Abstract
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.
Highlights
In humans (h), Human concentrative nucleoside transporter 1 (hCNT1) and hCNT2 are pyrimidine nucleoside-selective and purine nucleoside-selective, respectively, whereas hCNT3 transports both pyrimidine and purine nucleosides (9 –11)
Topological investigations suggest that hCNT1–3 and other eukaryote CNT family members have a 13-transmembrane helix (TM) architecture, and multiple alignments reveal strong sequence similarities within the C-terminal half of the proteins [20]
Using hCNT1 as the template, we report here the consequences of individually mutating 10 aspartate and glutamate residues that are highly conserved in mammalian CNTs
Summary
Site-directed Mutagenesis and DNA Sequencing— hCNT1 cDNA (GenBankTM accession number U62968) in the Xenopus expression vector pGEM-HE [34] provided the template for construction of hCNT1 mutants by the oligonucleotide-directed technique [35], using reagents from the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer’s directions. For influx in the presence of Naϩ, significant differences in mediated uridine uptake (p Ͻ 0.05) compared with wild-type hCNT1 are indicated by *. Xenopus oocytes and assayed for uridine transport activity (10 M uridine influx, 1 min fluxes) in the presence and absence of Naϩ as described under “Experimental Procedures.”. The time course of uridine uptake by wild-type hCNT1 shown in Fig. 2A demonstrates that the measured fluxes corresponded to initial rates of transport. Purified oocyte plasma membranes using polyclonal antibodies [22] directed against amino acid residues 31–55 at the N terminus of the protein (Fig. 3A). Wild-type hCNT1 and transporters with mutations at positions 308 and 322 were present in similar amounts, indicating that these single amino acid substitutions resulted in loss of intrinsic hCNT1 transport activity without altering surface quantities in the oocyte plasma membrane.
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