ENT1

ENT1 (equilibrative nucleoside transporter 1)

Aliases: None
Gene name: Solute carrier family 29 member 1 (SLC29A1)

Summary

ENT1 (SLC29A1) is a sodium-independent transporter for purine and pyrimidine nucleosides and for some nucleobases. Nucleoside transporters are classified into two major classes, equilibrative bi-directional facilitators (ENTs) and Na+-dependent concentrative transporters (CNTs) [1]. Human ENT1 (hENT1, SLC29A1) was cloned from the placenta in 1996 and thus the first member to be characterized in the SLC29 family [2]. ENT1 is a glycosylated protein that contains 456 amino acid residues (50 kDa). Its gene is located at the chromosomal region 6p21.1-21.2. ENTs share a hypothetical 11-transmembrane (TM) helix topology, with an intracellular amino terminus and extracellular carboxyl terminus [3]. The region between TM domains 3 and 6 is responsible for the sensitivity to different inhibitors such as dipyridamole, dilazep, or S-(4-Nitrobenzyl)-6-thioinosine (NBMPR). ENT1 is inhibited by nanomolar concentrations of NBMPR and is thus also referred to as the NBMPR-sensitive equilibrative nucleoside transporter (es). The recently determined crystal structure of human ENT1 has revealed the formation of ENT1 homooligomers as well as ENT1/ENT2 heterooligomers.
ENT1 has orthologs in many eukaryotes including mammals, yeast, nematodes, plants and protozoa.

Localization

ENT1 is widely distributed throughout the body: it is present in erythrocytes as well as the liver, heart, spleen, kidney, lung, intestine, brain and placenta, although its abundance varies between tissues [2-8]. Expression of ENT1 was quantifiable by LC-MS/MS-based proteomics in microsomal fractions of liver and kidney [9]. Interestingly, in the blood brain barrier it is localized in the apical membrane and was quantified by LC-MS/MS-based proteomics [10]. ENT1 is expressed in HepaRG cells, a cellular hepatocyte model frequently used in ADMETox research [11]. Ent1 is expressed in rabbit cornea [12] and was shown to mediate nose-to-brain delivery in rats [13]. In addition to the plasma membrane ENT1 is also expressed and functional in the mitochondrial membrane and mitochondrial transport of fialuridine by human but not rodent ENT1 is thought to partly explain the toxicity of fialuridine [14, 15].
Most mammalian cells express more than one type of nucleoside transporter. Tissue-specific regulation of nucleoside transporters, along with their tissue distribution, can provide valuable information about their biology and pharmacological role [16].

Function, physiology, and clinically significant polymorphisms

Based on its tissue distribution, ENT1 is thought to play a critical role in the provision of nucleosides, derived from the diet or produced by tissues such as the liver, for salvage pathways of nucleotide synthesis in cells lacking de novo biosynthetic pathways. Besides, ENT1 is also involved in the absorption, distribution and excretion of xenobiotics.
Adenosine is a potent endogenous physiological and pharmacological regulator of numerous functions. Cellular signaling by adenosine occurs through four known G-protein-coupled adenosine receptors A1, A2A, A2B, and A3. By influencing the concentration of adenosine available to these receptors, ENTs fulfil important regulatory roles in different physiological processes. ENT1 has been reported to be a player in the modulation of coronary blood flow, myocardial O2 supply-demand balance, inflammation, and neurotransmission [4, 17-22].
Song et al. have recently provided human genetic evidence that soluble CD73-depedent elevation of plasma adenosine signaling via phosphorylation, ubiquitination and proteasomal degradation of ENT1 in the erythrocytes enhances adenosine response to hypoxia, and thereby counteracts hypoxia-induced maladaptation [23].
Several single nucleotide polymorphisms (SNPs) resulting in nonsynonymous variants of the human ENT1 transporter have been identified [24]. Those that have been characterized functionally show normal nucleoside and nucleoside drug uptake kinetics [24]. Daniels et al. reported a nonsynonymous SNP in SLC29A1 (rs45458701) which is responsible for the Augustine-negative (At(a-)) blood type [25]. The most frequent genetic polymorphism (rs45573936) causes an amino acid change at codon 216 from isoleucine to threonine with a minor allele frequency of 2%, almost exclusively found in Northern Europe [24, 26]. ENT1 Ile216Thr appears to contribute to the genetic predisposition to alcoholism with an increased risk of withdrawal-related seizures. Decreased adenosine activity observed in ENT1 null mice appears to be similar to that of chronic ethanol-induced increase of uptake activity in ENT1-216Thr, indicating that increased uptake function of the ENT1-216Thr variant upon alcohol exposure could reduce extracellular or synaptic adenosine levels [27].

