2.A.57 The Equilibrative Nucleoside Transporter (ENT) Family
Several members of the ENT family (Pfam CLN3) have been functionally characterized (Engel et al., 2004; Griffiths et al., 1997b; Mäser et al., 1999; Sundaram et al., 1998; Vasudevan et al., 1998). The hENT1 is of human placental origin, is 456 amino acyl residues long and possesses 11 TMSs. It has an N-terminal mitochondrial targetting sequence and is expressed in the mitochondria and other organelles of many human tissues. Homologues have been sequenced from yeast, protozoa, plants, nematodes and mammals. Most characterized plant (and probably lower eukaryotic) ENTs act in a concentrative manner, defying their name (Girke et al. 2015). C. elegans possesses at least five such homologues. Among these are the two smaller nucleolar ''''delayed early response'''' gene products, HNP36, sequenced from humans and mice (Williams and Lanahan, 1995). The hENT1 and rENT1 proteins appear to exhibit broad specificity for purine and pyrimidine nucleosides and cytotoxic nucleoside analogues used in cancer and viral chemotherapy. Some are sensitive and others are insensitive to inhibition by nitrobenzyl thioinosine. hENT2 has higher affinity for adenosine, inosine and hypoxanthine than hENT1 but lower affinity for other nucleosides. Both human and rat isoforms of hENT1 are cell surface and organellar localized being found in mitochondria, nuclear envelopes and lysosomes. One, PMAT (TC #2.A.57.1.5), transports monoamines, probably by an H+ symport mechanism. Nucleoside drug analogues and inhibitors used in cancer chemotherapy include docetaxel, uridine-furane and S-(4-nitrobenzyl)-6-thioinosine (Drápela et al. 2018).
Nucleoside transporters have been identified in Trypanosoma brucei and Leishmania donovani. They transport adenosine and probably other nucleosides and nucleobases as well as several drugs. When reconstituted in yeast, one (called TbAT1) catalyzes adenosine uptake and confers susceptibility to melaminophenyl arsenicals. Tyrpanocide drug-resistant tyrpanosomes have a mutated TbAT1 gene. These protozoan proteins are 460-500 residues long and exhibit 10 putative TMSs. The three Leishmania donovani paralogues (NT1.1, NT1.2 and NT2) are all electrogenic proton symporters (Stein et al., 2003).
The 7 known human nucleosides transporters (hNTs) exhibit varying permeant selectivities and are found into 2 protein families: the solute carrier (SLC) 29 (SLC29A1, SLC29A2, SLC29A3, SLC29A4) and SLC28 (SLC28A1, SLC28A2, SLC28A3) proteins, otherwise known, respectively, as the human equilibrative NTs (hENTs, hENT1, hENT2, hENT3, hENT4) and human concentrative NTs (hCNTs, hCNT1, hCNT2, hCNT3) (Elwi et al., 2006). The well characterized hENTs (hENT1 and hENT2) are bidirectional facilitative diffusion transporters in plasma membranes; hENT3 and hENT4 are much less well known, although hENT3, found in lysosomal membranes, transports nucleosides and is pH dependent. hENT4-PMAT is a H+/adenosine cotransporter as well as a monoamine-organic cation transporter. The 3 hCNTs are unidirectional secondary active Na+/nucleoside cotransporters. In renal epithelial cells, hCNT1, hCNT2, and hCNT3, at apical membranes, and hENT1 and hENT2 at basolateral membranes, apparently work in concert to mediate reabsorption of nucleosides from lumen to blood, driven by Na+ gradients. Secretion of some physiological nucleosides, therapeutic nucleoside analog drugs, and nucleotide metabolites of therapeutic nucleoside and nucleobase drugs likely occurs through various xenobiotic transporters in renal epithelia, including organic cation transporters, organic anion transporters, multidrug resistance related proteins, and multidrug resistance proteins. Mounting evidence suggests that hENT1 may have a presence at both apical and basolateral membranes of renal epithelia, and thus may participate in both selective secretory and reabsorptive fluxes of nucleosides (Elwi et al., 2006).
Juvenile neuronal ceroid lipofuscinosis (JNCL) is a fatal childhood-onset neurodegenerative disorder caused by mutations in ceroid lipofuscinosis neuronal-3 (CLN3), a transmembrane protein of unresolved function. There may be blood-brain barrier (BBB) defects in JNCL. Cln3 is expressed in mouse brain endothelium. Tecedor et al. 2013 showed that CLN3 is necessary for normal trafficking of the microdomain-associated proteins caveolin-1, syntaxin-6, and multidrug resistance protein 1 (MDR1) in brain endothelial cells. CLN3-null cells have reduced caveolae, and impaired caveolae- and MDR1-related functions including endocytosis, drug efflux, and cell volume regulation. They also detected an abnormal blood-brain barrier response to osmotic stress in vivo and proposed that CLN3 facilitates golgi-to-plasma membrane transport of microdomain-associated proteins.
