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J. Biol. Chem., Vol. 280, Issue 12, 11025-11034, March 25, 2005

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Identification and Mutational Analysis of Amino Acid Residues Involved in Dipyridamole Interactions with Human and Caenorhabditis elegans Equilibrative Nucleoside Transporters^*

Frank Visser¶||, Stephen A. Baldwin**, R. Elwyn Isaac, James D. Young¶¶, and Carol E. Cass¶||||

From the Membrane Protein Research Group, Departments of Oncology and Physiology, University of Alberta, and the ^¶Cross Cancer Institute, Edmonton, Alberta T6G 1Z2, Canada and the Schools of ^**Biochemistry and Microbiology and of Biology, University of Leeds, Leeds LS2 9JT, United Kingdom

The equilibrative nucleoside transporters, hENT1 and CeENT1 from humans and Caenorhabditis elegans, respectively, are inhibited by nanomolar concentrations of dipyridamole and share a common 11-transmembrane helix (TM) topology. Random mutagenesis and screening by functional complementation in yeast for clones with reduced sensitivities to dipyridamole yielded mutations at Ile⁴²⁹ in TM 11 of CeENT1 and Met³³ in TM 1 of hENT1. Mutational analysis of the corresponding residues of both proteins suggested important roles for these residues in competitive inhibition of hENT1 and CeENT1 by dipyridamole. To verify the roles of these residues in dipyridamole interactions, hENT2, which naturally exhibits low dipyridamole sensitivity, was mutated to contain side chains favorable for high affinity dipyridamole binding (i.e. a Met at the TM 1 and/or an Ile at the TM 11 positions). The single mutants exhibited increased hENT2 sensitivity to inhibition by dipyridamole, and the double mutant was the most sensitive, with an IC₅₀ value that was only 2% of that of wild type. Functional analysis of the TM 1 and 11 mutants of hENT1 and CeENT1 revealed that Ala and Thr in the TM 1 and 11 positions, respectively, impaired uridine and adenosine transport and that Leu⁴⁴² of hENT1 was involved in permeant selectivity. Mechanistic and structural models of dipyridamole interactions with the TM 1 and 11 residues are proposed. This study demonstrated that the corresponding residues in TMs 1 and 11 of hENT1, hENT2, and CeENT1 are important for dipyridamole interactions and nucleoside transport.

Received for publication, September 9, 2004 , and in revised form, December 23, 2004.

^* This work was supported 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.), and the Wellcome Trust and Medical Research Council UK (to R. E. I. and S. A. B.). 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.

^|| Supported by a studentship from the Alberta Heritage Foundation for Medical Research and the Endowed Ph.D. Studentship in Oncology.

^¶¶ Heritage Scientist of the Alberta Heritage Foundation for Medical Research.

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

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