Bidirectional transport of 2-chloroadenosine by equilibrative nucleoside transporter 4 (hENT4): Evidence for allosteric kinetics at acidic pH.

Adenosine has been reported to be transported by equilibrative nucleoside transporter 4 (ENT4), encoded by the SLC29A4 gene, in an acidic pH-dependent manner. This makes hENT4 of interest as a therapeutic target in acidic pathologies where adenosine is protective (e.g. vascular ischaemia). We examined the pH-sensitivity of nucleoside influx and efflux by hENT4 using a recombinant transfection model that lacks the confounding influences of other nucleoside transporters (PK15-NTD). We established that [3H]2-chloroadenosine, which is resistant to metabolism by adenosine deaminase, is a substrate for hENT4. Transport of [3H]2-chloroadenosine at a pH of 6.0 in PK15-NTD cells stably transfected with SLC29A4 was biphasic, with a low capacity (Vmax ~ 30 pmol/mg/min) high-affinity component (Km ~ 50 µM) apparent at low substrate concentrations, which shifted to a high capacity (Vmax ~ 500 pmol/mg/min) low affinity system (Km > 600 µM) displaying positive cooperativity at concentrations above 200 µM. Only the low affinity component was observed at a neutral pH of 7.5 (Km ~ 2 mM). Efflux of [3H]2-chloroadenosine from these cells was also enhanced by more than 4-fold at an acidic pH. Enhanced influx and efflux of nucleosides by hENT4 under acidic conditions supports its potential as a therapeutic target in pathologies such as ischaemia-reperfusion injury.

Current Progress on Equilibrative Nucleoside Transporter Function and Inhibitor Design.

Physiological nucleosides are used for the synthesis of DNA, RNA, and ATP in the cell and serve as universal mammalian signaling molecules that regulate physiological processes such as vasodilation and platelet aggregation by engaging with cell surface receptors. The same pathways that allow uptake of physiological nucleosides mediate the cellular import of synthetic nucleoside analogs used against cancer, HIV, and other viral diseases. Physiological nucleosides and nucleoside drugs are imported by two families of nucleoside transporters: the SLC28 concentrative nucleoside transporters (CNTs) and SLC29 equilibrative nucleoside transporters (ENTs). The four human ENT paralogs are expressed in distinct tissues, localize to different subcellular sites, and transport a variety of different molecules. Here we provide an overview of the known structure-function relationships of the ENT family with a focus on ligand binding and transport in the context of a new hENT1 homology model. We provide a generic residue numbering system for the different ENTs to facilitate the interpretation of mutational data produced using different ENT homologs. The discovery of paralog-selective small-molecule modulators is highly relevant for the design of new therapies and for uncovering the functions of poorly characterized ENT family members. Here, we discuss recent developments in the discovery of new paralog-selective small-molecule ENT inhibitors, including new natural product-inspired compounds. Recent progress in the ability to heterologously produce functional ENTs will allow us to gain insight into the structure and functions of different ENT family members as well as the rational discovery of highly selective inhibitors.

Malaria parasite type 4 equilibrative nucleoside transporters (ENT4) are purine transporters with distinct substrate specificity.

Malaria, caused by Plasmodia parasites, affects hundreds of millions of people. As purine auxotrophs, Plasmodia use transporters to import host purines for subsequent metabolism by the purine salvage pathway. Thus purine transporters are attractive drug targets. All sequenced Plasmodia genomes encode four ENTs (equilibrative nucleoside transporters). During the pathogenic intraerythrocytic stages, ENT1 is a major route of purine nucleoside/nucleobase transport. Another plasma membrane purine transporter exists because Plasmodium falciparum ENT1-knockout parasites survive at supraphysiological purine concentrations. The other three ENTs have not been characterized functionally. Codon-optimized Pf- (P. falciparum) and Pv- (Plasmodium vivax) ENT4 were expressed in Xenopus laevis oocytes and substrate transport was determined with radiolabelled substrates. ENT4 transported adenine and 2'-deoxyadenosine at the highest rate, with millimolar-range apparent affinity. ENT4-expressing oocytes did not accumulate hypoxanthine, a key purine salvage pathway substrate, or AMP. Micromolar concentrations of the plant hormone cytokinin compounds inhibited both PfENT4 and PvENT4. In contrast with PfENT1, ENT4 interacted with the immucillin compounds in the millimolar range and was inhibited by 10 µM dipyridamole. Thus ENT4 is a purine transporter with unique substrate and inhibitor specificity. Its role in parasite physiology remains uncertain, but is likely to be significant because of the strong conservation of ENT4 homologues in Plasmodia genomes.

Molecular analysis and structure-activity relationship modeling of the substrate/inhibitor interaction site of plasma membrane monoamine transporter.

