A proton-mediated conformational shift identifies a mobile pore-lining cysteine residue (Cys-561) in human concentrative nucleoside transporter 3

The Journal of Biological Chemistry
Melissa D SlugoskiJames D Young

Abstract

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 PCM...Continue Reading

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Citations

Jun 6, 2009·The Journal of Biological Chemistry·Danielle E JohnsonJoseph R Casey
Jul 16, 2008·The Journal of Biological Chemistry·Melissa D SlugoskiJames D Young
Feb 3, 2011·Medicinal Research Reviews·Pedro Cano-Soldado, Marçal Pastor-Anglada
Aug 17, 2010·Biochimica Et Biophysica Acta·Cheng-Chang ChenWolfgang B Fischer
Feb 26, 2015·The Journal of Biological Chemistry·Jung-Suk ChoiAnthony J Berdis
Jul 22, 2017·The Journal of Biological Chemistry·Md Fazlur RahmanRajgopal Govindarajan

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