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J. Biol. Chem., Vol. 280, Issue 19, 19177-19184, May 13, 2005

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Substrate Binding Stoichiometry and Kinetics of the Norepinephrine Transporter^*

Joel W. Schwartz, Gaia Novarino, David W. Piston¶, and Louis J. DeFelice||**

From the Center for Molecular Neuroscience, Departments of ^||Pharmacology and ^¶Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee 37232-8548 and the Dipartimento di Biologia Cellulare e dello Sviluppo, Università Roma 1 "La Sapienza," P.le Aldo Moro 5, 00185 Roma, Italy

The human norepinephrine (NE) transporter (hNET) attenuates neuronal signaling by rapid NE clearance from the synaptic cleft, and NET is a target for cocaine and amphetamines as well as therapeutics for depression, obsessive-compulsive disorder, and post-traumatic stress disorder. In spite of its central importance in the nervous system, little is known about how NET substrates, such as NE, 1-methyl-4-tetrahydropyridinium (MPP⁺), or amphetamine, interact with NET at the molecular level. Nor do we understand the mechanisms behind the transport rate. Previously we introduced a fluorescent substrate similar to MPP⁺, which allowed separate and simultaneous binding and transport measurement (Schwartz, J. W., Blakely, R. D., and DeFelice, L. J. (2003) J. Biol. Chem. 278, 9768–9777). Here we use this substrate, 4-(4-(dimethylamino)styrl)-N-methyl-pyridinium (ASP⁺), in combination with green fluorescent protein-tagged hNETs to measure substrate-transporter stoichiometry and substrate binding kinetics. Calibrated confocal microscopy and fluorescence correlation spectroscopy reveal that hNETs, which are homomultimers, bind one substrate molecule per transporter subunit. Substrate residence at the transporter, obtained from rapid on-off kinetics revealed in fluorescence correlation spectroscopy, is 526 µs. Substrate residence obtained by infinite dilution is 1000 times slower. This novel examination of substrate-transporter kinetics indicates that a single ASP⁺ molecule binds and unbinds thousands of times before being transported or ultimately dissociated from hNET. Calibrated fluorescent images combined with mass spectroscopy give a transport rate of 0.06 ASP⁺/hNET-protein/s, thus 36,000 on-off binding events (and 36 actual departures) occur for one transport event. Therefore binding has a low probability of resulting in transport. We interpret these data to mean that inefficient binding could contribute to slow transport rates.

Received for publication, November 15, 2004 , and in revised form, March 7, 2005.

^* This work was supported by National Institutes of Health NS-34075 and DA-6338 (to L. J. D.), CA68485 and DK20593 (to D. W. P.), and MH 58921 (to J. W. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** 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{at}vanderbilt.edu.

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