Plasma membrane monoamine transporter (PMAT) is a new polyspecific transporter that interacts with a wide range of structurally diverse organic cations. To map the physicochemical descriptors of cationic compounds that allow interaction with PMAT, we systematically analyzed the interactions between PMAT and three series of structural analogs of known organic cation substrates including phenylalkylamines, n-tetraalkylammonium (n-TAA) compounds, and ß-carbolines. Our results showed that phenylalkylamines with a distance between the aromatic ring and the positively charged amine nitrogen atom of ∼6.4 Å confer optimal interactions with PMAT, whereas studies with n-TAA compounds revealed an excellent correlation between IC(50) values and hydrophobicity. The five ß-carbolines that we tested, which possess a pyridinium-like structure and are structurally related to the neurotoxin 1-methyl-4-phenylpyridinium, inhibited PMAT with high affinity (IC(50) values of 39.1-65.5 µM). Cytotoxicity analysis further showed that cells expressing PMAT are 14- to 15-fold more sensitive to harmalan and norharmanium, suggesting that these two ß-carbolines are also transportable substrates of PMAT. We then used computer-aided modeling to generate qualitative and quantitative three-dimensional pharmacophore models on the basis of 23 previously reported and currently identified PMAT inhibitors and noninhibitors. These models are characterized by a hydrogen bond donor and two to three hydrophobic features with distances between the hydrogen bond donor and hydrophobic features ranging between 5.20 and 7.02 Å. The consistency between the mapping results and observed PMAT affinity of a set of test compounds indicates that the models performed well in inhibitor prediction and could be useful for future virtual screening of new PMAT inhibitors.

Tyrosine 112 is essential for organic cation transport by the plasma membrane monoamine transporter.

Plasma membrane monoamine transporter (PMAT) is a polyspecific organic cation transporter in the solute carrier 29 (SLC29) family. Previous studies suggested that the major substrate recognition sites are located within transmembrane domains (TM) 1-6, and interaction of PMAT with organic cations may involve aromatic residues. In this study, we analyzed the roles of tyrosine and tryptophan residues located within TM1-6 with a goal of identifying potential residues involved in substrate recognition and translocation. The six tyrosines and one tryptophan in this region were each mutated to alanine followed by analysis of the mutant's membrane localization and transport activity toward 1-methyl-4-phenylpyridinium (MPP(+)), serotonin (5-HT), and dopamine. Two mutants, Y85A and Y112A, exhibited normal cell surface expressions but lost their transport activities toward organic cations. At position Y85, aromatic substitution with phenylalanine or tryptophan fully restored organic cation transport activity. Interestingly, at position Y112, phenylalanine substitution was not allowed. Tryptophan substitution at Y112 partially restored transport activity toward 5-HT and dopamine but severely impaired MPP(+) transport. Detailed kinetic analyses revealed that tryptophan substitution at Y85 and Y112 affected the apparent binding affinity (K(m)) and maximal transport velocity (V(max)) in a substrate-dependent manner. Together, our data suggest that Y85 and Y112 are important molecular determinants for PMAT function, and Y112 is indispensable for optimal interaction with organic cation substrates. Our analyses also suggest the involvement of transmembrane domains 1 and 2 in forming the substrate permeation pathway of PMAT.

Characterization of mammalian equilibrative nucleoside transporters (ENTs) by mass spectrometry.

Equilibrative nucleoside transporters (ENTs) are integral membrane proteins that facilitate the movement of nucleosides and hydrophilic nucleoside analog (NA) drugs across cell membranes. ENTs are also targets for cardioprotectant drugs, which block re-uptake of the purine nucleoside adenosine, thereby enhancing purinergic receptor signaling pathways. ENTs are therefore important contributors to drug bioavailability and efficacy. Despite this important clinical role, very little is known about the structure and regulation of ENTs. Biochemical and structural studies on ENT proteins have been limited by their low endogenous expression levels, hydrophobicity and labile nature. To address these issues, we developed an approach whereby tagged mammalian ENT1 protein was over-expressed in mammalian cell lines, confirmed to be functional and isolated by affinity purification to sufficient levels to be analyzed using MALDI-TOF and tandem MS mass spectrometry. This proteomic approach will allow for a more detailed analysis of the structure, function and regulation of ENTs in the future.

Human equilibrative nucleoside transporter (ENT) family of nucleoside and nucleobase transporter proteins.

1. The human (h) SLC29 family of integral membrane proteins is represented by four members, designated equilibrative nucleoside transporters (ENTs) because of the properties of the first-characterized family member, hENT1. They belong to the widely distributed eukaryotic ENT family of equilibrative and concentrative nucleoside/nucleobase transporter proteins. 2. A predicted topology of eleven transmembrane helices has been experimentally confirmed for hENT1. The best-characterized members of the family, hENT1 and hENT2, possess similar broad permeant selectivities for purine and pyrimidine nucleosides, but hENT2 also efficiently transports nucleobases. hENT3 has a similar broad permeant selectivity for nucleosides and nucleobases and appears to function in intracellular membranes, including lysosomes. 3. hENT4 is uniquely selective for adenosine, and also transports a variety of organic cations. hENT3 and hENT4 are pH sensitive, and optimally active under acidic conditions. ENTs, including those in parasitic protozoa, function in nucleoside and nucleobase uptake for salvage pathways of nucleotide synthesis and, in humans, are also responsible for the cellular uptake of nucleoside analogues used in the treatment of cancers and viral diseases. 4. By regulating the concentration of adenosine available to cell surface receptors, mammalian ENTs additionally influence physiological processes ranging from cardiovascular activity to neurotransmission.

