Conserved glutamate residues Glu-343 and Glu-519 provide mechanistic insights into cation/nucleoside cotransport by human concentrative nucleoside transporter hCNT3.
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.
Slugoski
Melissa D
MD
From the Departments of Physiology, Edmonton, Alberta T6G 2H7, Canada.
Smith
Kyla M
KM
From the Departments of Physiology, Edmonton, Alberta T6G 2H7, Canada.
Ng
Amy M L
AML
From the Departments of Physiology, Edmonton, Alberta T6G 2H7, Canada.
Yao
Sylvia Y M
SYM
From the Departments of Physiology, Edmonton, Alberta T6G 2H7, Canada.
Karpinski
Edward
E
From the Departments of Physiology, Edmonton, Alberta T6G 2H7, Canada.
Cass
Carol E
CE
Oncology, Membrane Protein Research Group, University of Alberta, Edmonton, Alberta T6G 2H7, Canada; Cross Cancer Institute, Edmonton, Alberta T6G 1Z2, Canada.
Baldwin
Stephen A
SA
Astbury Centre for Structural Molecular Biology, Institute of Membrane and Systems Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
Young
James D
JD
From the Departments of Physiology, Edmonton, Alberta T6G 2H7, Canada. Electronic address: james.young@ualberta.ca.
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D017124:Conserved Sequence; D005260:Female; D018698:Glutamic Acid / Q000737:chemistry; D006151:Guanosine / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D017136:Ion Transport; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009705:Nucleosides / Q000378:metabolism; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology; D014529:Uridine / Q000378:metabolism; D014981:Xenopus
D026901:Membrane Transport Proteins; D009705:Nucleosides; D011994:Recombinant Proteins; C065171:cif nucleoside transporter; D006151:Guanosine; D018698:Glutamic Acid; D002731:4-Chloromercuribenzenesulfonate; D012964:Sodium; D014529:Uridine