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Allosterically Linked Binding Sites in Serotonin Transporter Revealed by Single Molecule Force Spectroscopy

Crystal structures and experiments relying on the tools of molecular pharmacology reported conflicting results on ligand binding sites in neurotransmitter/sodium symporters (NSS). We explored the number and functionality of ligand binding sites of NSS in a physiological setting by designing novel to...

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Autores principales: Zhu, Rong, Sandtner, Walter, Ahiable, Joan E. A., Newman, Amy Hauck, Freissmuth, Michael, Sitte, Harald H., Hinterdorfer, Peter
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7325972/
https://www.ncbi.nlm.nih.gov/pubmed/32656227
http://dx.doi.org/10.3389/fmolb.2020.00099
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author Zhu, Rong
Sandtner, Walter
Ahiable, Joan E. A.
Newman, Amy Hauck
Freissmuth, Michael
Sitte, Harald H.
Hinterdorfer, Peter
author_facet Zhu, Rong
Sandtner, Walter
Ahiable, Joan E. A.
Newman, Amy Hauck
Freissmuth, Michael
Sitte, Harald H.
Hinterdorfer, Peter
author_sort Zhu, Rong
collection PubMed
description Crystal structures and experiments relying on the tools of molecular pharmacology reported conflicting results on ligand binding sites in neurotransmitter/sodium symporters (NSS). We explored the number and functionality of ligand binding sites of NSS in a physiological setting by designing novel tools for atomic force microscopy (AFM). These allow for directly measuring the interaction forces between the serotonin transporter (SERT) and the antidepressant S-citalopram (S-CIT) on the single molecule level: the AFM cantilever tips were functionalized with S-CIT via a flexible polyethylene glycol (PEG) linker. The tip chemistry was validated by specific force measurements and recognition imaging on CHO cells. Two distinct populations of characteristic binding strengths of S-CIT binding to SERT were revealed in Na(+)-containing buffer. In contrast, in Li(+)-containing buffer, SERT showed only low force interactions. Conversely, the vestibular mutant SERT-G402H merely displayed the high force population. These observations provide physical evidence for the existence of two binding sites in SERT. The dissociation rate constant of both binding sites was extracted by varying the dynamics of the force-probing experiments. Competition experiments revealed that the two sites are allosterically coupled and exert reciprocal modulation.
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spelling pubmed-73259722020-07-09 Allosterically Linked Binding Sites in Serotonin Transporter Revealed by Single Molecule Force Spectroscopy Zhu, Rong Sandtner, Walter Ahiable, Joan E. A. Newman, Amy Hauck Freissmuth, Michael Sitte, Harald H. Hinterdorfer, Peter Front Mol Biosci Molecular Biosciences Crystal structures and experiments relying on the tools of molecular pharmacology reported conflicting results on ligand binding sites in neurotransmitter/sodium symporters (NSS). We explored the number and functionality of ligand binding sites of NSS in a physiological setting by designing novel tools for atomic force microscopy (AFM). These allow for directly measuring the interaction forces between the serotonin transporter (SERT) and the antidepressant S-citalopram (S-CIT) on the single molecule level: the AFM cantilever tips were functionalized with S-CIT via a flexible polyethylene glycol (PEG) linker. The tip chemistry was validated by specific force measurements and recognition imaging on CHO cells. Two distinct populations of characteristic binding strengths of S-CIT binding to SERT were revealed in Na(+)-containing buffer. In contrast, in Li(+)-containing buffer, SERT showed only low force interactions. Conversely, the vestibular mutant SERT-G402H merely displayed the high force population. These observations provide physical evidence for the existence of two binding sites in SERT. The dissociation rate constant of both binding sites was extracted by varying the dynamics of the force-probing experiments. Competition experiments revealed that the two sites are allosterically coupled and exert reciprocal modulation. Frontiers Media S.A. 2020-06-03 /pmc/articles/PMC7325972/ /pubmed/32656227 http://dx.doi.org/10.3389/fmolb.2020.00099 Text en Copyright © 2020 Zhu, Sandtner, Ahiable, Newman, Freissmuth, Sitte and Hinterdorfer. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Molecular Biosciences
Zhu, Rong
Sandtner, Walter
Ahiable, Joan E. A.
Newman, Amy Hauck
Freissmuth, Michael
Sitte, Harald H.
Hinterdorfer, Peter
Allosterically Linked Binding Sites in Serotonin Transporter Revealed by Single Molecule Force Spectroscopy
title Allosterically Linked Binding Sites in Serotonin Transporter Revealed by Single Molecule Force Spectroscopy
title_full Allosterically Linked Binding Sites in Serotonin Transporter Revealed by Single Molecule Force Spectroscopy
title_fullStr Allosterically Linked Binding Sites in Serotonin Transporter Revealed by Single Molecule Force Spectroscopy
title_full_unstemmed Allosterically Linked Binding Sites in Serotonin Transporter Revealed by Single Molecule Force Spectroscopy
title_short Allosterically Linked Binding Sites in Serotonin Transporter Revealed by Single Molecule Force Spectroscopy
title_sort allosterically linked binding sites in serotonin transporter revealed by single molecule force spectroscopy
topic Molecular Biosciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7325972/
https://www.ncbi.nlm.nih.gov/pubmed/32656227
http://dx.doi.org/10.3389/fmolb.2020.00099
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