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J Biol Chem. 2002 Jul 12;277(28):24938-48. Epub 2002 May 2.

Functional and molecular characterization of nucleobase transport by recombinant human and rat equilibrative nucleoside transporters 1 and 2. Chimeric constructs reveal a role for the ENT2 helix 5-6 region in nucleobase translocation.

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  • 1Membrane Protein Research Group, Department of Physiology, University of Alberta, Edmonton, Canada.

Abstract

The human (h) and rat (r) equilibrative (Na(+)-independent) nucleoside transporters (ENTs) hENT1, rENT1, hENT2, and rENT2 belong to a family of integral membrane proteins with 11 transmembrane domains (TMs) and are distinguished functionally by differences in sensitivity to inhibition by nitrobenzylthioinosine and coronary vasoactive drugs. Structurally, the proteins have a large glycosylated loop between TMs 1 and 2 and a large cytoplasmic loop between TMs 6 and 7. In the present study, hENT1, rENT1, hENT2, and rENT2 were produced in Xenopus laevis oocytes and investigated for their ability to transport pyrimidine and purine nucleobases. hENT2 and rENT2 efficiently transported radiolabeled hypoxanthine, adenine, guanine, uracil, and thymine (apparent K(m) values 0.7-2.6 mm), and hENT2, but not rENT2, also transported cytosine. These findings were independently confirmed by hypoxanthine transport experiments with recombinant hENT2 produced in purine-cytosine permease (FCY2)-deficient Saccharomyces cerevisiae and provide the first direct demonstration that the ENT2 isoform is a dual mechanism for the cellular uptake of nucleosides and nucleobases, both of which are physiologically important salvage metabolites. In contrast, recombinant hENT1 and rENT1 mediated negligible oocyte fluxes of hypoxanthine relative to hENT2 and rENT2. Chimeric experiments between rENT1 and rENT2 using splice sites at rENT1 residues 99 (end of TM 2), 171 (between TMs 4 and 5), and 231 (end of TM 6) identified TMs 5-6 of rENT2 (amino acid residues 172-231) as a determinant of nucleobase transport activity, suggesting that this domain forms part(s) of the ENT2 substrate translocation channel.

PMID:
12006583
[PubMed - indexed for MEDLINE]
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