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Originally published In Press as doi:10.1074/jbc.M209824200 on December 23, 2002

J. Biol. Chem., Vol. 278, Issue 11, 9768-9777, March 14, 2003
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Binding and Transport in Norepinephrine Transporters
REAL-TIME, SPATIALLY RESOLVED ANALYSIS IN SINGLE CELLS USING A FLUORESCENT SUBSTRATE*

Joel W. Schwartz, Randy D. Blakely, and Louis J. DeFeliceDagger

From the Department of Pharmacology, Center for Molecular Neuroscience, Vanderbilt University Medical Center, Nashville, Tennessee 37232-8548

Monoamine transporters, the molecular targets for drugs of abuse and antidepressants, clear norepinephrine, dopamine, or serotonin from the synaptic cleft. Neurotransmitters, amphetamines, and neurotoxins bind before being transported, whereas cocaine and antidepressants bind to block transport. Although binding is crucial to transport, few assays separate binding from transport, nor do they provide adequate temporal or spatial resolution to describe real-time kinetics or localize sites of active uptake. Here, we report a new method that distinguishes substrate binding from substrate transport using single-cell, space-resolved, real-time fluorescence microscopy. For these studies we use a fluorescent analogue of 1-methyl-4-phenylpyridinium, a neurotoxic metabolite and known substrate of monoamine transporters, to assess binding and transport with 50-ms, sub-micron resolution. We show that ASP+ (4-(4-(dimethylamino)styrl)-N-methylpyridinium) has micromolar potency for the human norepinephrine transporter, that ASP+ accumulation is Na+-, Cl--, cocaine-, and desipramine-sensitive and temperature-dependent, and that ASP+ competes with norepinephrine uptake. Using this method we demonstrate that norepinephrine transporters are efficient buffers for substrate, with binding rates exceeding transport rates by 100-fold. Furthermore, substrates bind deep within the transporter, isolated from both the bath and the lipid bilayer. Although transport per se depends on Na+ and Cl-, binding is independent of Na+ and actually increases in low Cl-. We further demonstrate that ASP+ interacts with transporters not only in transfected cells but in cultured neurons. ASP+ is also a substrate for dopamine and serotonin transporters and therefore represents a powerful new technique for studying the biophysical properties of monoamine transporters, an approach also amenable to high throughput assays for drug discovery.

* This work was supported by National Institutes of Health (NIH) Grants NS-34075 (to L. J. D.) and MH 58921 (to R. D. B.). Analyses were performed in part in the Vanderbilt University Medical Center Cell Imaging Core Resource under the supervision of Dr. Sam Wells (supported by NIH Grant CA68485).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232-8548. Tel.: 615-343-6278; Fax: 615-343-1679; E-mail: lou.defelice@vanderbilt.edu.

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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