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Angew Chem Int Ed Engl. 2016 Jan 26;55(5):1719-22. doi: 10.1002/anie.201508755. Epub 2015 Dec 22.

Nanopharmacological Force Sensing to Reveal Allosteric Coupling in Transporter Binding Sites.

Author information

1
Institute for Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020, Linz, Austria.
2
Christian Doppler Laboratory for Nanoscopic Methods in Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020, Linz, Austria.
3
Center of Physiology and Pharmacology, Medical University of Vienna, Waehringerstrasse 13a, 1090, Vienna, Austria.
4
Medicinal Chemistry Section, Molecular Targets and Medications Discovery Branch, Intramural Research Program, National Institute on Drug Abuse, Baltimore, MD, 21224, USA.
5
Keysight Technologies Austria GmbH, Mooslackengasse 17, 1190, Vienna, Austria.
6
Center for Advanced Bioanalysis, Gruberstrasse 40, 4020, Linz, Austria.
7
Institute for Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020, Linz, Austria. peter.hinterdorfer@jku.at.
8
Christian Doppler Laboratory for Nanoscopic Methods in Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020, Linz, Austria. peter.hinterdorfer@jku.at.
9
Center for Advanced Bioanalysis, Gruberstrasse 40, 4020, Linz, Austria. peter.hinterdorfer@jku.at.

Abstract

Controversy regarding the number and function of ligand binding sites in neurotransmitter/sodium symporters arose from conflicting data in crystal structures and molecular pharmacology. Here, we have designed novel tools for atomic force microscopy that directly measure the interaction forces between the serotonin transporter (SERT) and the S- and R-enantiomers of citalopram on the single molecule level. This approach is based on force spectroscopy, which allows for the extraction of dynamic information under physiological conditions thus inaccessible via X-ray crystallography. Two distinct populations of characteristic binding strengths of citalopram 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 when accessed in a physiological context. Competition experiments revealed that these two sites are allosterically coupled and exert reciprocal modulation.

KEYWORDS:

allostery; binding sites; citalopram; nanopharmacology; serotonin transporter

PMID:
26695726
PMCID:
PMC4932834
DOI:
10.1002/anie.201508755
[Indexed for MEDLINE]
Free PMC Article

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