We developed a yeast-based assay for selection of hENT1 (human equilibrative nucleoside transporter 1) mutants that have altered affinity for hENT1 inhibitors and substrates. In this assay, expression of hENT1 in a yeast strain deficient in adenine biosynthesis (ade2) permits yeast growth on a plate lacking adenine but containing adenosine, a hENT1 substrate. This growth was prevented when inhibitors of hENT1 {e.g. NBMPR [S6-(4-nitrobenzyl)-mercaptopurine riboside], dilazep or dipyridamole} were included in the media. To identify hENT1 mutants resistant to inhibition by these compounds, hENT1 was randomly mutagenized and introduced into this strain. Mutation(s) that allowed growth of yeast cells in the presence of these inhibitors were then identified and characterized. Mutants harbouring amino acid changes at Leu92 exhibited resistance to NBMPR and dilazep but not dipyridamole. The IC50 values of NBMPR and dilazep for [3H]adenosine transport by one of these mutants L92Q (Leu92→Gln) were approx. 200- and 4-fold greater when compared with the value for the wild-type hENT1, whereas that for dipyridamole remained unchanged. Additionally, when compared with the wild-type transporter, [3H]adenosine transport by L92Q transporter was significantly resistant to inhibition by inosine and guanosine but not by adenosine or pyrimidines. The Km value for inosine transport was approx. 4-fold greater for the L92Q mutant (260±16 µM) when compared with the wild-type transporter (65±7.8 µM). We have identified for the first time an amino acid residue (Leu92) of hENT1 that, when mutated, selectively alters the affinity of hENT1 to transport the nucleosides inosine and guanosine and its sensitivity to the inhibitors NBMPR and dilazep.
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May 15 2004
Mutation of leucine-92 selectively reduces the apparent affinity of inosine, guanosine, NBMPR [S6-(4-nitrobenzyl)-mercaptopurine riboside] and dilazep for the human equilibrative nucleoside transporter, hENT1 Available to Purchase
Christopher J. ENDRES;
Christopher J. ENDRES
Department of Pharmaceutics, University of Washington, Box 357610, Seattle, WA 98195, U.S.A.
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Dhruba J. SENGUPTA;
Dhruba J. SENGUPTA
Department of Pharmaceutics, University of Washington, Box 357610, Seattle, WA 98195, U.S.A.
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Jashvant D. UNADKAT
Jashvant D. UNADKAT
1
Department of Pharmaceutics, University of Washington, Box 357610, Seattle, WA 98195, U.S.A.
1To whom correspondence should be addressed (e-mail [email protected]).
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Publisher: Portland Press Ltd
Received:
December 04 2003
Revision Received:
January 30 2004
Accepted:
February 03 2004
Accepted Manuscript online:
February 03 2004
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2004
2004
Biochem J (2004) 380 (1): 131–137.
Article history
Received:
December 04 2003
Revision Received:
January 30 2004
Accepted:
February 03 2004
Accepted Manuscript online:
February 03 2004
Citation
Christopher J. ENDRES, Dhruba J. SENGUPTA, Jashvant D. UNADKAT; Mutation of leucine-92 selectively reduces the apparent affinity of inosine, guanosine, NBMPR [S6-(4-nitrobenzyl)-mercaptopurine riboside] and dilazep for the human equilibrative nucleoside transporter, hENT1. Biochem J 15 May 2004; 380 (1): 131–137. doi: https://doi.org/10.1042/bj20031880
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