Ekaitz Errasti-Murugarren

SLC28 genes and concentrative nucleoside transporter (CNT) proteins

The human concentrative nucleoside transporter (hCNT) protein family has three members, hCNT1, 2, and 3, encoded by SLC28A1, A2, and A3 genes, respectively. hCNT1 and hCNT2 translocate pyrimidine- and purine-nucleosides, respectively, by a sodium-dependent mechanism, whereas hCNT3 shows broad substrate selectivity and the unique ability of translocating nucleosides both in a sodium- and a proton-coupled manner. hCNT proteins are also responsible for the uptake of most nucleoside-derived antiviral and anticancer drugs. Thus, hCNTs are key pharmacological targets. This review focuses on several crucial aspects of hCNT biology and pharmacology: protein structure–function, structural determinants for transportability, pharmacogenetics of hCNT-encoding genes, role of hCNT proteins in nucleoside-based therapeutics, and finally hCNT physiology.

Role of CNT3 in the transepithelial flux of nucleosides and nucleoside‐derived drugs

We examined the role of the concentrative nucleoside transporter CNT3 in the establishment of a transepithelial flux of natural nucleosides and their pharmacologically active derivatives in renal epithelial cell lines. Murine PCT cells grown on a transwell dish showed endogenous CNT3 activity at their apical membrane that was responsible for the sodium‐dependent transepithelial flux of both purine and pyrimidine nucleosides. hCNT3 was also identified in human kidney and its role in the transport of nucleosides was tested. To this end, MDCK cells, lacking endogenous CNT3 activity, were genetically engineered to express the human orthologue of CNT3 (hCNT3‐MDCK cells). In these cells, hCNT3 was inserted into the apical membrane, thus generating, as for PCT cells, a transepithelial flux of both nucleosides and nucleoside‐derived drugs. Apical‐to‐basolateral transepithelial flux was present in all cells expressing a functional CNT3 transporter and was significantly higher than that found either in PCT cells in absence of sodium or in mock‐transfected MDCK cells. Nevertheless in all cases a significant amount of the transported nucleoside was retained and transformed inside cells. However release to the opposite compartment was CNT3 dependent, not only in terms of absolute flux (much higher when an apical CNT3 transporter was active) but also regarding metabolic transformations of the apically absorbed nucleosides. These results underline a critical role of CNT3 in the renal reabsorption of nucleosides and their derivatives as well as in their intracellular metabolism.

Drug transporter pharmacogenetics in nucleoside-based therapies

This article focuses on the different types of transporter proteins that have been implicated in the influx and efflux of nucleoside-derived drugs currently used in the treatment of cancer, viral infections (i.e., AIDS) and other conditions, including autoimmune and inflammatory diseases. Genetic variations in nucleoside-derived drug transporter proteins encoded by the gene families SLC15, SLC22, SLC28, SLC29, ABCB, ABCC and ABCG will be specifically considered. Variants known to affect biological function are summarized, with a particular emphasis on those for which clinical correlations have already been established. Given that relatively little is known regarding the genetic variability of the players involved in determining nucleoside-derived drug bioavailability, it is anticipated that major challenges will be faced in this area of research.

Concentrative nucleoside transporters (CNTs) in epithelia: from absorption to cell signaling.

Concentrative and Equilibrative Nucleoside Transporter proteins (CNT and ENT, respectively) are encoded by gene families SLC28 and SLC29. They mediate the uptake of natural nucleosides and a variety of nucleoside-derived drugs, mostly used in anticancer therapy. CNT and ENT proteins are mostly localized in the apical and basolateral sides, respectively, in (re)absorptive epithelia. This anatomic distribution determines nucleoside and nucleoside-derived vectorial flux. CNT expression (particularly CNT2) is associated with differentiation and is also nutritionally regulated in intestinal epithelia, whereas ENT protein amounts (mostly ENT1) are increased when cells are exposed to proliferative stimuli such as EGF, TGF-α or wounding. Although all these features suggest a role for NT proteins in nucleoside salvage and (re)absorption, recent data demonstrate that CNT2 might be under purinergic control, in a manner that is dependent on energy metabolism. A physiological link between CNT2 function and intracellular metabolism is also supported by the evidence that extracellular adenosine can activate the AMP-dependent kinase (AMPK), by a mechanism which relies upon adenosine transport and phosphorylation. Thus the complex pattern of NT isoform expression in mammalian cells can fulfill physiological roles other than salvage.

A splice variant of the SLC28A3 gene encodes a novel human concentrative nucleoside transporter-3 (hCNT3) protein localized in the endoplasmic reticulum

Nucleoside transporters are plasma membrane proteins essential for nucleoside salvage. Among them, human concentrative nucleoside transporter 3 (hCNT3, SLC28A3) plays an essential role in this process because of its broader substrate selectivity and higher concentrative ability than the other members of the SLC28 protein family, hCNT1 and hCNT2. The aim of this study was to characterize an isoform of hCNT3, encoded by an alternatively spliced SLC28A3‐related mRNA, the first identified for a CNT protein. This variant, named hCNT3ins, is the result of the insertion of 176 bp corresponding to an intron located between exons 2 and 3 of the gene. This insertion results in a shift of the reading frame, yielding a protein lacking 69 residues of the N terminus. hCNT3 and hCNT3ins mRNAs are simultaneously expressed both in normal and transformed cells and are differentially regulated by activation and differentiation. Because of the N‐terminal deletion, hCNT3ins is retained in the endo‐plasmic reticulum, where it shows a typical hCNT3‐related activity. hCNT3ins exhibits a shorter half‐life than its plasma membrane counterpart, being degraded via a proteasome‐dependent pathway. We suggest that this novel hCNT3 isoform would be involved in the salvage of intracellular nucleosides from the lumen of the endoplasmic reticulum to the cytoplasm.—Errasti‐Murugarren, E., Molina‐Arcas, M., Casado, F. J., Pastor‐Anglada, M. A splice variant of the SLC28A3 gene encodes a novel human concentrative nucleoside trans‐porter‐3 (hCNT3) protein localized in the endoplasmic reticulum. FASEBJ. 23, 172‐182 (2009)

