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J. Biol. Chem., Vol. 282, Issue 5, 3188-3195, February 2, 2007

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Molecular Determinants of Substrate Selectivity of a Novel Organic Cation Transporter (PMAT) in the SLC29 Family^*

From the Department of Pharmaceutics, University of Washington, Seattle, Washington 98195

Plasma membrane monoamine transporter (PMAT or ENT4) is a newly cloned transporter assigned to the equilibrative nucleoside transporter (ENT) family (SLC29). Unlike ENT1–3, PMAT mainly functions as a polyspecific organic cation transporter. In this study, we investigated the molecular mechanisms underlying the unique substrate selectivity of PMAT. By constructing chimeras between human PMAT and ENT1, we showed that a chimera consisting of transmembrane domains (TM) 1–6 of PMAT and TM7–11 of hENT1 behaved like PMAT, transporting 1-methyl-4-phenylpyridinium (MPP⁺, an organic cation) but not uridine (a nucleoside), suggesting that TM1–6 contains critical domains responsible for substrate recognition. To identify residues important for the cation selectivity of PMAT, 10 negatively charged residues were chosen and substituted with alanine. Five of the alanine mutants retained PMAT activity, and four were non-functional due to impaired targeting to the plasma membrane. However, alanine substitution at Glu²⁰⁶ in TM5 abolished PMAT activity without affecting cell surface expression. Eliminating the charge at Glu²⁰⁶ (E206Q) resulted in loss of organic cation transport activity, whereas conserving the negative charge (E206D) restored transporter function. Interestingly, mutant E206Q, which possesses the equivalent residue in ENT1, gained uridine transport activity. Thr²²⁰, another residue in TM5, also showed an effect on PMAT activity. Helical wheel analysis of TM5 revealed a distinct amphipathic pattern with Glu²⁰⁶ and Thr²²⁰ clustered in the center of the hydrophilic face. In summary, our results suggest that Glu²⁰⁶ functions as a critical charge sensor for cationic substrates and TM5 forms part of the substrate permeation pathway in PMAT.

Received for publication, October 5, 2006 , and in revised form, November 20, 2006.

^* This work was supported by National Institutes of Health Grant GM66233. 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.

¹ Supported in part by a predoctoral fellowship from the Eli Lilly and Company Foundation.

² Present address: Merck KGaA, Institute of Drug Metabolism and Pharmacokinetics, Am Feld 32, D-85567 Grafing, Germany.

³ To whom correspondence should be addressed: H272J, Health Sciences Bldg., Seattle, WA 98195-7610. Tel.: 206-221-6561; Fax: 206-543-3204; E-mail: jowang{at}u.washington.edu.

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