| Autor: |
Slugoski MD; From the Departments of Physiology, Edmonton, Alberta T6G 2H7, Canada., Smith KM; From the Departments of Physiology, Edmonton, Alberta T6G 2H7, Canada., Ng AML; From the Departments of Physiology, Edmonton, Alberta T6G 2H7, Canada., Yao SYM; From the Departments of Physiology, Edmonton, Alberta T6G 2H7, Canada., Karpinski E; From the Departments of Physiology, Edmonton, Alberta T6G 2H7, Canada., Cass CE; Oncology, Membrane Protein Research Group, University of Alberta, Edmonton, Alberta T6G 2H7, Canada; Cross Cancer Institute, Edmonton, Alberta T6G 1Z2, Canada., Baldwin SA; Astbury Centre for Structural Molecular Biology, Institute of Membrane and Systems Biology, University of Leeds, Leeds LS2 9JT, United Kingdom., Young JD; From the Departments of Physiology, Edmonton, Alberta T6G 2H7, Canada. Electronic address: james.young@ualberta.ca. |
| Abstrakt: |
Human concentrative nucleoside transporter 3 (hCNT3) utilizes electrochemical gradients of both Na(+) and H(+) to accumulate pyrimidine and purine nucleosides within cells. We have employed radioisotope flux and electrophysiological techniques in combination with site-directed mutagenesis and heterologous expression in Xenopus oocytes to identify two conserved pore-lining glutamate residues (Glu-343 and Glu-519) with essential roles in hCNT3 Na(+)/nucleoside and H(+)/nucleoside cotransport. Mutation of Glu-343 and Glu-519 to aspartate, glutamine, and cysteine severely compromised hCNT3 transport function, and changes included altered nucleoside and cation activation kinetics (all mutants), loss or impairment of H(+) dependence (all mutants), shift in Na(+):nucleoside stoichiometry from 2:1 to 1:1 (E519C), complete loss of catalytic activity (E519Q) and, similar to the corresponding mutant in Na(+)-specific hCNT1, uncoupled Na(+) currents (E343Q). Consistent with close-proximity integration of cation/solute-binding sites within a common cation/permeant translocation pore, mutation of Glu-343 and Glu-519 also altered hCNT3 nucleoside transport selectivity. Both residues were accessible to the external medium and inhibited by p-chloromercuribenzene sulfonate when converted to cysteine. |
