| Autor: |
Slugoski MD; Membrane Protein Research Group, Department of Physiology, University of Alberta, Edmonton, Alberta, Canada., Ng AM, Yao SY, Smith KM, Lin CC, Zhang J, Karpinski E, Cass CE, Baldwin SA, Young JD |
| Jazyk: |
angličtina |
| Zdroj: |
The Journal of biological chemistry [J Biol Chem] 2008 Mar 28; Vol. 283 (13), pp. 8496-507. Date of Electronic Publication: 2008 Jan 16. |
| DOI: |
10.1074/jbc.M710433200 |
| Abstrakt: |
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. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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