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J. Biol. Chem., Vol. 278, Issue 41, 40324-40329, October 10, 2003

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-Conopeptide MrIA Partially Overlaps Desipramine and Cocaine Binding Sites on the Human Norepinephrine Transporter^*

Lesley J. Bryan-Lluka , Heinz Bönisch   ¶ and Richard J. Lewis || **

From the ^||Institute for Molecular Bioscience, The University of Queensland, Brisbane 4072, Queensland, Australia, Department of Physiology and Pharmacology, School of Biomedical Sciences, The University of Queensland, Brisbane 4072, Queensland, Australia, and Department of Pharmacology and Toxicology, University of Bonn, Reuterstrasse 2b, D-53113 Bonn, Germany

The interactions of -conopeptide MrIA with the human norepinephrine transporter (hNET) were investigated by determining the effects of hNET point mutations on the inhibitory potency of MrIA. The mutants were produced by site-directed mutagenesis and expressed in COS-7 cells. The potency of MrIA was greater for inhibition of uptake by hNET of [³H]norepinephrine (K_i 1.89 µM) than [³H]dopamine (K_i 4.33 µM), and the human dopamine transporter and serotonin transporter were not inhibited by MrIA (to 7 µM). Of 18 mutations where hNET amino acid residues were exchanged with those of the human dopamine transporter, MrIA had increased potency for inhibition of [³H]norepinephrine uptake for three mutations (in predicted extracellular loops 3 and 4 and transmembrane domain (TMD) 8) and decreased potency for one mutation (in TMD6 and intracellular loop (IL) 3). Of the 12 additional mutations in TMDs 2, 4, 5, and 11 and IL1, three mutations (in TMD2 and IL1) had reduced MrIA inhibitory potency. All of the other mutations tested had no influence on MrIA potency. A comparison of the results with previous data for desipramine and cocaine inhibition of norepinephrine uptake by the mutant hNETs reveals that MrIA binding to hNET occurs at a site that is distinct from but overlaps with the binding sites for tricyclic antidepressants and cocaine.

Received for publication, December 23, 2002 , and in revised form, May 23, 2003.

^* This research was supported by grants from the National Health and Medical Research Council of Australia. 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.

^¶ Recipient of a University of Queensland Travel Grant for International Collaborative Research.

^** To whom correspondence should be addressed. Tel.: 617-3346-2984; Fax: 617-3346-2101; E-mail: r.lewis{at}imb.uq.edu.au.

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