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. 2001 Jan 15;353(Pt 2):387–393. doi: 10.1042/0264-6021:3530387

Identification of Cys140 in helix 4 as an exofacial cysteine residue within the substrate-translocation channel of rat equilibrative nitrobenzylthioinosine (NBMPR)-insensitive nucleoside transporter rENT2.

S Y Yao 1, M Sundaram 1, E G Chomey 1, C E Cass 1, S A Baldwin 1, J D Young 1
PMCID: PMC1221582  PMID: 11139404

Abstract

The human and rat equilibrative nucleoside transporter proteins hENT1, rENT1, hENT2 and rENT2 belong to a family of integral membrane proteins with 11 potential transmembrane segments (TMs), and are distinguished functionally by differences in transport of nucleobases and sensitivity to inhibition by nitrobenzylthioinosine (NBMPR) and vasoactive drugs. In the present study, we have produced recombinant hENT1, rENT1, hENT2 and rENT2 in Xenopus oocytes and investigated uridine transport following exposure to the impermeant thiol-reactive reagent p-chloromercuriphenyl sulphonate (PCMBS). PCMBS caused reversible inhibition of uridine influx by rENT2, but had no effect on hENT1, hENT2 or rENT1. This difference correlated with the presence in rENT2 of a unique Cys residue (Cys(140)) in the outer half of TM4 that was absent from the other ENTs. Mutation of Cys(140) to Ser produced a functional protein (rENT2/C140S) that was insensitive to inhibition by PCMBS, identifying Cys(140) as the exofacial Cys residue in rENT2 responsible for PCMBS inhibition. Uridine protected wild-type rENT2 against PCMBS inhibition, suggesting that Cys(140) in TM4 lies within or is closely adjacent to the substrate-translocation channel of the transporter. TM4 has been shown previously to be within a structural domain (TMs 3-6) responsible for interactions with NBMPR, vasoactive drugs and nucleobases.

