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J. Biol. Chem., Vol. 278, Issue 41, 40317-40323, October 10, 2003

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Inhibition of the Norepinephrine Transporter by the Venom Peptide -MrIA

SITE OF ACTION, Na⁺ DEPENDENCE, AND STRUCTURE-ACTIVITY RELATIONSHIP^*

Iain A. Sharpe  , Elka Palant ¶, Christina I. Schroeder , David M. Kaye ||, David J. Adams , Paul F. Alewood  ¶ and Richard J. Lewis  ¶ **

From the Institute for Molecular Bioscience, The University of Queensland, St. Lucia 4072, Queensland, Australia, the School of Biomedical Sciences, The University of Queensland, St. Lucia 4072, Queensland, Australia, ^¶Xenome Ltd, 50 Meiers Road, Indooroopilly 4068, Queensland, Australia, and the ^||Baker Heart Research Institute, Commercial Road, Prahran 3181, Victoria, Australia

-Conopeptide MrIA (-MrIA) is a 13-residue peptide contained in the venom of the predatory marine snail Conus marmoreus that has been found to inhibit the norepinephrine transporter (NET). We investigated whether -MrIA targeted the other members of the monoamine transporter family and found no effect of the peptide (100 µM) on the activity of the dopamine transporter and the serotonin transporter, indicating a high specificity of action. The binding of the NET inhibitors, [³H]nisoxetine and [³H]mazindol, to the expressed rat and human NET was inhibited by -MrIA with the conopeptide displaying a slight preference toward the rat isoform. For both radioligands, saturation binding studies showed that the inhibition by -MrIA was competitive in nature. It has previously been demonstrated that -MrIA does not compete with norepinephrine, unlike classically described NET inhibitors such as nisoxetine and mazindol that do. This pattern of behavior implies that the binding site for -MrIA on the NET overlaps the antidepressant binding site and is wholly distinct from the substrate binding site. The inhibitory effect of -MrIA was found to be dependent on Na⁺ with the conopeptide becoming a less effective blocker of [³H]norepinephrine by the NET under the conditions of reduced extracellular Na⁺. In this respect, -MrIA is similar to the antidepressant inhibitors of the NET. The structure-activity relationship of -MrIA was investigated by alanine scanning. Four residues in the first cysteine-bracketed loop of -MrIA and a His in loop 2 played a dominant role in the interaction between -MrIA and the NET. H chemical shift comparisons indicated that side-chain interactions at these key positions were structurally perturbed by the replacement of Gly-6. From these data, we present a model of the structure of -MrIA that shows the relative orientation of the key binding residues. This model provides a new molecular caliper for probing the structure of the NET.

Received for publication, December 20, 2002 , and in revised form, July 2, 2003.

The atomic coordinates and structure factors (code 1IEO) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was provided by 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.

^** To whom correspondence should be addressed. Tel.: 61-7-3346-2984; Fax: 617-3346-2101; E-mail: r.lewis{at}imb.uq.edu.au.

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