Functional Characterization of a Nucleoside-Derived Drug Transporter Variant (hCNT3_C602R) Showing Altered Sodium-Binding Capacity

Abstract

A novel cloned polymorphism of the human concentrative nucleoside transporter hCNT3 was described and functionally characterized. This variant consists of a T/C transition leading to the substitution of cysteine 602 by an arginine residue in the core of transmembrane domain 13. The resulting hCNT3 C602R protein has the same selectivity and affinity for natural nucleosides and nucleoside-derived drugs as hCNT3 but much lower concentrative capacity. The insertion of the transporter into a polarized membrane seems unaffected in the variant. In a preliminary survey of a typical Spanish population, this variant showed an allelic frequency of 1%. The functional impairment of the hCNT3 C602R polymorphism is attributable to the presence of an arginine rather than the loss of a cysteine at position 602, because an engineered hCNT3 protein with a serine residue at this position (hCNT3 C602S ) and hCNT3 have similar kinetic parameters. The sodium activation kinetic analysis of both transporters revealed a variation in the affinity for sodium and a shift in the Hill coefficient that could be consistent with a stoichiometry of 2:1 and 1:1 sodium/nucleoside, for hCNT3 and hCNT3 C602R , respectively. In conclusion, the presence of an arginine residue in the core of transmembrane domain 13 is responsible for the different sodium affinity showed by the polymorphic transporter compared with the reference transporter. Individuals with the hCNT3 C602R variant might show a lower nucleoside and nucleoside analog concentrative capacity, which could be clinically relevant.

The Human Concentrative Nucleoside Transporter-3 C602R Variant Shows Impaired Sorting to Lipid Rafts and Altered Specificity for Nucleoside-Derived Drugs

“…Expression of the wild-type transporter resulted in sodium-dependent nucleoside transport activity with broad selectivity, similar to that described previously and consistent with hCNT3-type transporters (Ritzel et al, 2001;Hu et al, 2006). The hCNT3C602R transporter variant showed lower transport rates, as described previously (Errasti-Murugarren et al, 2008). In contrast to results obtained with natural nucleosides, the magnitude of this decrease (23, 0.25, 22, 13, and 44% of hCNT3 activity for gemcitabine, 5Ј-DFUR, fludarabine, AZT, and ribavirin, respectively) was not similar among nucleoside-derived drugs (Fig.…”

“…As noted above, the hCNT3C602R variant has the same selectivity and affinity for natural nucleosides as does hCNT3, but it has a much lower transport capacity (Errasti-Murugarren et al, 2008). To address whether this substitution affects hCNT3 interaction with some commonly used antiviral and antineoplastic nucleosidederived drugs, we measured drug uptake in HeLa cells transiently transfected with expression constructs of either hCNT3 (GenBank accession number AF305210) or hCNT3C602R.…”

“…As a consequence of this substitution, the resulting hCNT3 protein shows changes in kinetic behavior consistent with a shift from a 2:1 to a 1:1 substrate translocation stoichiometry and unaltered affinity for natural substrates (Errasti-Murugarren et al, 2008). The profound effects of this substitution predict a central role for this transmembrane domain in transporter function (Zhang et al, 2006;Errasti-Murugarren et al, 2008).…”

“…In this regard, we have characterized recently a novel, nonsynonymous polymorphism, C602R, that is located in the middle of the hCNT3 transmembrane domain 13 and shows an allelic frequency of 1% in a typical Spanish population (Errasti-Murugarren et al, 2008). As a consequence of this substitution, the resulting hCNT3 protein shows changes in kinetic behavior consistent with a shift from a 2:1 to a 1:1 substrate translocation stoichiometry and unaltered affinity for natural substrates (Errasti-Murugarren et al, 2008).…”

Host-Based Ribavirin Resistance Influences Hepatitis C Virus Replication and Treatment Response

“…Posttranslational modifications of ENT1 protein or mutations in the ENT1 sequence could account for altered ENT1 transporter activity, while maintaining similar protein and RNA levels. A polymorphism within the transmembrane domain of human CNT3 has been shown to alter transport specificity for several nucleoside-derived drugs (11,12). Several single-nucleotide polymorphisms (SNPs) within the ENT1 gene (SLC29A1) have been reported (38).…”