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J. Biol. Chem., Vol. 283, Issue 36, 24922-24934, September 5, 2008

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A Conformationally Mobile Cysteine Residue (Cys-561) Modulates Na⁺ and H⁺ Activation of Human CNT3^*

Melissa D. Slugoski¹, Kyla M. Smith, Ras Mulinta, Amy M. L. Ng, Sylvia Y. M. Yao, Ellen L. Morrison, Queenie O. T. Lee, Jing Zhang^¶, Edward Karpinski, Carol E. Cass^¶2, Stephen A. Baldwin^||, and James D. Young³

From the Membrane Protein Research Group, Departments of Physiology and Oncology, University of Alberta, Edmonton, Alberta, T6G 2H7, and the ^¶Cross Cancer Institute, Edmonton, Alberta T6G 1Z2, Canada, and the ^||Astbury Centre for Structural Molecular Biology, Institute of Membrane and Systems Biology, University of Leeds, Leeds LS2 9JT, United Kingdom

In humans, the SLC28 concentrative nucleoside transporter (CNT) protein family is represented by three Na⁺-coupled members; human CNT1 (hCNT1) and hCNT2 are pyrimidine and purine nucleoside-selective, respectively, whereas hCNT3 transports both purine and pyrimidine nucleosides and nucleoside drugs. Belonging to a phylogenetic CNT subfamily distinct from hCNT1/2, hCNT3 also mediates H⁺/nucleoside cotransport. Using heterologous expression in Xenopus oocytes, we have characterized a cysteineless version of hCNT3 (hCNT3C-). Processed normally to the cell surface, hCNT3C-exhibited hCNT3-like transport properties, but displayed a decrease in apparent affinity specific for Na⁺ and not H⁺. Site-directed mutagenesis experiments in wild-type and hCNT3C-backgrounds identified intramembranous Cys-561 as the residue responsible for this altered Na⁺-binding phenotype. Alanine at this position restored Na⁺ binding affinity, whereas substitution with larger neutral amino acids (threonine, valine, and isoleucine) abolished hCNT3 H⁺-dependent nucleoside transport activity. Independent of these findings, we have established that Cys-561 is located in a mobile region of the hCNT3 translocation pore adjacent to the nucleoside binding pocket and that access of p-chloromercuribenzene sulfonate to this residue reports a specific H⁺-induced conformational state of the protein ( Slugoski, M. D., Ng, A. M. L., Yao, S. Y. M., Smith, K. M., Lin, C. C., Zhang, J., Karpinski, E., Cass, C. E., Baldwin, S. A., and Young, J. D. (2008) J. Biol. Chem. 283, 8496-8507[Abstract/Free Full Text] ). The present investigation validates hCNT3C- as a template for substituted cysteine accessibility method studies of CNTs and reveals a pivotal functional role for Cys-561 in Na⁺- as well as H⁺-coupled modes of hCNT3 nucleoside transport.

Received for publication, March 5, 2008 , and in revised form, July 7, 2008.

^* This work was supported in part by the National Cancer Institute of Canada, with funds from the Canadian Cancer Society and the Alberta Cancer Board. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S3.

¹ Supported by a Studentship from the Alberta Heritage Foundation for Medical Research.

² Canada Research Chair in Oncology.

³ Heritage Scientist of the Alberta Heritage Foundation for Medical Research. To whom correspondence should be addressed: Dept. of Physiology, 7-55 Medical Sciences Bldg., University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Tel.: 780-492-5895; Fax: 780-492-7566; E-mail: james.young{at}ualberta.ca.

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