Exploring the K+ binding site and its coupling to transport in the neurotransmitter:sodium symporter LeuT
- Laboratory for Membrane Protein Dynamics, Department of Neuroscience, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark
- Membrane Transport Biophysics Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, United States
Figures
Figure 1 with 1 supplement
K+ competitively inhibits Na+-mediated [3H]leucine binding in LeuT.
(A) Na+-mediated [3H]leucine (10× Kd) binding to purified LeuT in the absence (red triangles) or presence of 200 mM (light blue diamond) or 800 mM (blue squares) K+. Data are normalized to Bmax and …
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Figure 1—source data 1
Excel file containing data for (A) Na+-mediated [3H]leucine binding to purified LeuT in the absence of K+, and (B) K+-dependent displacement of Na+-mediated [3H]leucine binding.
- https://cdn.elifesciences.org/articles/87985/elife-87985-fig1-data1-v1.zip
Figure 1—figure supplement 1
K+ inhibition is preserved for LeuT in low ionic strength.
(A) Displacement of Na+-mediated [3H]leucine (10× Kd) binding to LeuT by 0–200 mM K+, using Ch+ (red squares) or N-methyl-D-glucamine (NMDG+) (blue triangles) as the counter ion. Displacement with …
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Figure 1—figure supplement 1—source data 1
Excel file containing data for displacement of Na+-mediated [3H]leucine binding to LeuT by K+, using Ch+ or N-methyl-D-glucamine (NMDG+) as the counter ion.
- https://cdn.elifesciences.org/articles/87985/elife-87985-fig1-figsupp1-data1-v1.zip
Figure 2 with 1 supplement
K+ shifts the conformational equilibrium toward an outward-closed state.
(A) Left: cartoon representation of the superimposed structures of LeuT in the outward-open Na+-bound state (orange; PDB-ID 3TT1) and inward-open state (turquoise; PDB-ID 3TT3), viewed parallel to …
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Figure 2—source data 1
Excel file containing data for Figure 2B and C providing transition metal ion FRET (tmFRET) efficiencies obtained for by the added ions in 10 mM Ni2+.
- https://cdn.elifesciences.org/articles/87985/elife-87985-fig2-data1-v1.zip
Figure 2—figure supplement 1
Transition metal ion FRET (tmFRET) principle and characterization of LeuTtmFRET.
(A) Theoretical FRET efficiencies (1 – F/Fno site) (left axis), converted to distances by the FRET equation (right axis), as a function of the relative population of the outward-closed (OC) state. …
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Figure 2—figure supplement 1—source data 1
Excel file containing data for transition metal ion FRET (tmFRET) efficiencies as (B) a function of increasing Ni2+ in ions, (C) for Rb+, and (D) background FRET signal.
- https://cdn.elifesciences.org/articles/87985/elife-87985-fig2-figsupp1-data1-v1.zip
Figure 3 with 1 supplement
K+ modulates LeuT-mediated [3H]alanine transport.
(A) Schematic of LeuT reconstituted into liposome containing buffer with various cations (X) and Cl- as corresponding anion. (B) Time-dependent [3H]alanine (2 µM) uptake in the presence of 200 mM Na+…
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Figure 3—source data 1
Word file containing data table for rate constants for time-dependent [3H]alanine transport by LeuT into PLs.
- https://cdn.elifesciences.org/articles/87985/elife-87985-fig3-data1-v1.docx
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Figure 3—source data 2
Word file containing data table for Vmax and Km of [3H]alanine transport by LeuT into PLs.
- https://cdn.elifesciences.org/articles/87985/elife-87985-fig3-data2-v1.docx
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Figure 3—source data 3
Word file containing data table for Vmax and Km of [3H]alanine transport as an effect by various K+ concentrations.
- https://cdn.elifesciences.org/articles/87985/elife-87985-fig3-data3-v1.docx
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Figure 3—source data 4
Excel file containing data for [3H]alanine uptake by LeuT reconstituted into PLs.
- https://cdn.elifesciences.org/articles/87985/elife-87985-fig3-data4-v1.xlsx
Figure 3—figure supplement 1
[3H]alanine uptake into proteoliposomes.
(A) Binding of a saturating concentration (20× Kd) of [3H]leucine to LeuT solubilized from the proteoliposomes. The amount of binding in counts per minute (c.p.m.) was taken as a measure of the …
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Figure 3—figure supplement 1—source data 1
Excel file containing data for [3H]alanine uptake into PLs with varying K+ or Na+ concentrations.