Clinical significance

ENT1 contributes to the disposition of nucleoside analog drugs used to treat various forms of cancer, cardiovascular disorders, neurological conditions as well as viral and parasitic infections [28-32]. These drugs act via incorporation into nucleic acids, by interference with nucleic acid synthesis or by interference with the metabolism of physiological nucleosides [33].
ENT proteins are important determinants of sensitivity to, and toxicity from, nucleoside and other related drugs. Many of the nucleoside analog drugs available are used in the treatment of cancer. The first nucleoside analog approved as a treatment for cancer was the pyrimidine analog cytarabine (AraC). AraC is primarily transported into the cell via ENT1 [28]. Ara-G, the active form of the prodrug nelarabine, is also an ENT1 substrate, and ENT1 expression along with deoxycytidine kinase (DCK) expression was suggested as a biomarker of response to nelarabine therapy in T-cell acute lymphoblastic leukemia [34]. Another pyrimidine analog is gemcitabine, which is used as the primary or adjuvant therapy in the chemotherapeutic treatment of several cancer types [35-37]. Of the nucleoside analog drugs currently approved for cancer treatment (e.g. cladribine, pentostatin, etc.), 13 have been shown to use ENT1 as a route of entry into cells. Increased ENT1 abundance may contribute to the relative selectivity of nucleoside chemotherapy for malignant cells; measurement of transporter abundance may therefore provide a predictive tool for guiding the appropriate use of such drugs in individual patients [38, 39]. ENT1 also transports nucleobases that include 5-fluorouracil, 6-mercaptopurine and several endogenous nucleobases [40].
Nucleoside analog drugs (e.g. zidovudine, emtricitabine [41], ribavirin [42], etc.) are also commonly used to treat human immunodeficiency virus (HIV), hepatitis B, and hepatitis C infections. The polar nucleoside drug ribavirin is part of the first-line treatment for chronic hepatitis C virus infection, and ENT1 seems to be the major transporter controlling the hepatic uptake of ribavirin [43]. The accumulation of ribavirin inside erythrocytes upon long-term administration also depends on ENT1 transport. The phosphorylated metabolites of ribavirin accumulate in erythrocytes and produce dose-limiting hemolytic anemia [44]. Entecavir, the anti-HBV drug is also an ENT1 substrate [45].
A variety of drugs such as dilazep, dipyridamole, and draflazine [46] interact with nucleoside transporters and alter adenosine levels, which can lead to cardioprotective or vasodilatory effects. It was also demonstrated that ENT1 and adenosine constitute biomarkers of the initial stages of neurodegeneration in Huntington disease [47]. It is known that adenosine is an important mediator of ethanol intoxication. ENT1 has a physiological role in ethanol-mediated behaviors and suggest that decreased A1 adenosine receptor function promotes alcohol consumption [48]. Interestingly, in male but not female mice constant light increased alcohol drinking and decreased accumbal expression of Ent1 and adenosine levels. Also, only male but not female Ent1 knockout mice exhibited greater alcohol intake upon exposure to constant light, suggesting decreased Ent1 expression leads to increased alcohol drinking in a sex-dependent manner [49]. Crystal structure of human ENT1 is now used to identify and characterize interaction of adenosine reuptake inhibitors with ENT1 [50].

Regulatory requirements

ENT1 is not currently recommended for investigation by regulatory guidelines. However, a white paper from the International Transporter Consortium (ITC) enlisted ENT1 and ENT2 as transporters of clinical importance [30]. Therefore, in vitro evaluations may be required on a case-by-case basis.

References

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