A proton-mediated conformational shift identifies a mobile pore-lining cysteine residue (Cys-561) in human concentrative nucleoside transporter 3
| Autor: | Amy M. L. Ng, Jing Zhang, Edward Karpinski, Colin C. Lin, Kyla M. Smith, Carol E. Cass, James D. Young, Sylvia Y.M. Yao, Stephen A. Baldwin, Melissa D. Slugoski |
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| Rok vydání: | 2008 |
| Předmět: |
Models
Molecular Patch-Clamp Techniques Molecular Sequence Data Biochemistry Concentrative nucleoside transporter Residue (chemistry) chemistry.chemical_compound Xenopus laevis Animals Humans Amino Acid Sequence Cysteine Molecular Biology Uridine Nucleoside binding Mesylates biology Membrane transport protein Membrane Transport Proteins Cell Biology Protein Structure Tertiary Electrophysiology Membrane Transport Structure Function and Biogenesis chemistry Mutation biology.protein Oocytes Protons Cotransporter Nucleoside 4-Chloromercuribenzenesulfonate Sequence Alignment |
| Zdroj: | The Journal of biological chemistry. 283(13) |
| ISSN: | 0021-9258 |
| Popis: | 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: | OpenAIRE |
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