ENT2

ENT2 (equilibrative nucleoside transporter 2)

Aliases: DER12, HNP36
Gene name: Solute carrier family 29 member 2 (SLC29A2)

Summary

ENT2 (SLC29A2) is a sodium-independent, nitrobenzylmercaptopurine riboside (NBMPR)-insensitive transporter that participates in the transport of purine and pyrimidine nucleosides and nucleobases. Nucleoside transporters are classified into two major classes, equilibrative bi-directional facilitators (ENTs) and Na+-dependent concentrative transporters (CNTs) [1]. ENT2 was initially cloned from a human placental cDNA library and it has also been cloned independently by functional complementation from nucleoside transport-deficient CEM human leukemia cells [2, 3]. ENT2 is a glycosylated transport protein that contains 456 amino acid residues with a molecular mass of 50 kDa; its gene is located on chromosome 11q13.2. ENT2 is 46% identical by amino acid sequence with ENT1. ENTs have a predicted topology of 11 transmembrane (TM) domains, with an intracellular amino terminus and extracellular carboxyl terminus [4]. The region between TM domains 3 and 6 is responsible for the sensitivity to inhibitors such as dipyridamole, dilazep, NBMPR.  

Localization

Expression of ENT2 is relatively ubiquitous but varies in abundance among tissues and cell types. It has been confirmed in the brain, heart, placenta, thymus, pancreas, prostate and kidney [2, 3]. Relative to other transporters, ENT2 has one of the highest mRNA expressions in skeletal muscle [4, 5].
Since most mammalian cells express more than one type of nucleoside transporter, the tissue-specific regulation of nucleoside transporters rather than tissue distribution alone can provide valuable information about nucleoside transporter biology and their pharmacological role [6].

Function, physiology, and clinically significant polymorphisms

The exact function of ENT2 remains unclear, but as a high capacity, low affinity transporter, ENT2 is assumed to play a key role in maintaining nucleoside homeostasis by transporting a wide range of purine and pyrimidine nucleobases (e.g. cytosine, hypoxanthine, adenine, guanine, uracil, and thymine) [7]. ENT2 has a higher affinity than ENT1 for inosine, a molecule known for its cytoprotective effects [8, 9]. The co-existence in many cell types of both ENT1 and ENT2, which exhibit similar nucleoside specificities, may reflect the importance of the ENT2 substrate hypoxanthine as a source of purines for salvage pathways of nucleotide synthesis in tissues and cells (including brain, muscle, erythrocytes, leukocytes, and bone marrow cells) deficient in de novo nucleotide biosynthetic pathways [10]. Adenosine is a potent endogenous physiologic and pharmacologic 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 play key regulatory roles.  ENT2 might also be important in transporting adenosine and its metabolites inosine and hypoxanthine in tissues such as skeletal muscle during muscle exercise and recovery, respectively [3, 8, 11]. Three isoforms of ENT2 produced by alternative splicing have been described. Splice variants of human ENT2 yield a protein of 36 kDa nucleolar protein designated HNP36, and another 32 kDa protein, ENT2A. Both are non-functional as nucleoside transporters. Several polymorphic sites in SLC29A2 have been identified from ethnically diverse populations.  The low genetic and functional variation observed in ENT2 suggests a critical physiological role similar to ENT1. Variants of ENT2 with altered function in inosine, uridine, hypoxanthine, gemcitabine and fludarabine transport have been observed but with very low frequencies [12-14].

Clinical significance

Most anticancer and antiviral drugs are substrates for multiple transporters. ENT2 is thought to play a crucial role in the disposition of anticancer [15] and antiviral nucleoside analogs [4, 16]. These drugs act via incorporation into nucleic acids, by interference with nucleic acid synthesis, or by interference with the metabolism of physiological nucleosides [17]. When gemcitabine is administered by hepatic arterial infusion (HAI), hepatic extraction of gemcitabine is predominantly mediated by ENT2, and at clinically relevant dose levels ENT2-mediated uptake is not completely saturated. Thus, expression of ENT2 in hepatic tissue may be a key determinant of the feasibility of dose escalation without severe adverse events in gemcitabine HAI [18]. Largely overlapping with the substrates of ENT1, ENT2 is capable of transporting AZT and also exhibits a much greater capacity to transport ddC and ddI [19, 20]. ENT2-mediated transport is only partially inhibited by NBMPR at micromolar concentrations and ENT2 is less sensitive to inhibition of transport by dilazep and dipyridamole [19]. ENT2 at the inner blood-retina barrier could be a potential route for delivering nucleoside drugs from the circulation to the retina [21-23]. Similarly, molecules that are substrates for muscle uptake transporters including ENT2 can provide concentration in muscle tissue [24]. A modified/optimized monoclonal antibody fragment 3E10 Fv that has intracellular-delivery capabilities has been shown to penetrate cells through ENT2 [25]. ENT proteins are important determinants of sensitivity to, and toxicity from, nucleoside and other related drugs. ENT function dictates the transportability of new therapeutic nucleosides and other potential ENT substrates, and influences nucleoside analog pharmacokinetics. Not to the least, ENTs themselves can serve as drug targets.

Regulatory requirements

ENT2 is not currently recommended for investigation by regulatory guidelines.

