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J. Biol. Chem., Vol. 282, Issue 19, 14148-14157, May 11, 2007

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Residues 334 and 338 in Transmembrane Segment 8 of Human Equilibrative Nucleoside Transporter 1 Are Important Determinants of Inhibitor Sensitivity, Protein Folding, and Catalytic Turnover^*

Frank Visser^¶12, Lijie Sun^||1, Vijaya Damaraju^¶, Tracey Tackaberry^¶, Yunshan Peng^**, Morris J. Robins^**3, Stephen A. Baldwin^||, James D. Young⁴, and Carol E. Cass^¶5

From the Membrane Protein Research Group, Departments of Oncology and Physiology, University of Alberta, and the ^¶Cross Cancer Institute, Edmonton, Alberta T6G 1Z2, Canada, the ^**Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602-5700, and the ^||Institute of Membrane and Systems Biology, University of Leeds, Leeds LS2 9JT, United Kingdom

Equilibrative nucleoside transporters (ENTs) are important for the metabolic salvage of nucleosides and the cellular uptake of antineoplastic and antiviral nucleoside analogs. Human equilibrative nucleoside transporter 1 (hENT1) is inhibited by nanomolar concentrations of structurally diverse compounds, including dipyridamole, dilazep, nitrobenzylmercaptopurine ribonucleoside (NBMPR), draflazine, and soluflazine. Random mutagenesis and screening by functional complementation for inhibitor-resistant mutants in yeast revealed mutations at Phe-334 and Asn-338. Both residues are predicted to lie in transmembrane segment 8 (TM 8), which contains residues that are highly conserved in the ENT family. F334Y displayed increased V_max values that were attributed to increased rates of catalytic turnover, and N338Q and N338C displayed altered membrane distributions that appeared to be because of protein folding defects. Mutations of Phe-334 or Asn-338 impaired interactions with dilazep and dipyridamole, whereas mutations of Asn-338 impaired interactions with draflazine and soluflazine. A helical wheel projection of TM 8 predicted that Phe-334 and Asn-338 lie in close proximity to other highly conserved and/or hydrophilic residues, suggesting that they form part of a structurally important region that influences interactions with inhibitors, protein folding, and rates of conformational change during the transport cycle.

Received for publication, February 28, 2007

^* This work was supported in part by grants from the Canadian Institutes of Health Research (to C. E. C. and J. D. Y.), the National Cancer Institute of Canada (to C. E. C.), the Wellcome Trust and Medical Research Council UK (to S. A. B.), pharmaceutical company unrestricted gift funds to Brigham Young University (to M. J. R.), and studentship funding from the Alberta Heritage Foundation for Medical Research (to F. V. and J. Z.), the Canadian Institutes of Health Research (to J. Z.), Overseas Research Scholarship and University of Leeds awards (to L. S.), and an R. K. Robins research fellowship (to Y. P.). 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 Both authors contributed equally to this work.

² Present address: Dept. of Biochemistry and Molecular Biology, University of Calgary, Heath Sciences Center, 3330 Hospital Dr. NW, Calgary, Alberta T2N 4N1, Canada.

³ The J. Rex Goates Professor of Chemistry at Brigham Young University.

⁴ Heritage Scientist of the Alberta Heritage Foundation for Medical Research.

⁵ Holds the Canada Research Chair in Oncology. To whom correspondence should be addressed: Dept. of Oncology, Cross Cancer Institute, 11560 University Ave., Edmonton, Alberta T6G 1Z2, Canada. Tel.: 780-432-8320; Fax: 780-432-8425; E-mail: carol.cass{at}cancerboard.ab.ca.

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