Conserved Glutamate Residues Glu-343 and Glu-519 Provide Mechanistic Insights into Cation/Nucleoside Cotransport by Human Concentrative Nucleoside Transporter hCNT3

Kyla M Smith,Sylvia Y.M Yao,James D Young,Edward Karpinski,Amy M.L Ng,Stephen A Baldwin,Melissa D Slugoski,Carol E Cass

doi:10.1074/jbc.m109.009613

Abstract

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.

Highlights

Determinant of the pharmacologic actions of nucleoside drugs [3,4,5,6]
The concentrative nucleoside transporter (CNT) protein family in humans is represented by three members, hCNT1, hCNT2, and Human concentrative nucleoside transporter 3 (hCNT3)
Belonging to a CNT subfamily phylogenetically distinct from hCNT1/2, hCNT3 utilizes electrochemical gradients of both Naϩ and Hϩ to accumulate a broad range of pyrimidine and purine nucleosides and nucleoside drugs within cells [10, 11]. hCNT1 and hCNT2, in contrast, are Naϩ-specific and transport pyrimidine and purine nucleosides, respectively [11,12,13]

Summary

EXPERIMENTAL PROCEDURES

Site-directed Mutagenesis of hCNT3 and Expression in Xenopus Oocytes— hCNT3 mutants were constructed using the QuikChange௡ site-directed mutagenesis kit (Stratagene) with hCNT3 cDNA (GenBankTM accession number AF305210) in the pGEM-HE vector [32] as the template for mutant construction. Uptake of 20 ␮M radiolabeled uridine, inosine, and thymidine in 100 mM NaCl, pH 7.5, was measured in oocytes producing hCNT3 mutants or wild-type hCNT3. NaCl, pH 7.5), Table 1 presents for each mutant initial rates of transport (1-min flux) of 20 ␮M radiolabeled uridine (a universal mammalian CNT permeant), inosine (a representative purine nucleoside), and thymidine (a representative pyrimidine nucleoside). The flux values shown in this figure (and in all subsequent experiments) depict mediated transport activity, defined as the difference in uptake between RNA transcript-injected and control water-injected oocytes. Mutants—To verify that the observed decreases in transport activities were not secondary to altered cell-surface expression, oocytes producing Glu-343 or Glu-519 mutants were subjected to cell-surface labeling with sulfosuccinimidyl-6-(biotinamido) hexanoate using immobilized streptavidin resin to separate cell-surface protein from that associated with intracellular

16 D E519D

24 F hCNT3

B E343D

10 E hCNT3

DISCUSSION