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J. Biol. Chem., Vol. 283, Issue 13, 8496-8507, March 28, 2008

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A Proton-mediated Conformational Shift Identifies a Mobile Pore-lining Cysteine Residue (Cys-561) in Human Concentrative Nucleoside Transporter 3^*

Melissa D. Slugoski¹, Amy M. L. Ng, Sylvia Y. M. Yao, Kyla M. Smith, Colin C. Lin, Jing Zhang^¶, Edward Karpinski, Carol E. Cass^¶2, Stephen A. Baldwin^||, and James D. Young, A Heritage Scientist of the Alberta Heritage Foundation for Medical Research³

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

The concentrative nucleoside transporter (CNT) protein family in humans is represented by three members, hCNT1, hCNT2, and hCNT3. Belonging to a CNT subfamily phylogenetically distinct from hCNT1/2, hCNT3 mediates transport of a broad range of purine and pyrimidine nucleosides and nucleoside drugs, whereas hCNT1 and hCNT2 are pyrimidine and purine nucleoside-selective, respectively. All three hCNTs are Na⁺-coupled. Unlike hCNT1/2, however, hCNT3 is also capable of H⁺-mediated nucleoside cotransport. Using site-directed mutagenesis in combination with heterologous expression in Xenopus oocytes, we have identified a C-terminal intramembranous cysteine residue of hCNT3 (Cys-561) that reversibly binds the hydrophilic thiol-reactive reagent p-chloromercuribenzene sulfonate (PCMBS). Access of this membrane-impermeant probe to Cys-561, as determined by inhibition of hCNT3 transport activity, required H⁺, but not Na⁺, and was blocked by extracellular uridine. Although this cysteine residue is also present in hCNT1 and hCNT2, neither transporter was affected by PCMBS. We conclude that Cys-561 is located in the translocation pore in a mobile region within or closely adjacent to the nucleoside binding pocket and that access of PCMBS to this residue reports a specific H⁺-induced conformational state of the protein.

Received for publication, December 21, 2007 , and in revised form, January 15, 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.

¹ Funded by a studentship from the Alberta Heritage Foundation for Medical Research.

² Canada Research Chair in Oncology.

³ 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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