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Nanopharmacological Force Sensing to Reveal Allosteric Coupling in Transporter Binding Sites
Author(s) -
Zhu Rong,
Sinwel Doris,
Hasenhuetl Peter S.,
Saha Kusumika,
Kumar Vivek,
Zhang Peng,
Rankl Christian,
Holy Marion,
Sucic Sonja,
Kudlacek Oliver,
Karner Andreas,
Sandtner Walter,
Stockner Thomas,
Gruber Hermann J.,
Freissmuth Michael,
Newman Amy Hauck,
Sitte Harald H.,
Hinterdorfer Peter
Publication year - 2016
Publication title -
angewandte chemie international edition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.831
H-Index - 550
eISSN - 1521-3773
pISSN - 1433-7851
DOI - 10.1002/anie.201508755
Subject(s) - allosteric regulation , transporter , coupling (piping) , biophysics , chemistry , biology , biochemistry , materials science , receptor , gene , metallurgy
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.