Conserved glutamate residues are critically involved in Na+/nucleoside cotransport by human concentrative nucleoside transporter 1 (hCNT1).
Human concentrative nucleoside transporter 1 (hCNT1), the first discovered of three human members of the SLC28 (CNT) protein family, is a Na+/nucleoside cotransporter with 650 amino acids. The potential functional roles of 10 conserved aspartate and glutamate residues in hCNT1 were investigated by site-directed mutagenesis and heterologous expression in Xenopus oocytes. Initially, each of the 10 residues was replaced by the corresponding neutral amino acid (asparagine or glutamine). Five of the resulting mutants showed unchanged Na+-dependent uridine transport activity (D172N, E338Q, E389Q, E413Q, and D565N) and were not investigated further. Three were retained in intracellular membranes (D482N, E498Q, and E532Q) and thus could not be assessed functionally. The remaining two (E308Q and E322Q) were present in normal quantities at cell surfaces but exhibited low intrinsic transport activities. Charge replacement with the alternate acidic amino acid enabled correct processing of D482E and E498D, but not of E532D, to cell surfaces and also yielded partially functional E308D and E322D. Relative to wild-type hCNT1, only D482E exhibited normal transport kinetics, whereas E308D, E308Q, E322D, E322Q, and E498D displayed increased K50(Na+) and/or Km(uridine) values and diminished Vmax(Na+) and Vmax(uridine) values. E322Q additionally exhibited uridine-gated uncoupled Na+ transport. Together, these findings demonstrate roles for Glu-308, Glu-322, and Glu-498 in Na+/nucleoside cotransport and suggest locations within a common cation/nucleoside translocation pore. Glu-322, the residue having the greatest influence on hCNT1 transport function, exhibited uridine-protected inhibition by p-chloromercuriphenyl sulfonate and 2-aminoethyl methanethiosulfonate when converted to cysteine.
Yao
Sylvia Y M
SY
Membrane Protein Research Group, Department of Physiology, University of Alberta and Cross Cancer Institute, Edmonton, Alberta T6G 2H7, Canada.
Ng
Amy M L
AM
Slugoski
Melissa D
MD
Smith
Kyla M
KM
Mulinta
Ras
R
Karpinski
Edward
E
Cass
Carol E
CE
Baldwin
Stephen A
SA
Young
James D
JD
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects / Q000502:physiology; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D007202:Indicators and Reagents / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009865:Oocytes / Q000166:cytology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012964:Sodium / Q000737:chemistry / Q000378:metabolism; D014529:Uridine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
D000596:Amino Acids; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; C065171:cif nucleoside transporter; C093171:methanethiosulfonate ethylammonium; D002731:4-Chloromercuribenzenesulfonate; D005020:Ethyl Methanesulfonate; D012964:Sodium; D014529:Uridine