The best-characterized members of the human Ent 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. 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. By regulating the concentration of adenosine available to cell surface receptors, mammalian ENTs additionally influence physiological processes ranging from cardiovascular activity to neurotransmission (Young et al. 2008).
Recessive inheritance of loss of function mutations in CLN3 (TC# 2.A.57.5.8) are responsible for juvenile neuronal ceroid lipofuscinosis (Batten disease, or CLN3 disease), a fatal childhood onset neurodegenerative disease causing vision loss, seizures, progressive dementia, motor function loss and premature death (Cotman and Lefrancois 2021). CLN3 localizes to endosomes and lysosomes, and defects in endocytosis, autophagy, and lysosomal function are common findings in CLN3-deficiency model systems. Cotman and Lefrancois 2021 summarized the understanding of the CLN3 protein interaction network and discuss how this knowledge is starting to delineate the molecular pathogenesis of CLN3 disease. Accumulating evidence points towards CLN3 playing a role in regulation of the cytoskeleton and cytoskeletal associated proteins to tether cellular membranes, regulation of membrane complexes such as channels/transporters, and modulating the functions of small GTPases to effectively mediate vesicular movement and membrane dynamics.
The generalized transport reaction catalyzed by well characterized ENT family members is:
Nucleoside (out) → Nucleoside (in)
Equilibrative nucleoside transporter (ENT1) (present in the membranes surrounding the cell as well as eukaryotic organelles) (Lee et al., 2006). Residues 334 and 338 in TMS8 determine the inhibitor sensitivity, protein folding and catalytic turnover (Visser et al., 2007). The porter is inhibited by nanomolar concentrations of various structurally distinct coronary vasodilator drugs, including dipyridamole, dilazep, draflazine, soluflazine and nitrobenzylmercaptopurine ribonucleoside (NBMPR) (Paproski et al., 2008). It transports the A1 adenosine receptor agonist, tecadenoson (Lepist et al. 2013) and mediates gemcitabine (GEM) and folfirinox uptake, chemotheraputic agents for patients with metastatic pancreatic cancer (Orlandi et al. 2016). The matricellular protein, cysteine-rich angiogenic inducer 61 (CYR61), negatively regulates synthesis of the nucleoside transporters hENT1 and hCNT3, both of which transport gemcitabine (Hesler et al. 2016). These two transporters as well as ENT2 (TC# 2.A.57.1.8) are able to take up the adenosine analogue, fludarabine (AraFA), used to treat cancer (lymphomas and leukemia) (Gorzkiewicz et al. 2018). NEM modification of Cys(416), which is located at the inner extremity of TM10, results in inhibition of hENT1 uridine transport and NBMPR binding by constraining the protein in its inward-facing conformation (Yao et al. 2018). Ent1 transports nucleosides and bases, like Ent2, but Ent1 is more important than Ent2 or CNT3 in determining plasma adenosine concentrations (Altaweraqi et al. 2020). The Thr residue at position 336 may help determine inhibitor and substrate sensitivity (Boakes et al. 2022).
SLC29A1 or Ent1 of Homo sapiens
ENT7 of 417 aas and 11 TMSs, an equilibrative nucleoside transporter in contrast to most plant ENT proteins which are concentrative, functioning by H+ symport (Girke et al. 2015). Binding of purine and pyrimidine nucleosides to the purified recombinant protein, and binding of nucleobases has been demonstrated (Girke et al. 2015).
ENT7 of Arabidopsis thaliana (Mouse-ear cress)
Putative equilibrative nucleoside transporter 1 isoform X1 of 432 aas and 11 TMSs.
ENT of Harp seal herpesvirus
Uncharacterized protein of 379 aas and 10 TMSs in a 5 + 5 TMS arrangement.
UP of Entamoeba histolytica
Putative nucleoside transporter of 481 aas and 12 TMSs.
UP of Entamoeba histolytica
Equilabrative nucleoside transporter of 445 aas and 11 TMSs. This system and the human isoforms are inhibited by nanomolar concentrations of dipyridamole, and residues involved in the binding have been identified (Visser et al. 2005).
Ent of Caenorhabditis elegans
Equilibrative nucleoside transporter, ENT1 of 423 aas and 11 TMSs.
