Revealing the location and dynamics of a concealed binding site in the dopamine transporter
Abstract The dopamine transporter (DAT) is linked to neuropsychiatric disorders including ADHD, Parkinson’s disease, and substance use disorders. Accordingly, DAT is the target of illicit drugs and clinically important medicines. However, the number and function of ligand binding sites in DAT is eni...
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Nature Portfolio
2025-05-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-025-59511-w |
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| author | Rong Zhu Walter Sandtner Thomas Stockner Alexander Heilinger Marion Holy Oliver Kudlacek Linda Wildling Kusumika Saha Anna Sophie Fröhlich Michael Bindl Paraskevi Tziortzouda Anna Haider Julia Gobl Saanfor Hubert Suh Jawad Akbar Khan Julia Bicher Nina Kastner Andreas Ebner Hermann J. Gruber Michael Freissmuth Amy Hauck Newman Harald H. Sitte Peter Hinterdorfer |
| author_facet | Rong Zhu Walter Sandtner Thomas Stockner Alexander Heilinger Marion Holy Oliver Kudlacek Linda Wildling Kusumika Saha Anna Sophie Fröhlich Michael Bindl Paraskevi Tziortzouda Anna Haider Julia Gobl Saanfor Hubert Suh Jawad Akbar Khan Julia Bicher Nina Kastner Andreas Ebner Hermann J. Gruber Michael Freissmuth Amy Hauck Newman Harald H. Sitte Peter Hinterdorfer |
| author_sort | Rong Zhu |
| collection | DOAJ |
| description | Abstract The dopamine transporter (DAT) is linked to neuropsychiatric disorders including ADHD, Parkinson’s disease, and substance use disorders. Accordingly, DAT is the target of illicit drugs and clinically important medicines. However, the number and function of ligand binding sites in DAT is enigmatic due to conflicting data from available structures and molecular pharmacology. Herein, we design force sensors with DAT ligands and measure their interaction forces with wild-type and mutated DATs, from which two distinct populations of unbinding strengths and off-rates are detected. The high-force population is reduced by V152I and S422A mutations, or by substituting Na+ with K+ or NMDG+. In contrast, several modifications including mutation G386H, acetylation of K92 and K384, mutation K92A, mutation K384A, or protonation of H477 decrease the low-force population. The present data delineate the threshold of binding strength, which may account for certain ligand binding sites to be imperceptible in crystal or cryo-EM structures. Furthermore, the force spectra provide the information on the position and kinetic rates of a herein detected ligand binding site in DAT. |
| format | Article |
| id | doaj-art-e6ab0b9acc66437e8d5968bfc4ab8749 |
| institution | OA Journals |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-e6ab0b9acc66437e8d5968bfc4ab87492025-08-20T01:49:39ZengNature PortfolioNature Communications2041-17232025-05-0116111210.1038/s41467-025-59511-wRevealing the location and dynamics of a concealed binding site in the dopamine transporterRong Zhu0Walter Sandtner1Thomas Stockner2Alexander Heilinger3Marion Holy4Oliver Kudlacek5Linda Wildling6Kusumika Saha7Anna Sophie Fröhlich8Michael Bindl9Paraskevi Tziortzouda10Anna Haider11Julia Gobl12Saanfor Hubert Suh13Jawad Akbar Khan14Julia Bicher15Nina Kastner16Andreas Ebner17Hermann J. Gruber18Michael Freissmuth19Amy Hauck Newman20Harald H. Sitte21Peter Hinterdorfer22Institute of Biophysics, Johannes Kepler University LinzInstitute of Pharmacology, Center of Physiology and Pharmacology, Medical University of ViennaInstitute of Pharmacology, Center of Physiology and Pharmacology, Medical University of ViennaInstitute of Biophysics, Johannes Kepler University LinzInstitute of Pharmacology, Center of Physiology and Pharmacology, Medical University of ViennaInstitute of Pharmacology, Center of Physiology and Pharmacology, Medical University of ViennaInstitute of Biophysics, Johannes Kepler University LinzInstitute of Pharmacology, Center of Physiology and Pharmacology, Medical University of ViennaInstitute of Biophysics, Johannes Kepler University LinzInstitute of Biophysics, Johannes Kepler University LinzInstitute of Biophysics, Johannes Kepler University LinzInstitute of Biophysics, Johannes Kepler University LinzInstitute of Biophysics, Johannes Kepler University LinzInstitute of Biophysics, Johannes Kepler University LinzInstitute of Pharmacology, Center of Physiology and Pharmacology, Medical University of ViennaInstitute of Pharmacology, Center of Physiology and Pharmacology, Medical University of ViennaInstitute of Pharmacology, Center of Physiology and Pharmacology, Medical University of ViennaInstitute of Biophysics, Johannes Kepler University LinzInstitute of Biophysics, Johannes Kepler University LinzInstitute of Pharmacology, Center of Physiology and Pharmacology, Medical University of ViennaMedicinal Chemistry Section, Molecular Targets and Medications Discovery Branch, National Institute on Drug Abuse-Intramural Research ProgramInstitute of Pharmacology, Center of Physiology and Pharmacology, Medical University of ViennaInstitute of Biophysics, Johannes Kepler University LinzAbstract The dopamine transporter (DAT) is linked to neuropsychiatric disorders including ADHD, Parkinson’s disease, and substance use disorders. Accordingly, DAT is the target of illicit drugs and clinically important medicines. However, the number and function of ligand binding sites in DAT is enigmatic due to conflicting data from available structures and molecular pharmacology. Herein, we design force sensors with DAT ligands and measure their interaction forces with wild-type and mutated DATs, from which two distinct populations of unbinding strengths and off-rates are detected. The high-force population is reduced by V152I and S422A mutations, or by substituting Na+ with K+ or NMDG+. In contrast, several modifications including mutation G386H, acetylation of K92 and K384, mutation K92A, mutation K384A, or protonation of H477 decrease the low-force population. The present data delineate the threshold of binding strength, which may account for certain ligand binding sites to be imperceptible in crystal or cryo-EM structures. Furthermore, the force spectra provide the information on the position and kinetic rates of a herein detected ligand binding site in DAT.https://doi.org/10.1038/s41467-025-59511-w |
| spellingShingle | Rong Zhu Walter Sandtner Thomas Stockner Alexander Heilinger Marion Holy Oliver Kudlacek Linda Wildling Kusumika Saha Anna Sophie Fröhlich Michael Bindl Paraskevi Tziortzouda Anna Haider Julia Gobl Saanfor Hubert Suh Jawad Akbar Khan Julia Bicher Nina Kastner Andreas Ebner Hermann J. Gruber Michael Freissmuth Amy Hauck Newman Harald H. Sitte Peter Hinterdorfer Revealing the location and dynamics of a concealed binding site in the dopamine transporter Nature Communications |
| title | Revealing the location and dynamics of a concealed binding site in the dopamine transporter |
| title_full | Revealing the location and dynamics of a concealed binding site in the dopamine transporter |
| title_fullStr | Revealing the location and dynamics of a concealed binding site in the dopamine transporter |
| title_full_unstemmed | Revealing the location and dynamics of a concealed binding site in the dopamine transporter |
| title_short | Revealing the location and dynamics of a concealed binding site in the dopamine transporter |
| title_sort | revealing the location and dynamics of a concealed binding site in the dopamine transporter |
| url | https://doi.org/10.1038/s41467-025-59511-w |
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