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Selected References

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  1. Agbanyo F. R., Cass C. E., Paterson A. R. External location of sites on pig erythrocyte membranes that bind nitrobenzylthioinosine. Mol Pharmacol. 1988 Mar;33(3):332–337. [PubMed] [Google Scholar]
  2. Baldwin S. A., Mackey J. R., Cass C. E., Young J. D. Nucleoside transporters: molecular biology and implications for therapeutic development. Mol Med Today. 1999 May;5(5):216–224. doi: 10.1016/S1357-4310(99)01459-8. [DOI] [PubMed] [Google Scholar]
  3. Che M., Ortiz D. F., Arias I. M. Primary structure and functional expression of a cDNA encoding the bile canalicular, purine-specific Na(+)-nucleoside cotransporter. J Biol Chem. 1995 Jun 9;270(23):13596–13599. doi: 10.1074/jbc.270.23.13596. [DOI] [PubMed] [Google Scholar]
  4. Crawford C. R., Patel D. H., Naeve C., Belt J. A. Cloning of the human equilibrative, nitrobenzylmercaptopurine riboside (NBMPR)-insensitive nucleoside transporter ei by functional expression in a transport-deficient cell line. J Biol Chem. 1998 Feb 27;273(9):5288–5293. doi: 10.1074/jbc.273.9.5288. [DOI] [PubMed] [Google Scholar]
  5. Fredholm B. B. Adenosine and neuroprotection. Int Rev Neurobiol. 1997;40:259–280. [PubMed] [Google Scholar]
  6. Griffiths M., Beaumont N., Yao S. Y., Sundaram M., Boumah C. E., Davies A., Kwong F. Y., Coe I., Cass C. E., Young J. D. Cloning of a human nucleoside transporter implicated in the cellular uptake of adenosine and chemotherapeutic drugs. Nat Med. 1997 Jan;3(1):89–93. doi: 10.1038/nm0197-89. [DOI] [PubMed] [Google Scholar]
  7. Griffiths M., Yao S. Y., Abidi F., Phillips S. E., Cass C. E., Young J. D., Baldwin S. A. Molecular cloning and characterization of a nitrobenzylthioinosine-insensitive (ei) equilibrative nucleoside transporter from human placenta. Biochem J. 1997 Dec 15;328(Pt 3):739–743. doi: 10.1042/bj3280739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Huang Q. Q., Harvey C. M., Paterson A. R., Cass C. E., Young J. D. Functional expression of Na(+)-dependent nucleoside transport systems of rat intestine in isolated oocytes of Xenopus laevis. Demonstration that rat jejunum expresses the purine-selective system N1 (cif) and a second, novel system N3 having broad specificity for purine and pyrimidine nucleosides. J Biol Chem. 1993 Sep 25;268(27):20613–20619. [PubMed] [Google Scholar]
  9. Huang Q. Q., Yao S. Y., Ritzel M. W., Paterson A. R., Cass C. E., Young J. D. Cloning and functional expression of a complementary DNA encoding a mammalian nucleoside transport protein. J Biol Chem. 1994 Jul 8;269(27):17757–17760. [PubMed] [Google Scholar]
  10. Jarvis S. M., McBride D., Young J. D. Erythrocyte nucleoside transport: asymmetrical binding of nitrobenzylthioinosine to nucleoside permeation sites. J Physiol. 1982 Mar;324:31–46. doi: 10.1113/jphysiol.1982.sp014099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jarvis S. M. Nitrobenzylthioinosine-sensitive nucleoside transport system: mechanism of inhibition by dipyridamole. Mol Pharmacol. 1986 Dec;30(6):659–665. [PubMed] [Google Scholar]
  12. Jarvis S. M., Young J. D. Nucleoside translocation in sheep reticulocytes and fetal erythrocytes: a proposed model for the nucleoside transporter. J Physiol. 1982 Mar;324:47–66. doi: 10.1113/jphysiol.1982.sp014100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jarvis S. M., Young J. D. Nucleoside transport in rat erythrocytes: two components with differences in sensitivity to inhibition by nitrobenzylthioinosine and p-chloromercuriphenyl sulfonate. J Membr Biol. 1986;93(1):1–10. doi: 10.1007/BF01871013. [DOI] [PubMed] [Google Scholar]
  14. Kwong F. Y., Fincham H. E., Davies A., Beaumont N., Henderson P. J., Young J. D., Baldwin S. A. Mammalian nitrobenzylthioinosine-sensitive nucleoside transport proteins. Immunological evidence that transporters differing in size and inhibitor specificity share sequence homology. J Biol Chem. 1992 Oct 25;267(30):21954–21960. [PubMed] [Google Scholar]
  15. Kwong F. Y., Wu J. S., Shi M. M., Fincham H. E., Davies A., Henderson P. J., Baldwin S. A., Young J. D. Enzymic cleavage as a probe of the molecular structures of mammalian equilibrative nucleoside transporters. J Biol Chem. 1993 Oct 15;268(29):22127–22134. [PubMed] [Google Scholar]
  16. Mackey J. R., Baldwin S. A., Young J. D., Cass C. E. Nucleoside transport and its significance for anticancer drug resistance. Drug Resist Updat. 1998;1(5):310–324. doi: 10.1016/s1368-7646(98)80047-2. [DOI] [PubMed] [Google Scholar]
  17. Ritzel M. W., Yao S. Y., Huang M. Y., Elliott J. F., Cass C. E., Young J. D. Molecular cloning and functional expression of cDNAs encoding a human Na+-nucleoside cotransporter (hCNT1). Am J Physiol. 1997 Feb;272(2 Pt 1):C707–C714. doi: 10.1152/ajpcell.1997.272.2.C707. [DOI] [PubMed] [Google Scholar]
  18. Ritzel M. W., Yao S. Y., Ng A. M., Mackey J. R., Cass C. E., Young J. D. Molecular cloning, functional expression and chromosomal localization of a cDNA encoding a human Na+/nucleoside cotransporter (hCNT2) selective for purine nucleosides and uridine. Mol Membr Biol. 1998 Oct-Dec;15(4):203–211. doi: 10.3109/09687689709044322. [DOI] [PubMed] [Google Scholar]
  19. Sarkar G., Sommer S. S. The "megaprimer" method of site-directed mutagenesis. Biotechniques. 1990 Apr;8(4):404–407. [PubMed] [Google Scholar]
  20. Shryock J. C., Belardinelli L. Adenosine and adenosine receptors in the cardiovascular system: biochemistry, physiology, and pharmacology. Am J Cardiol. 1997 Jun 19;79(12A):2–10. doi: 10.1016/s0002-9149(97)00256-7. [DOI] [PubMed] [Google Scholar]
  21. Sundaram M., Yao S. Y., Ng A. M., Griffiths M., Cass C. E., Baldwin S. A., Young J. D. Chimeric constructs between human and rat equilibrative nucleoside transporters (hENT1 and rENT1) reveal hENT1 structural domains interacting with coronary vasoactive drugs. J Biol Chem. 1998 Aug 21;273(34):21519–21525. doi: 10.1074/jbc.273.34.21519. [DOI] [PubMed] [Google Scholar]
  22. Tse C. M., Wu J. S., Young J. D. Evidence for the asymmetrical binding of p-chloromercuriphenyl sulphonate to the human erythrocyte nucleoside transporter. Biochim Biophys Acta. 1985 Sep 10;818(3):316–324. doi: 10.1016/0005-2736(85)90005-7. [DOI] [PubMed] [Google Scholar]
  23. Vickers M. F., Mani R. S., Sundaram M., Hogue D. L., Young J. D., Baldwin S. A., Cass C. E. Functional production and reconstitution of the human equilibrative nucleoside transporter (hENT1) in Saccharomyces cerevisiae. Interaction of inhibitors of nucleoside transport with recombinant hENT1 and a glycosylation-defective derivative (hENT1/N48Q). Biochem J. 1999 Apr 1;339(Pt 1):21–32. [PMC free article] [PubMed] [Google Scholar]
  24. Wang J., Su S. F., Dresser M. J., Schaner M. E., Washington C. B., Giacomini K. M. Na(+)-dependent purine nucleoside transporter from human kidney: cloning and functional characterization. Am J Physiol. 1997 Dec;273(6 Pt 2):F1058–F1065. doi: 10.1152/ajprenal.1997.273.6.F1058. [DOI] [PubMed] [Google Scholar]
  25. Yao S. Y., Ng A. M., Muzyka W. R., Griffiths M., Cass C. E., Baldwin S. A., Young J. D. Molecular cloning and functional characterization of nitrobenzylthioinosine (NBMPR)-sensitive (es) and NBMPR-insensitive (ei) equilibrative nucleoside transporter proteins (rENT1 and rENT2) from rat tissues. J Biol Chem. 1997 Nov 7;272(45):28423–28430. doi: 10.1074/jbc.272.45.28423. [DOI] [PubMed] [Google Scholar]
  26. Yao S. Y., Ng A. M., Ritzel M. W., Gati W. P., Cass C. E., Young J. D. Transport of adenosine by recombinant purine- and pyrimidine-selective sodium/nucleoside cotransporters from rat jejunum expressed in Xenopus laevis oocytes. Mol Pharmacol. 1996 Dec;50(6):1529–1535. [PubMed] [Google Scholar]

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