ENT1 of Oryza sativa subsp. japonica (Rice)
Equilibrative nuceloside transporter 2, ENT2, of 418 aas and 11 TMSs. OsENT2 transports adenosine and uridine with high affinity (adenosine, Km = 3.0 μM; uridine, Km = 0.7 μm) (Hirose et al. 2005). Purine or pyrimidine nucleosides and 2'-deoxynucleosides strongly inhibit adenosine transport via OsENT2, suggesting that it possesses broad substrate specificity. OsENT2-mediated adenosine transport is resistant to the typical inhibitors of mammalian ENTs, nitrobenzylmercaptopurine ribonucleoside, dilazep, and dipyridamole. Transport activity is maximal at pH 5.0. In competition experiments with various cytokinins, adenosine transport is 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 is predominantly expressed in roots. The OsENT2 promoter drives expression in the scutellum during germination and in vascular tissues in germinated plants, suggesting participation in the retrieval of endosperm-derived nucleosides by the germinating embryo and in the long-distance transport of nucleosides in growing plants, respectively (Hirose et al. 2005). Three other ENTs have been partially characterized, Ent4, Ent6 and Ent7; all three proteins exhibit broad substrate specificity and transport the purine nucleosides adenosine and guanosine, as well as the pyrimidine nucleosides cytidine and uridine. The apparent Km values were in the range 3-94 μM, and transport was inhibited most strongly by deoxynucleosides, and to a lesser extent by nucleobases. Ent6 is in the plasma membrane (Wormit et al. 2004). Ent2 transports both nucleosides and bases, and is less important than Ent1 (Altaweraqi et al. 2020).
ENT2 of Oryza sativa, Japonica group
Equilibrative nitrobenzylmercaptopurine riboside (NBMPR)-insensitive nucleoside transporter of 456 aas, ENT2 (Slc29a2). In humans, the same gene product is the nucleolar protein, HNP36 (function unknown).
ENT2 (HNP36) of Mus musculus (Q61672)
Equilibrative high affinity nucleoside transporter (nitrobenzyl-thioinosine-sensitive) (transports thymidine, adenosine, cytosine, and guanosine; inosine and hypoxanthine are poorly transported). Uridine uptake in the basolateral membrane of sertoli cells is selectively inhibited by 100 nM nitrobenzylmercaptopurine riboside (NBMPR, 6-S-[(4-nitrophenyl)methyl]-6-thioinosine) (Klein et al. 2013).
rENT1 of Rattus norvegicus
The brain plasma membrane monoamine transporter, PMAT, Slc29A4 or ENT4, a polyspecific orgnaocation transporter. (transports serotonin [Km=110 μM), dopamine (Km=330 μM), metformin (Km=1.3 mM) and the neurotoxin, 1-methyl-4-phenylpyridinium (Km=33 μM)) (Nucleosides and nucleobases are not transported; transport is sensitive to the membrane potential, but is Na+ and Cl- independent.) (Engel et al., 2004). Also expressed in kidney apical membranes where it transports MPP+ by a ΔΨ-dependent process (Xia et al., 2007). TMSs 1 - 6 bear the substrate recognition site, and Glu206 in TMS5 determines the catioin specificiity. An E206Q mutant lost cation selectivity and transported uridine (Zhou et al. 2007). Residues Ile89 and thr220 influence its organic cation transport activity and sensitivity to inhibition by dilazep (Ho et al., 2012). May play a role in insulin secretion in β-cells (Kobayashi et al. 2016).
SLC29A4 of Homo sapiens
Equilibrative (Na+-independent) low affinity nucleoside transporter, hENT3 or SLC29A3 (transports nucleosides with broad selectivity and low affinity; also transports adenine). Relatively low sensitivity to classical nucleoside transport inhibitors, nitrobenzylthioinosine, dipyridamole, and dilazep. pH optimum=5.5; present in acidic intracellular compartments (Baldwin et al., 2005). (Present largely in the lysosomes). May cause histiocytosis, perturb lysosome function and upset macrophage homeostasis when defective (Hsu et al., 2012; Farooq et al., 2012). A single nucleotide polymorphism (SNP) in ENT3 may be a risk factor for squamous cell carcioma (Li et al. 2010). Mutations cause H syndrome, an autosomal recessive genodermatosis characterized by hyperpigmented and hypertrichotic skin (Liu et al. 2015).
SLC29A3 of Homo sapiens
The fluorouridine insensitive 1 (Fur1) or Ent3 pyrimidine nucleoside transporter (Traub et al., 2007). AtENT4, AtENT6 and AtENT7). Three paralogs, Ent4, Ent6 and Ent7, exhibited broad substrate specificity and transported the purine nucleosides adenosine and guanosine, as well as the pyrimidine nucleosides cytidine and uridine (Wormit et al. 2004). The apparent Km values were in the range 3-94 μM, and transport was inhibited strongly by deoxynucleosides, and to a smaller extent by nucleobases.