- https://cdn.elifesciences.org/articles/87985/elife-87985-fig3-figsupp1-data1-v1.zip
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Figure 3—figure supplement 1—source data 2
Files of original uncropped sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel shown in Figure 3—figure supplement 1D.
- https://cdn.elifesciences.org/articles/87985/elife-87985-fig3-figsupp1-data2-v1.zip
Figure 4
Na1 site mutants of LeuT display altered ion selectivity.
(A) Cartoon representation of the Na1 site from the outward-occluded LeuT structure (PDB ID: 2A65) with the endogenous Na1 site coordinating residues along with the substrate leucine (yellow) and …
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Figure 4—source data 1
Excel file containing data for Na+-mediated [3H]leucine binding for Na1 site mutations and how that is inhibited by K+.
- https://cdn.elifesciences.org/articles/87985/elife-87985-fig4-data1-v1.zip
Figure 5
Na+-mediated [3H]leucine binding and inhibition by K+ by the LeuT mutants.
(A–F) Na+-mediated [3H]leucine (10× Kd) binding for the Na1 site mutants performed in the absence (red triangles) and presence of 200 mM (light blue diamond) and 800 (blue squares) mM K+. (A) A22V, …
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Figure 5—source data 1
Excel file containing data for Na+-mediated [3H]leucine binding for the Na1 site mutants and in the presence of K+.
- https://cdn.elifesciences.org/articles/87985/elife-87985-fig5-data1-v1.zip
Figure 6 with 1 supplement
The Na1 site LeuT mutants exhibit different conformational equilibria.
(A–E) Transition metal ion FRET (tmFRET) efficiencies (1 – F/Fno site) for N286QtmFRET (orange, A), N27QtmFRET (red, B), A22VtmFRET (green, C), A22StmFRET (purple, D), and T254StmFRET (blue, E) …
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Figure 6—source data 1
Excel file containing data for transition metal ion FRET (tmFRET) efficiencies for all mutants in N-methyl-D-glucamine (NMDG+), Na+, leucine, or K+.
- https://cdn.elifesciences.org/articles/87985/elife-87985-fig6-data1-v1.zip
Figure 6—figure supplement 1
[3H]alanine uptake by selected Na1 mutants.
(A) [3H]alanine activity over time by LeuT A22V in the presence of 200 mM Na+. LeuT A22V is reconstituted into proteoliposomes with either 200 mM Na+ (open diamond), N-methyl-D-glucamine (NMDG+) …
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Figure 6—figure supplement 1—source data 1
Excel file containing data for [3H]alanine transport activity over time by selected Na1 mutants.
- https://cdn.elifesciences.org/articles/87985/elife-87985-fig6-figsupp1-data1-v1.zip
Figure 7
Proposed role of K+ in the translocation cycle.
Here, exemplified for alanine (Ala) as substrate. LeuT apo-form will likely reside in an equilibrium between its outward- (Tout) and inward-facing (Tin) conformation. After Na+ and Ala are bound (1) …
Author response image 1
Tables
Table 1
LeuT mutant affinities for leucine and alanine.
| Leucine affinityKd (nM) | n | Alanine affinityKi (µM) | n | |
|---|---|---|---|---|
| WT | 6.9 [6.7; 7.2]A | 4 | 1.61 [1.58; 1.65] | 4 |
| A22V | 5.4 [4.9; 5.9]A | 3 | 1.02 [0.97; 1.08] | 4 |
| A22S | 8.4 [7.7; 9.1]A | 3 | 2.11 [2.05; 2.17] | 4 |
| N27Q | 1590 [1530; 1650] | 3 | 255 [248; 263] | 3 |
| T254S | 5.6 [5.4; 5.8]A | 3 | 1.17 [1.12; 1.21] | 3 |
| N286Q | 1460 [1390; 1540] | 3 | 287 [277; 297] | 3 |
| E290Q | 193 [169; 221] | 4 | 30.0 [28.9; 31.2] | 3 |
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Table 1—source data 1
Pdf file with affinity curves for leucine and alanine binding to LeuT and mutants.
- https://cdn.elifesciences.org/articles/87985/elife-87985-table1-data1-v1.docx
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Table 1—source data 2
Excel file containing data for leucine and alanine affinities for LeuT WT and all investigated mutants.
- https://cdn.elifesciences.org/articles/87985/elife-87985-table1-data2-v1.xlsx
Table 2
Na+-mediated [3H]leucine binding and the effect of K+ addition, and K+-dependent Na+/[3H]leucine displacement in Na1 site mutants.
