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Probing Binding Pocket of Serotonin Transporter by Single Molecular Force Spectroscopy on Living Cells

The serotonin transporter (SERT) terminates neurotransmission by removing serotonin from the synaptic cleft. In addition, it is the site of action of antidepressants (which block the transporter) and of amphetamines (which induce substrate efflux). The interaction energies involved in binding of suc...

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Autores principales: Wildling, Linda, Rankl, Christian, Haselgrübler, Thomas, Gruber, Hermann J., Holy, Marion, Newman, Amy Hauck, Zou, Mu-Fa, Zhu, Rong, Freissmuth, Michael, Sitte, Harald H., Hinterdorfer, Peter
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Biochemistry and Molecular Biology 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3249061/
https://www.ncbi.nlm.nih.gov/pubmed/22033932
http://dx.doi.org/10.1074/jbc.M111.304873
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author Wildling, Linda
Rankl, Christian
Haselgrübler, Thomas
Gruber, Hermann J.
Holy, Marion
Newman, Amy Hauck
Zou, Mu-Fa
Zhu, Rong
Freissmuth, Michael
Sitte, Harald H.
Hinterdorfer, Peter
author_facet Wildling, Linda
Rankl, Christian
Haselgrübler, Thomas
Gruber, Hermann J.
Holy, Marion
Newman, Amy Hauck
Zou, Mu-Fa
Zhu, Rong
Freissmuth, Michael
Sitte, Harald H.
Hinterdorfer, Peter
author_sort Wildling, Linda
collection PubMed
description The serotonin transporter (SERT) terminates neurotransmission by removing serotonin from the synaptic cleft. In addition, it is the site of action of antidepressants (which block the transporter) and of amphetamines (which induce substrate efflux). The interaction energies involved in binding of such compounds to the transporter are unknown. Here, we used atomic force microscopy (AFM) to probe single molecular interactions between the serotonin transporter and MFZ2-12 (a potent cocaine analog) in living CHOK1 cells. For the AFM measurements, MFZ2-12 was immobilized on AFM tips by using a heterobifunctional cross-linker. By varying the pulling velocity in force distance cycles drug-transporter complexes were ruptured at different force loadings allowing for mapping of the interaction energy landscape. We derived chemical rate constants from these recordings and compared them with those inferred from inhibition of transport and ligand binding: k(off) values were in good agreement with those derived from uptake experiments; in contrast, the k(on) values were scaled down when determined by AFM. Our observations generated new insights into the energy landscape of the interaction between SERT and inhibitors. They thus provide a useful framework for molecular dynamics simulations by exploring the range of forces and energies that operate during the binding reaction.
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spelling pubmed-32490612012-01-04 Probing Binding Pocket of Serotonin Transporter by Single Molecular Force Spectroscopy on Living Cells Wildling, Linda Rankl, Christian Haselgrübler, Thomas Gruber, Hermann J. Holy, Marion Newman, Amy Hauck Zou, Mu-Fa Zhu, Rong Freissmuth, Michael Sitte, Harald H. Hinterdorfer, Peter J Biol Chem Molecular Biophysics The serotonin transporter (SERT) terminates neurotransmission by removing serotonin from the synaptic cleft. In addition, it is the site of action of antidepressants (which block the transporter) and of amphetamines (which induce substrate efflux). The interaction energies involved in binding of such compounds to the transporter are unknown. Here, we used atomic force microscopy (AFM) to probe single molecular interactions between the serotonin transporter and MFZ2-12 (a potent cocaine analog) in living CHOK1 cells. For the AFM measurements, MFZ2-12 was immobilized on AFM tips by using a heterobifunctional cross-linker. By varying the pulling velocity in force distance cycles drug-transporter complexes were ruptured at different force loadings allowing for mapping of the interaction energy landscape. We derived chemical rate constants from these recordings and compared them with those inferred from inhibition of transport and ligand binding: k(off) values were in good agreement with those derived from uptake experiments; in contrast, the k(on) values were scaled down when determined by AFM. Our observations generated new insights into the energy landscape of the interaction between SERT and inhibitors. They thus provide a useful framework for molecular dynamics simulations by exploring the range of forces and energies that operate during the binding reaction. American Society for Biochemistry and Molecular Biology 2012-01-02 2011-10-27 /pmc/articles/PMC3249061/ /pubmed/22033932 http://dx.doi.org/10.1074/jbc.M111.304873 Text en © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Author's Choice—Final version full access. Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) applies to Author Choice Articles
spellingShingle Molecular Biophysics
Wildling, Linda
Rankl, Christian
Haselgrübler, Thomas
Gruber, Hermann J.
Holy, Marion
Newman, Amy Hauck
Zou, Mu-Fa
Zhu, Rong
Freissmuth, Michael
Sitte, Harald H.
Hinterdorfer, Peter
Probing Binding Pocket of Serotonin Transporter by Single Molecular Force Spectroscopy on Living Cells
title Probing Binding Pocket of Serotonin Transporter by Single Molecular Force Spectroscopy on Living Cells
title_full Probing Binding Pocket of Serotonin Transporter by Single Molecular Force Spectroscopy on Living Cells
title_fullStr Probing Binding Pocket of Serotonin Transporter by Single Molecular Force Spectroscopy on Living Cells
title_full_unstemmed Probing Binding Pocket of Serotonin Transporter by Single Molecular Force Spectroscopy on Living Cells
title_short Probing Binding Pocket of Serotonin Transporter by Single Molecular Force Spectroscopy on Living Cells
title_sort probing binding pocket of serotonin transporter by single molecular force spectroscopy on living cells
topic Molecular Biophysics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3249061/
https://www.ncbi.nlm.nih.gov/pubmed/22033932
http://dx.doi.org/10.1074/jbc.M111.304873
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