Functional characterization and expression analysis of a gene, OsENT2, encoding an equilibrative nucleoside transporter in rice suggest a function in cytokinin transport.

We identified four genes for potential equilibrative nucleoside transporters (ENTs) from rice (Oryza sativa; designated OsENT1 through OsENT4). Growth analysis of budding yeast (Saccharomyces cerevisiae) cells expressing OsENTs showed that OsENT2 transported adenosine and uridine with high affinity (adenosine, K(m) = 3.0 microm; uridine, K(m) = 0.7 microm). Purine or pyrimidine nucleosides and 2'-deoxynucleosides strongly inhibited adenosine transport via OsENT2, suggesting that OsENT2 possesses broad substrate specificity. OsENT2-mediated adenosine transport was resistant to the typical inhibitors of mammalian ENTs, nitrobenzylmercaptopurine ribonucleoside, dilazep, and dipyridamole. The transport activity was maximal at pH 5.0 and decreased slightly at lower as well as higher pH. In competition experiments with various cytokinins, adenosine transport by OsENT2 was inhibited by isopentenyladenine riboside (iPR). Direct measurements with radiolabeled cytokinins demonstrated that OsENT2 mediated uptake of iPR (K(m) = 32 microm) and trans-zeatin riboside (K(m) = 660 microm), suggesting that OsENT2 participates in iPR transport in planta. In mature plants, OsENT2 was predominantly expressed in roots. The OsENT2 promoter drove the expression of the beta-glucuronidase reporter gene in the scutellum during germination and in vascular tissues in germinated plants, suggesting a participation of OsENT2 in the retrieval of endosperm-derived nucleosides by the germinating embryo and in the long-distance transport of nucleosides in growing plants, respectively.

The equilibrative nucleoside transporter family, SLC29.

The human SLC29 family of proteins contains four members, designated equilibrative nucleoside transporters (ENTs) because of the properties of the first-characterised family member, hENT1. They belong to the widely-distributed eukaryotic ENT family of equilibrative and concentrative nucleoside/nucleobase transporters and are distantly related to a lysosomal membrane protein, CLN3, mutations in which cause neuronal ceroid lipofuscinosis. A predicted topology of 11 transmembrane helices with a cytoplasmic N-terminus and an extracellular C-terminus has been experimentally confirmed for hENT1. The best-characterised members of the family, hENT1 and hENT2, possess similar broad substrate specificities for purine and pyrimidine nucleosides, but hENT2 in addition efficiently transports nucleobases. The ENT3 and ENT4 isoforms have more recently also been shown to be genuine nucleoside transporters. All four isoforms are widely distributed in mammalian tissues, although their relative abundance varies: ENT2 is particularly abundant in skeletal muscle. In polarised cells ENT1 and ENT2 are found in the basolateral membrane and, in tandem with concentrative transporters of the SLC28 family, may play a role in transepithelial nucleoside transport. The transporters play key roles in nucleoside and nucleobase uptake for salvage pathways of nucleotide synthesis, and are also responsible for the cellular uptake of nucleoside analogues used in the treatment of cancers and viral diseases. In addition, by regulating the concentration of adenosine available to cell surface receptors, they influence many physiological processes ranging from cardiovascular activity to neurotransmission.

Molecular evolution of the equilibrative nucleoside transporter family: identification of novel family members in prokaryotes and eukaryotes.

Equilibrative nucleoside transporters (ENTs) are integral membrane proteins which enable the movement of hydrophilic nucleosides and nucleoside analogs down their concentration gradients across cell membranes. ENTs were only recently characterized at the molecular level, and little is known about the tertiary structure or distribution of these proteins in nonmammalian organisms. To identify conserved regions, residues, and motifs of ENTs that may indicate functionally important parts of the protein and to better understand the evolutionary history of this protein family, we conducted an exhaustive analysis to characterize and compare ENTs in taxonomically diverse organisms. We have identified novel ENT family members in humans, mice, fish, tunicates, slime molds, and bacteria. This greatly extends our knowledge on the distribution of the ENTs in eukaryotes, and we have identified, for the first time, family members in bacteria. The prokaryote ENTs are attractive models for future studies on transporter tertiary structure and mechanism of substrate translocation. Using sequence similarities, we have identified regions, residues, and motifs that are conserved across all family members. These areas are presumably correlated with function and therefore are important targets for future analysis. Finally, we propose an evolutionary history for the ENT family which clarifies the origin(s) of multiple isoforms in different taxa.