title: Nanopharmacological Force Sensing to Reveal Allosteric Coupling in Transporter Binding Sites.
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.
authors.affiliation: Institute for Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020, Linz, Austria.
authors.forename: Rong
authors.initials: R
authors.lastname: Zhu
authors.affiliation: Institute for Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020, Linz, Austria.|Christian Doppler Laboratory for Nanoscopic Methods in Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020, Linz, Austria.
authors.forename: Doris
authors.initials: D
authors.lastname: Sinwel
authors.affiliation: Center of Physiology and Pharmacology, Medical University of Vienna, Waehringerstrasse 13a, 1090, Vienna, Austria.
authors.forename: Peter S
authors.initials: PS
authors.lastname: Hasenhuetl
authors.affiliation: Center of Physiology and Pharmacology, Medical University of Vienna, Waehringerstrasse 13a, 1090, Vienna, Austria.
authors.forename: Kusumika
authors.initials: K
authors.lastname: Saha
authors.affiliation: Medicinal Chemistry Section, Molecular Targets and Medications Discovery Branch, Intramural Research Program, National Institute on Drug Abuse, Baltimore, MD, 21224, USA.
authors.forename: Vivek
authors.initials: V
authors.lastname: Kumar
authors.affiliation: Medicinal Chemistry Section, Molecular Targets and Medications Discovery Branch, Intramural Research Program, National Institute on Drug Abuse, Baltimore, MD, 21224, USA.
authors.forename: Peng
authors.initials: P
authors.lastname: Zhang
authors.affiliation: Keysight Technologies Austria GmbH, Mooslackengasse 17, 1190, Vienna, Austria.
authors.forename: Christian
authors.initials: C
authors.lastname: Rankl
authors.affiliation: Center of Physiology and Pharmacology, Medical University of Vienna, Waehringerstrasse 13a, 1090, Vienna, Austria.
authors.forename: Marion
authors.initials: M
authors.lastname: Holy
authors.affiliation: Center of Physiology and Pharmacology, Medical University of Vienna, Waehringerstrasse 13a, 1090, Vienna, Austria.
authors.forename: Sonja
authors.initials: S
authors.lastname: Sucic
authors.affiliation: Center of Physiology and Pharmacology, Medical University of Vienna, Waehringerstrasse 13a, 1090, Vienna, Austria.
authors.forename: Oliver
authors.initials: O
authors.lastname: Kudlacek
authors.affiliation: Institute for Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020, Linz, Austria.|Center for Advanced Bioanalysis, Gruberstrasse 40, 4020, Linz, Austria.
authors.forename: Andreas
authors.initials: A
authors.lastname: Karner
authors.affiliation: Center of Physiology and Pharmacology, Medical University of Vienna, Waehringerstrasse 13a, 1090, Vienna, Austria.
authors.forename: Walter
authors.initials: W
authors.lastname: Sandtner
authors.affiliation: Center of Physiology and Pharmacology, Medical University of Vienna, Waehringerstrasse 13a, 1090, Vienna, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Stockner
authors.affiliation: Institute for Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020, Linz, Austria.
authors.forename: Hermann J
authors.initials: HJ
authors.lastname: Gruber
authors.affiliation: Center of Physiology and Pharmacology, Medical University of Vienna, Waehringerstrasse 13a, 1090, Vienna, Austria.
authors.forename: Michael
authors.initials: M
authors.lastname: Freissmuth
authors.affiliation: Medicinal Chemistry Section, Molecular Targets and Medications Discovery Branch, Intramural Research Program, National Institute on Drug Abuse, Baltimore, MD, 21224, USA.
authors.forename: Amy Hauck
authors.initials: AH
authors.lastname: Newman
authors.affiliation: Center of Physiology and Pharmacology, Medical University of Vienna, Waehringerstrasse 13a, 1090, Vienna, Austria.
authors.forename: Harald H
authors.initials: HH
authors.lastname: Sitte
authors.affiliation: Institute for Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020, Linz, Austria. peter.hinterdorfer@jku.at.|Christian Doppler Laboratory for Nanoscopic Methods in Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020, Linz, Austria. peter.hinterdorfer@jku.at.|Center for Advanced Bioanalysis, Gruberstrasse 40, 4020, Linz, Austria. peter.hinterdorfer@jku.at.
authors.forename: Peter
authors.initials: P
authors.lastname: Hinterdorfer
mesh_terms: D000494:Allosteric Regulation; D001665:Binding Sites; D018360:Crystallography, X-Ray; D036103:Nanotechnology; D050486:Serotonin Plasma Membrane Transport Proteins / Q000378:metabolism*
chemical_list: D050486:Serotonin Plasma Membrane Transport Proteins
keywords: allostery; binding sites; citalopram; nanopharmacology; serotonin transporter