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 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: koff values were in good agreement with those derived from uptake experiments; in contrast, the kon 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.
Institute for Biophysics, Johannes Kepler University Linz, Altenbergerstrasse 69, 4040 Linz, Austria.
Linda
L
Wildling
Agilent Technologies, Austria GmbH, Aubrunnerweg 11, 4040 Linz, Austria.
Christian
C
Rankl
Center for Advanced Bioanalysis, Scharitzerstrasse 6-8, 4020 Linz, Austria.
Thomas
T
Haselgrübler
Institute for Biophysics, Johannes Kepler University Linz, Altenbergerstrasse 69, 4040 Linz, Austria.
Hermann J
HJ
Gruber
Center of Physiology and Pharmacology, Medical University of Vienna, Waehringerstrasse 13a, 1090 Vienna, Austria.
Marion
M
Holy
Medicinal Chemistry Section, Molecular Targets and Medications Discovery Branch, Intramural Research Program, National Institute on Drug Abuse, Baltimore, Maryland 21224.
Amy Hauck
AH
Newman
Medicinal Chemistry Section, Molecular Targets and Medications Discovery Branch, Intramural Research Program, National Institute on Drug Abuse, Baltimore, Maryland 21224.
Mu-Fa
MF
Zou
Institute for Biophysics, Johannes Kepler University Linz, Altenbergerstrasse 69, 4040 Linz, Austria.
Rong
R
Zhu
Center of Physiology and Pharmacology, Medical University of Vienna, Waehringerstrasse 13a, 1090 Vienna, Austria.
Michael
M
Freissmuth
Center of Physiology and Pharmacology, Medical University of Vienna, Waehringerstrasse 13a, 1090 Vienna, Austria.
Harald H
HH
Sitte
Institute for Biophysics, Johannes Kepler University Linz, Altenbergerstrasse 69, 4040 Linz, Austria; Center for Advanced Bioanalysis, Scharitzerstrasse 6-8, 4020 Linz, Austria. Electronic address: peter.hinterdorfer@jku.at.
Peter
P
Hinterdorfer
D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008024:Ligands; D018625:Microscopy, Atomic Force; D011485:Protein Binding; D050486:Serotonin Plasma Membrane Transport Proteins / Q000378:metabolism*; D017367:Selective Serotonin Reuptake Inhibitors / Q000378:metabolism; D013816:Thermodynamics; D014326:Tropanes / Q000378:metabolism
D008024:Ligands; D050486:Serotonin Plasma Membrane Transport Proteins; D017367:Selective Serotonin Reuptake Inhibitors; D014326:Tropanes; C434646:2-carbomethoxy-3-(3',4'-dichlorophenyl)tropane