Location Endogenous substrates In vitro substrates used experimentally Substrate drugs Inhibitors
ubiquitous adenosine, inosine, hypoxanthine, guanine, uridine, guanosine, thymine, thymidine, cytosine adenosine, hypoxanthine

2-chloroadenosine, formycin B, tubercidin, gemcitabine,fludarabine, cladribine, vidarabine, zidovudine, cytarabine

NBMPR, dipyridamole, dilazep, draflazine, soluflazine, mioflazine

 

References


1.    Cass, C.E., J.D. Young, and S.A. Baldwin, Recent advances in the molecular biology of nucleoside transporters of mammalian cells. Biochem Cell Biol, 1998. 76(5): p. 761-70.
2.    Griffiths, M., et al., Molecular cloning and characterization of a nitrobenzylthioinosine-insensitive (ei) equilibrative nucleoside transporter from human placenta. Biochem J, 1997. 328 ( Pt 3): p. 739-43.
3.    Crawford, C.R., et al., Cloning of the human equilibrative, nitrobenzylmercaptopurine riboside (NBMPR)-insensitive nucleoside transporter ei by functional expression in a transport-deficient cell line. J Biol Chem, 1998. 273(9): p. 5288-93.
4.    Baldwin, S.A., et al., The equilibrative nucleoside transporter family, SLC29. Pflugers Arch, 2004. 447(5): p. 735-43.
5.    Govindarajan, R., et al., In situ hybridization and immunolocalization of concentrative and equilibrative nucleoside transporters in the human intestine, liver, kidneys, and placenta. Am J Physiol Regul Integr Comp Physiol, 2007. 293(5): p. R1809-22.
6.    Pressacco, J., et al., Modulation of the equilibrative nucleoside transporter by inhibitors of DNA synthesis. Br J Cancer, 1995. 72(4): p. 939-42.
7.    Yao, S.Y., et al., Functional and molecular characterization of nucleobase transport by recombinant human and rat equilibrative nucleoside transporters 1 and 2. Chimeric constructs reveal a role for the ENT2 helix 5-6 region in nucleobase translocation. J Biol Chem, 2002. 277(28): p. 24938-48.
8.    Ward, J.L., et al., Kinetic and pharmacological properties of cloned human equilibrative nucleoside transporters, ENT1 and ENT2, stably expressed in nucleoside transporter-deficient PK15 cells. Ent2 exhibits a low affinity for guanosine and cytidine but a high affinity for inosine. J Biol Chem, 2000. 275(12): p. 8375-81.
9.    Modis, K., et al., Adenosine and inosine exert cytoprotective effects in an in vitro model of liver ischemia-reperfusion injury. Int J Mol Med, 2013. 31(2): p. 437-46.
10.    Tattersall, M.H., P. Slowiaczek, and A. De Fazio, Regional variation in human extracellular purine levels. J Lab Clin Med, 1983. 102(3): p. 411-20.
11.    Robillard, K.R., D.B. Bone, and J.R. Hammond, Hypoxanthine uptake and release by equilibrative nucleoside transporter 2 (ENT2) of rat microvascular endothelial cells. Microvasc Res, 2008. 75(3): p. 351-7.
12.    Leabman, M.K., et al., Natural variation in human membrane transporter genes reveals evolutionary and functional constraints. Proc Natl Acad Sci U S A, 2003. 100(10): p. 5896-901.
13.    Owen, R.P., et al., Functional characterization and haplotype analysis of polymorphisms in the human equilibrative nucleoside transporter, ENT2. Drug Metab Dispos, 2006. 34(1): p. 12-5.
14.    Mangravite, L.M., G. Xiao, and K.M. Giacomini, Localization of human equilibrative nucleoside transporters, hENT1 and hENT2, in renal epithelial cells. Am J Physiol Renal Physiol, 2003. 284(5): p. F902-10.
15.    Zhang, J., et al., The role of nucleoside transporters in cancer chemotherapy with nucleoside drugs. Cancer Metastasis Rev, 2007. 26(1): p. 85-110.
16.    Rahn, J.J., et al., Modulation of the metabolism of beta-L-(-)-2',3'-dideoxy-3'-thiacytidine by thymidine, fludarabine, and nitrobenzylthioinosine. Antimicrob Agents Chemother, 1997. 41(5): p. 918-23.
17.    Galmarini, C.M., J.R. Mackey, and C. Dumontet, Nucleoside analogues and nucleobases in cancer treatment. Lancet Oncol, 2002. 3(7): p. 415-24.
18.    Shimada, T., et al., Saturable Hepatic Extraction of Gemcitabine Involves Biphasic Uptake Mediated by Nucleoside Transporters Equilibrative Nucleoside Transporter 1 and 2. J Pharm Sci, 2015. 104(9): p. 3162-9.
19.    Yao, S.Y., et al., Transport of antiviral 3'-deoxy-nucleoside drugs by recombinant human and rat equilibrative, nitrobenzylthioinosine (NBMPR)-insensitive (ENT2) nucleoside transporter proteins produced in Xenopus oocytes. Mol Membr Biol, 2001. 18(2): p. 161-7.
20.    Sato, K., et al., Influx mechanism of 2',3'-dideoxyinosine and uridine at the blood-placenta barrier. Placenta, 2009. 30(3): p. 263-9.
21.    Hosoya, K. and M. Tachikawa, Inner blood-retinal barrier transporters: role of retinal drug delivery. Pharm Res, 2009. 26(9): p. 2055-65.
22.    Nagase, K., et al., Functional and molecular characterization of adenosine transport at the rat inner blood-retinal barrier. Biochim Biophys Acta, 2006. 1758(1): p. 13-9.
23.    Isakovic, A.J., N.J. Abbott, and Z.B. Redzic, Brain to blood efflux transport of adenosine: blood-brain barrier studies in the rat. J Neurochem, 2004. 90(2): p. 272-86.
24.    Ebner, D.C., et al., Strategies for skeletal muscle targeting in drug discovery. Curr Pharm Des, 2015. 21(10): p. 1327-36.
25.    Hansen, J.E., et al., Intranuclear protein transduction through a nucleoside salvage pathway. J Biol Chem, 2007. 282(29): p. 20790-3.

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