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J. Biol. Chem., Vol. 280, Issue 27, 25436-25449, July 8, 2005

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The Broadly Selective Human Na⁺/Nucleoside Cotransporter (hCNT3) Exhibits Novel Cation-coupled Nucleoside Transport Characteristics^*

Kyla M. Smith, Melissa D. Slugoski¶, Shaun K. Loewen¶, Amy M. L. Ng, Sylvia Y. M. Yao, Xing-Zhen Chen, Edward Karpinski, Carol E. Cass||**, Stephen A. Baldwin, and James D. Young¶¶

From the Membrane Protein Research Group, Departments of Physiology and^|| Oncology, University of Alberta, and the^** Cross Cancer Institute, Edmonton, Alberta T6G 2H7, Canada and the School of Biochemistry and Microbiology, University of Leeds, Leeds LS2 9JT, United Kingdom

The concentrative nucleoside transporter (CNT) protein family in humans is represented by three members, hCNT1, hCNT2, and hCNT3. hCNT3, a Na⁺/nucleoside symporter, transports a broad range of physiological purine and pyrimidine nucleosides as well as anticancer and antiviral nucleoside drugs, and belongs to a different CNT subfamily than hCNT1/2. H⁺-dependent Escherichia coli NupC and Candida albicans CaCNT are also CNT family members. The present study utilized heterologous expression in Xenopus oocytes to investigate the specificity, mechanism, energetics, and structural basis of hCNT3 cation coupling. hCNT3 exhibited uniquely broad cation interactions with Na⁺, H⁺, and Li⁺ not shared by Na⁺-coupled hCNT1/2 or H⁺-coupled NupC/CaCNT. Na⁺ and H⁺ activated hCNT3 through mechanisms to increase nucleoside apparent binding affinity. Direct and indirect methods demonstrated cation/nucleoside coupling stoichiometries of 2:1 in the presence of Na⁺ and both Na⁺ plus H⁺, but only 1:1 in the presence of H⁺ alone, suggesting that hCNT3 possesses two Na⁺-binding sites, only one of which is shared by H⁺. The H⁺-coupled hCNT3 did not transport guanosine or 3'-azido-3'-deoxythymidine and 2',3'-dideoxycytidine, demonstrating that Na⁺- and H⁺-bound versions of hCNT3 have significantly different conformations of the nucleoside binding pocket and/or translocation channel. Chimeric studies between hCNT1 and hCNT3 located hCNT3-specific cation interactions to the C-terminal half of hCNT3, setting the stage for site-directed mutagenesis experiments to identify the residues involved.

Received for publication, August 17, 2004 , and in revised form, March 24, 2005.

^* This work was supported in part by the National Cancer Institute of Canada, with funds from the Canadian Cancer Society, the Alberta Cancer Board, the Heart and Stroke Foundation, Canada, and the Medical Research Council, UK. 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.

These authors contributed equally to this work.

^¶ Funded by Studentships from the Alberta Heritage Foundation for Medical Research.

A Canada Research Chair in Oncology.

^¶¶ A Heritage Scientist of the Alberta Heritage Foundation for Medical Research. To whom correspondence should be addressed: Dept. of Physiology, 7-55 Medical Sciences Bldg., University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Tel.: 780-492-5895; Fax: 780-492-7566; E-mail: james.young{at}ualberta.ca.

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