Probing the binding pocket of the serotonin transporter by single molecular force spectroscopy on living cells
Sprache des Titels:
Englisch
Original Kurzfassung:
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
crosslinker. 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.
Forschungs-