TY - JOUR

T1 - Structural and functional probing of the biogenic amine transporters by fluorescence spectroscopy

AU - Rasmussen, Søren G F

AU - Adkins, Erika M

AU - Carroll, F Ivy

AU - Maresch, Martin J

AU - Gether, Ulrik

PY - 2003/10/31

Y1 - 2003/10/31

N2 - 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.

AB - 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.

KW - Animals

KW - Binding Sites

KW - Biogenic Monoamines

KW - Humans

KW - Membrane Transport Proteins

KW - Molecular Structure

KW - Spectrometry, Fluorescence

M3 - Journal article

C2 - 14612134

VL - 479

SP - 13

EP - 22

JO - European Journal of Pharmacology

JF - European Journal of Pharmacology

SN - 0014-2999

IS - 1-3

ER -