Abstract
The human SLC28 family of integral membrane CNT (concentrative nucleoside transporter) proteins has three members, hCNT1, hCNT2, and hCNT3. Na(+)-coupled hCNT1 and hCNT2 transport pyrimidine and purine nucleosides, respectively, whereas hCNT3 mediates transport of both pyrimidine and purine nucleosides utilizing Na(+) and/or H(+) electrochemical gradients. These and other eukaryote CNTs are currently defined by a putative 13-transmembrane helix (TM) topology model with an intracellular N terminus and a glycosylated extracellular C terminus. Recent mutagenesis studies, however, have provided evidence supporting an alternative 15-TM membrane architecture. In the absence of CNT crystal structures, valuable information can be gained about residue localization and function using substituted cysteine accessibility method analysis with thiol-reactive reagents, such as p-chloromercuribenzene sulfonate. Using heterologous expression in Xenopus oocytes and the cysteineless hCNT3 protein hCNT3C-, substituted cysteine accessibility method analysis with p-chloromercuribenzene sulfonate was performed on the TM 11-13 region, including bridging extramembranous loops. The results identified residues of functional importance and, consistent with a new revised 15-TM CNT membrane architecture, suggest a novel membrane-associated topology for a region of the protein (TM 11A) that includes the highly conserved CNT family motif (G/A)XKX(3)NEFVA(Y/M/F).
Highlights
Macologically nucleoside analogs are used as chemotherapeutic agents in the treatment of cancer and antiviral diseases (1, 2)
The hENT2 isoform is capable of nucleobase transport (9). hCNTs, in contrast, are inwardly directed Naϩ-dependent nucleoside transporters found predominantly in intestinal and renal epithelial and other specialized cell types (10, 11). hCNT1 and hCNT2 are pyrimidine and purine nucleoside-selective, respectively, and couple Naϩ/nucleoside cotransport with 1:1 stoichiometry (12–18)
HCNT3 is broadly selective for both pyrimidine and purine nucleosides and couples Naϩ/nucleoside cotransport with 2:1 stoichiometry (10, 18, 19). hCNT3 is capable of Hϩ/nucleoside cotransport with a coupling stoichiometry of 1:1, whereby one of the two Naϩ binding sites functionally interacts with Hϩ (18, 19)
Summary
Construction of Cysteineless hCNT3CϪ cDNA— hCNT3 cDNA (GenBankTM accession number AF305210) provided the template for the construction of a cysteineless version of hCNT3 (hCNT3CϪ). Defolliculated stage VI Xenopus oocytes were microinjected with 20 nl of water or 20 nl of water containing capped RNA transcripts (20 ng) and incubated in modified Barth’s medium (changed daily) at 18 °C for 72 h prior to assay of transport activity. At the end of the incubation period, extracellular label was removed by seven rapid washes in ice-cold Naϩ-free (choline chloride) transport medium (100 mM ChCl, pH 7.5), and individual oocytes were dissolved in 1% (w/v) SDS for quantitation of oocyte-associated radioactivity by liquid scintillation counting (LS 6000 IC; Beckman). The flux values shown represent mediated transport, corrected for basal uridine uptake measured in control water-injected oocytes, and are the means Ϯ S.E. of 10 –12 oocytes. 9 of the 133 residues investigated fell into this category (6.8%), and in every case, the mutation to cysteine resulted in a protein with low functional activity in both Naϩ-contain-. (Pierce) and developed with enhanced chemiluminescence dues were as follows: Met[496] and Gly[498] in TM 11; Glu[519] in TM reagents (Pierce)
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