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J. Biol. Chem., Vol. 277, Issue 28, 24938-24948, July 12, 2002
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From the Membrane Protein Research Group, Departments of
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 Km 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.
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*
,,,
, and**
Physiology and § Oncology, University of
Alberta, Edmonton, Alberta T6G 2H7, Canada and the School of
Biochemistry and Molecular Biology, University of Leeds,
Leeds LS2 9JT, United Kingdom
*
This work was supported in part by the Canadian Institutes
of Health Research, the Alberta Cancer Board, the Wellcome Trust, and
the Medical Research Council of the United Kingdom.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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