Structural and functional probing of the biogenic amine transporters by fluorescence spectroscopy.
Fluorescence spectroscopy techniques have proven extremely powerful for probing the molecular structure and function of membrane proteins. In this review, it will be described how we have applied a series of these techniques to the biogenic amine transporters, which are responsible for the clearance of dopamine, norepinephrine, and serotonin from the synaptic cleft. In our studies, we have focused on the serotonin transporter (SERT) for which we have established a purification procedure upon expression of the transporter in Sf-9 insect cells. Importantly, the purified transporter displays pharmacological properties in detergent micelles similar to that observed in membranes suggesting that the overall tertiary structure is preserved upon purification. Using this purified SERT preparation and the fluorescent cocaine analogue RTI-233 as a molecular reporter, we have been able to characterize the microenvironment of the cocaine-binding pocket. In current follow-up studies, we are attempting to map the relative position of this binding pocket using fluorescence resonance energy transfer (FRET) between RTI-233 and an acceptor fluorophore covalently attached to endogenous cysteines in the transporter. Finally, it will be described how we recently initiated the implementation of single-molecule confocal fluorescence spectroscopy techniques in our studies of the SERT.
Rasmussen
Søren G F
SG
The Molecular Neuropharmacology Group, Department of Pharmacology 18-6, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark.
Adkins
Erika M
EM
Carroll
F Ivy
FI
Maresch
Martin J
MJ
Gether
Ulrik
U
D000818:Animals; D001665:Binding Sites / Q000502:physiology; D015306:Biogenic Monoamines / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D015394:Molecular Structure; D013050:Spectrometry, Fluorescence / Q000379:methods
D015306:Biogenic Monoamines; D026901:Membrane Transport Proteins