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J. Biol. Chem., Vol. 283, Issue 25, 17691-17701, June 20, 2008

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An Intracellular Interaction Network Regulates Conformational Transitions in the Dopamine Transporter^*

Julie Kniazeff¹², Lei Shi¹, Claus J. Loland, Jonathan A. Javitch^¶, Harel Weinstein³, and Ulrik Gether⁴

From the Molecular Neuropharmacology Group and Center for Pharmacogenomics, Department of Neuroscience and Pharmacology, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark, the Department of Physiology and Biophysics and the HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Medical College of Cornell University, New York, New York 10021, and the ^¶Center for Molecular Recognition and Departments of Psychiatry and Pharmacology, Columbia University College of Physicians and Surgeons, New York, New York 10032

Neurotransmitter:sodium symporters (NSS)¹ mediate sodium-dependent reuptake of neurotransmitters from the synaptic cleft and are targets for many psychoactive drugs. The crystal structure of the prokaryotic NSS protein, LeuT, was recently solved at high resolution; however, the mechanistic details of regulation of the permeation pathway in this class of proteins remain unknown. Here we combine computational modeling and experimental probing in the dopamine transporter (DAT) to demonstrate the functional importance of a conserved intracellular interaction network. Our data suggest that a salt bridge between Arg-60 in the N terminus close to the cytoplasmic end of transmembrane segment (TM) 1 and Asp-436 at the cytoplasmic end of TM8 is stabilized by a cation- interaction between Arg-60 and Tyr-335 at the cytoplasmic end of TM6. Computational probing illustrates how the interactions may determine the flexibility of the permeation pathway, and mutagenesis within the network and results from assays of transport, as well as the state-dependent accessibility of a substituted cysteine in TM3, support the role of this network in regulating access between the substrate binding site and the intracellular milieu. The mechanism that emerges from these findings may be unique to the NSS family, where the local disruption of ionic interactions modulates the transition of the transporter between the outward- and inward-facing conformations.

Received for publication, January 18, 2008 , and in revised form, April 2, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant P01 DA 12408. This work was also supported by the Danish Health Science Research Council, the Lundbeck Foundation, the Novo Nordisk Foundation, and the Maersk Foundation. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2 and Movies I and II.

¹ Both authors contributed equally to this work.

² Recipient of a European Molecular Biology Organization long term fellowship.

³ To whom correspondence may be addressed: Rm. E-509, 1300 York Ave., New York, NY 10065. Tel.: 212-746-6358, Fax: 212-746-8690; E-mail: haw2002{at}med.cornell.edu.

⁴ To whom correspondence may be addressed. Tel.: 45-3532-7548; Fax: 45-3532-7610; E-mail: gether{at}sund.ku.dk.

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