For LeuT Na1 site mutants (and K398C), Na+-mediated [3H]leucine binding was assayed using 10× Kd of [3H]leucine in the presence of 0, 200, and 800 mM K+. For K+-dependent displacement of Na+-mediated…
| EC50 Na+ (mM) | IC50 K+ (mM) | EC50 Na++200 mM K+ (mM) | EC50 Na++800 mM K+ (mM) | EC50800/EC50 | |
|---|---|---|---|---|---|
| WT | 7.7 [7.3; 8.1] | 235 [228; 241] | 19.4 [19.1; 19.8] | 48.5 [47.5; 49.5] | ~6 |
| A22V | 2.0 [2.0; 2.1] | 49.0 [47.7; 50.4] | 23.0 [21.7; 24.5] | 70.7 [69.2; 72.2] | ~35 |
| A22S | 53.3 [45.5; 62.5] | >1900 | 72.4 [62.3; 84.2] | 117 [100; 137] | ~2 |
| N27Q | 2.4 [2.3; 2.5] | >2,300 | n.d. | 4.4 [4.0; 4.8] | ~2 |
| T254S | 15.1 [14.2; 16.0] | 219 [209; 229] | 43.5 [37.4; 50.5] | 90.2 [73.8; 110] | ~6 |
| N286Q | 54.6 [47.5; 62.6] | >2000 | n.d. | 121 [111; 133] | ~2 |
| E290Q | 9.6 [8.8; 10.4] | n.d. | n.d. | 144 [132; 157] | ~15 |
| K398C | 7.7 [6.8; 8.8] | n.d | n.d. | 51.5 [48.1; 55.1] | ~7 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Aquifex aeolicus) | LeuT gene | https://doi.org/10.1038/ncomms12755 | ||
| Strain, strain background (Escherichia coli) | BL21(DE3) | Sigma-Aldrich | CMC0016 | Electrocompetent cells |
| Strain, strain protein expression (Escherichia coli) | C41(DE3) | Sigma-Aldrich | CMC0017 | Cells for protein expression |
| Recombinant DNA reagent | LeuT; LeuT WT (plasmid) | https://doi.org/10.1038/ncomms12755 | pET16b backbone | |
| Recombinant DNA reagent | LeuTtmFRET; LeuT A313H-A317H-K398C (plasmid) | https://doi.org/10.1038/ncomms12755 | pET16b backbone | |
| Recombinant DNA reagent | LeuTK398C; LeuT K398C (plasmid) | https://doi.org/10.1038/ncomms12755 | pET16b backbone | |
| Sequence-based reagent | Primers for generation of LeuT mutants | This paper | See list of primers in Appendix. Primers were synthesized by Eurofins Genomics | |
| Chemical compound, drug | fluorescein-5-maleimide; F5M | Thermo Fischer Scientific | F150 | |
| Chemical compound, drug | [3H]leucine | PerkinElmer | NET1166001 | |
| Chemical compound, drug | E. coli polar lipid extract | Avanti Polar Lipids | 100600 C | |
| Chemical compound, drug | [3H]alanine | Moravek Biochemicals | Made on request | |
| Chemical compound, drug | Gel stain InstantBlue | abcam | Ab119211 | |
| Software, algorithm | GraphPad Prism 9.0 | GraphPad Software, Boston, Massachusetts USA | ||
| Other | Yttrium Silicate Copper (YSi-Cu) His-tag SPA beads; YSi-Cu His-tag SPA beads | PerkinElmer | RPNQ0096 | SPA beads used for scintillation proximity assays - See: Pharmacological characterization of LeuT mutants. In Materials & Methods Section |
| Other | thrombin | Cytiva | 27084601 | Protease used to cleave the His-tag from LeuT |
| Other | Filtermat B – GF/B | PerkinElmer | 1450–521 | Filters used to trap proteoliposomes for scintillation counting of [3 H]ligand |
| Other | MeltiLex B/HS | PerkinElmer | 1450–442 | Scintillation plates to add on Filtermats for [3 H] counting |
| Other | Nuclepore Track-Etch Membrane polycarbonate filter of pore size 400 nm | Sigma-Aldrich | WHA10417104 | For extrusion of PLs. |
| Other | SM-2 Bio-Beads | Bio-Rad Laboratories | 1523920 | For detergent extraction from the specimen to promote reconstitution of LeuT into PLs |