Functional Characterization of a Nucleoside-Derived Drug Transporter Variant (hCNT3C602R) Showing Altered Sodium-Binding Capacity

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 hCNT3C602R 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 hCNT3C602R 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 (hCNT3C602S) 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 hCNT3C602R, 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 hCNT3C602R 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

The human concentrative nucleoside transporter-3 C602R (hCNT3C602R), a recently identified human concentrative nucleoside transporter-3 (hCNT3) variant, has been shown to interact with natural nucleosides with apparent Km values similar to those of the wild-type transporter, although binding of one of the two sodium ions required for nucleoside translocation is impaired, resulting in decreased Vmax values (Mol Pharmacol 73:379–386, 2008). We have further analyzed the properties of this hCNT3 variant by determining its localization in plasma membrane lipid domains and its interaction with nucleoside-derived drugs used in anticancer and antiviral therapies. When expressed heterologously in HeLa cells, wild-type hCNT3 localized to both lipid raft and nonlipid raft domains. Treatment of cells with the cholesterol-depleting agent methyl-β-cyclodextrin resulted in a marked decrease in hCNT3-related transport activity that was associated with the loss of wild-type hCNT3 from lipid rafts. It is noteworthy that although exogenously expressed hCNT3C602R was present in nonlipid raft domains at a level similar to that of the wild-type transporter, the mutant transporter was present at much lower amounts in lipid rafts. A substrate profile analysis showed that interactions with a variety of nucleoside-derived drugs were altered in the hCNT3C602R variant and revealed that sugar hydroxyl residues are key structural determinants for substrate recognition by the hCNT3C602R variant.

Expression and Distribution of Nucleoside Transporter Proteins in the Human Syncytiotrophoblast

The plasma membrane distribution and related biological activity of nucleoside transporter proteins (NTs) were investigated in human syncytiotrophoblast from term placenta using a variety of approaches, including nucleoside uptake measurements into vesicles from selected plasma membrane domains, NT immunohistochemistry, and subcellular localization (basal, heavy, and light apical membranes as well as raft-enriched membranes from the apical domain). In contrast with other epithelia, in this epithelium, we have identified the high-affinity pyrimidine-preferring human concentrative nucleoside transporter (hCNT) 1 as the only hCNT-type protein expressed at both the basal and apical membranes. hCNT1 localization in lipid rafts is also dependent on its subcellular localization in the apical plasma membrane, suggesting a complex cellular and regional expression. Overall, this result favors the view that the placenta is a pyrimidine-preferring nucleoside sink from both maternal and fetal sides, and hCNT1 plays a major role in promoting pyrimidine salvage and placental growth. This finding may be of pharmacological relevance, because hCNT1 is known to interact with anticancer nucleoside-derived drugs and other molecules, such as nicotine and caffeine, for which a great variety of harmful effects on placental and fetal development, including intrauterine growth retardation, have been reported.

Different N-Terminal Motifs Determine Plasma Membrane Targeting of the Human Concentrative Nucleoside Transporter 3 in Polarized and Nonpolarized Cells

Human concentrative nucleoside transporter 3 (hCNT3) is a broad-selectivity, high-affinity protein implicated in the uptake of most nucleoside-derived anticancer and antiviral drugs. Regulated trafficking of hCNT3 has been recently postulated as a suitable way to improve nucleoside-based therapies. Moreover, the recent identification of a putative novel hCNT3-type transporter lacking the first 69 amino acids and retained at the endoplasmic reticulum anticipated that the N terminus of hCNT3 contains critical motifs implicated in trafficking. In the current study, we have addressed this issue by using deletions and site-directed mutagenesis and plasma membrane expression and nucleoside uptake kinetic analysis. Data reveal that 1) a segment between amino acids 50 and 62 contains plasma membrane-sorting determinants in nonpolarized cells; 2) in particular, the Val57-Thr58-Val59 tripeptide seems to be the core of the export signal, whereas acidic motifs upstream and downstream of it seem to be important for the kinetics of the process; and 3) in polarized epithelia, the β-turn-forming motif 17VGFQ20 is necessary for proper apical expression of the protein.

Role of the Transporter Regulator Protein (RS1) in the Modulation of Concentrative Nucleoside Transporters (CNTs) in Epithelia

SLC28 genes encode three plasma membrane transporter proteins, human concentrative nucleoside transporter (CNT)1, CNT2, and CNT3, all of which are implicated in the uptake of natural nucleosides and a variety of nucleoside analogs used in the chemotherapy of cancer and viral and inflammatory diseases. Mechanisms determining their trafficking toward the plasma membrane are not well known, although this might eventually become a target for therapeutic intervention. The transporter regulator RS1, which was initially identified as a short-term, post-transcriptional regulator of the high-affinity, Na+-coupled, glucose transporter sodium-dependent glucose cotransporter 1, was evaluated in this study as a candidate for coordinate regulation of membrane insertion of human CNT-type proteins. With a combination of studies with mammalian cells, Xenopus laevis oocytes, and RS1-null mice, evidence that RS1 down-regulates the localization and activity at the plasma membrane of the three members of this protein family (CNT1, CNT2, and CNT3) is provided, which indicates the biochemical basis for coordinate regulation of nucleoside uptake ability in epithelia and probably in other RS1-expressing cell types.