences.org/articles/83464/elife-83464-fig5-figsupp1-data1-v3.xlsx
Figure 6 with 1 supplement
Alanine serine cysteine transporter 2 (ASCT2) amino acid-induced anion current is partially inhibited by UCPH-101.
(A) Typical serine-induced ASCT2 anion current increases with increasing serine concentrations. The extracellular solution contained 140 mM sodium methanesulfonate (NaMes), with 130 mM NaSCN, and 10 …
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Figure 6—source data 1
Original current traces for Figure 6A and B, and the original data for panels (C-E).
- https://cdn.elifesciences.org/articles/83464/elife-83464-fig6-data1-v3.xlsx
Figure 6—figure supplement 1
ASCT2F136Y/I237M anion current is inhibited by UCPH-101 independent of voltage.
Voltage dependence of ASCT2F136Y/I237M anion currents under forward transport conditions, activated using 500 μM extracellular Ser (n=13) (A) or 500 μM Ser in the presence of 200 μM UCPH-101 (B). …
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Figure 6—figure supplement 1—source data 1
Current traces for Figure 6—figure supplement 1A and B, and the original data for panel (C).
- https://cdn.elifesciences.org/articles/83464/elife-83464-fig6-figsupp1-data1-v3.xlsx
Figure 7
The F136Y/I237M ASCT2 double mutation restores complete inhibition of anion current by UCPH-101.
(A) Predicted binding pose of UCPH-101 (cyan sticks) to the F136Y/I237M-double-mutant ASCT2 transporter (the trimerization and transport domains are highlighted in dark green and dark orange, …
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Figure 7—source data 1
Current traces for Figure 7B, and the original data for panels (C-E).
- https://cdn.elifesciences.org/articles/83464/elife-83464-fig7-data1-v3.xlsx
Figure 8
UCPH-101 slows kinetics of alanine serine cysteine transporter 2 (ASCT2) current onset and recovery after amino acid removal.
(A) Anion currents in response to two pulses of rapid serine application (1 mM), with varying inter-pulse interval (pulse protocol shown at the top) under homo-exchange conditions. The intracellular …
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Figure 8—source data 1
Original current traces for Figure 8A,B,C,E,F and G, and the original data for panels (D) and (H).
- https://cdn.elifesciences.org/articles/83464/elife-83464-fig8-data1-v3.xlsx
Figure 9 with 1 supplement
UCPH-101 slows kinetics of ASCT2F136Y/I237M current onset and recovery after amino acid removal.
(A) Anion currents in response to two pulses of rapid serine application (1 mM), with varying inter-pulse interval (pulse protocol shown at the top) under homo-exchange conditions in an ASCT2F136Y/I2…
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Figure 9—source data 1
Original current traces for Figure 9A,B,C,E,F and G, and the original data for panels (D) and (H).
- https://cdn.elifesciences.org/articles/83464/elife-83464-fig9-data1-v3.xlsx
Figure 9—figure supplement 1
UCPH-101 does not eliminate, but reduces capacitive charge movement caused by Na+ binding to the apo-form of ASCT2F136Y and ASCT2F136Y/I237M.
Voltage jumps (protocol shown in top panel) were used to disturb the Na+ binding equilibrium in the apo-form of ASCT2F136Y/I237M (absence of amino acid substrate). (A) and (B) show the transient …
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Figure 9—figure supplement 1—source data 1
Original current traces for Figure 9—figure supplement 1A and B, and the original data for panels (C) and (D).
- https://cdn.elifesciences.org/articles/83464/elife-83464-fig9-figsupp1-data1-v3.xlsx
Figure 10
Novel Cmpd #302, identified by docking analysis, is a partial inhibitor of alanine serine cysteine transporter 2 (ASCT2) transient transport current.
(A) Chemical structure of compound #302. (B) Predicted docking pose of compound #302 in the ASCT2 allosteric binding site. (C) Anion currents in response to two pulses of rapid serine application (1 …
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Figure 10—source data 1
Original current traces for Figure 10C,D,E and J, and the original data for panels (F-I).
- https://cdn.elifesciences.org/articles/83464/elife-83464-fig10-data1-v3.xlsx
Figure 11
Proposed simplified mechanism of inhibitors (U) targeting the allosteric binding site interacting with alanine serine cysteine transporter 2 (ASCT2) and excitatory amino acid transporter 1 (EAAT1) transporters (T).
The amino acid substrate is abbreviated as AA. Potential charges of the cation (Na+) and the transporter/substrate were omitted for simplicity, and only two of the three Na+ binding steps are shown. …
Tables
Table 1
Substrate selectivity of all alanine serine cysteine transporter 2 (ASCT2) mutants.
| Substrate binding affinity (μM) | Ala | Ser | Gln |
|---|---|---|---|
| WT | 290±50 | 280±40 | 190±30 |
| F135Y | 300±60 | 180±10 | 190±50 |
| F136Y | 280±70 | 790±130 | Insensitive |
| I237M | 230±20 | 300±70 | 270±50 |
| F136Y/I237M | 300±130 | 310±100 | Insensitive |
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Table 1—source data 1
Original data for all Km values listed in Table 1.
- https://cdn.elifesciences.org/articles/83464/elife-83464-table1-data1-v3.xlsx
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/83464/elife-83464-mdarchecklist1-v3.pdf
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Source code 1
UCPH-101 parameter file.
- https://cdn.elifesciences.org/articles/83464/elife-83464-code1-v3.zip
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Source code 2
UCPH-101 rtf file.
- https://cdn.elifesciences.org/articles/83464/elife-83464-code2-v3.zip