Ent3 of Arabidopsis thaliana (Q9M0Y3)
Equilibrative nucleoside transporter 2 (36 kDa nucleolar protein HNP36) (Delayed-early response protein 12) (Equilibrative nitrobenzylmercaptopurine riboside-insensitive nucleoside transporter) (Equilibrative NBMPR-insensitive nucleoside transporter) (Hydrophobic nucleolar protein, 36 kDa) (Nucleoside transporter, ei-type) (Solute carrier family 29 member 2). Takes up the adenosine analogue, fludarabine (AraFA), used to treat cancer (lymphomas and leukemia) (Gorzkiewicz et al. 2018).
SLC29A2 of Homo sapiens
Uncharacterized protein of 359 aas and 9 TMSs.
UP of Chondrus crispus
Concentrative nucleoside (adenosine, uridine, cytosine, tubercidin):H+ symporter, NT1.1 (The Leishmania major orthologue, NT1.1 (Q4QF58), also transports tubercidin) (Stein et al., 2003). TMS 5 is an amphipathic helix that forms part of the nucleoside translocation pathway (Valdés et al. 2004). The intracellular and extracellular gates have been defined by modeling FucP (Valdés et al. 2012; Valdés et al. 2014).
NT1.1 of Leishmania donovani
Nucleoside/nucleobase transporter 1, AT-A or NT11.1, of 482 aas and 11 TMSs. Transports adenine, xanthine and hypoxanthine as well as the drug, pentamidine (Schmidt et al. 2018; Zhang et al. 2022).
AT-A of Trypanosoma brucei
Putative nucleoside transporter, NT3 or ENT3, of 437 aas and 11 TMSs in a 6 + 5 TMS arrangement.
NT3 or Plasmodium falciparum
Nucleoside (nucleobase, drug) transporter of 463 aas, AT1 or P2. Transports adenosine and adenine as well as the drugs, eflornithine, melarsoprol, pentamidine, diminazene and cordycepin (Schmidt et al. 2018; Zhang et al. 2022); Kasozi et al. 2022).
TbAT1 of Trypanosoma brucei
High affinity, concentrative nucleoside (inosine, formycin, guanosine):H+ symporter, NT2 (Stein et al., 2003). Mutations confer drug (formycin) resistance and drug transport deficiency (Galazka et al. 2006). Asp389 is critical for transporter function without affecting ligand affinity or plasma membrane targeting, but Asn175 and Asp389 (when mutated as a second site mutation) lie in close proximity to each other; second-site suppressor mutations cluster to one region of the transporter, suggested that Asp175 is conformationally sensitive (Arastu-Kapur et al. 2005). Development of glutamatergic/cholinergic postmitotic human neurons is induced by short-term treatment with nucleoside analogues such as cytosine β-D-arabinofuranoside, 2'-O-methyl substituted 2-deoxy-β-D-ribofuranosyl residues as glyconic moieties, and cladribine (Raasch et al. 2015; González-Burguera et al. 2016).
NT2 of Leishmania donovani
Nucleotide transporter 2, NT2, specific for inosine and guanosine, but mutations in TMS 4 which may line the channel allow uptake of adenosine (Arendt & Ullman et al., 2010). (Most similar to 2.A.57.2.3).
NT2 of Crithidia fasciculata (Q9GTP4)
High affinity adenosine-specific nucleoside transporter (Arendt 2013). Similar to 2.A.547.2.1. A lysine residue in TMS4 plays an important role in substrate affinity.
Adenosine transporter of Crithidia fasciculata
Uncharacterized protein of 436 aas and 11 TMSs
UP of Meliniomyces bicolor
Uncharacterized protein of 746 aas and 7 TMSs. This protein has its TMSs in the N-terminal half of the protein, and the C-terminal hydrophilic half shows statistically meaningful seqence similarity with cytochrome P450 isoforms (e.g., P10632) (Mustafa et al. 2020); M Saier, unpublished observation.
UP of Aspergillus fumigatus
The parasite plasma membrane equilibrative nucleoside transporter, PfNT1 or PfENT1, of 422 aas and 11 or 12 TMSs in a 6 + 5 TMS arrangement. Both L- and D-nucleosides of both purines and pyrimidines are transported; L-nucleosides are transported with low affinity (transports adenosine, inosine and thymidine (KM values=1-2mM) (Downie et al., 2006)). ENT1 is the primary uptake transporter for purines, and transmembrane segments 2, 10 and 11 appear to line the purine permeation pathway (Riegelhaupt et al., 2010, Nishtala et al. 2018).
PfNT1 of Plasmodium falciparum
The intracellular (endoplasmic reticulum) nucleoside transporter, NT2, of 585 aas and 11 TMSs in a 6 + 5 TMS arrangement. Transports purine nucleosides and 5-fluorouridine (PfNT-2; Downie et al., 2010)
PfNT2 of Plasmodium falciparum (Q8IB78)
Uncharacterized protein of 443 aas and 11 TMSs.
UP of Eimeria maxima
Adenine/adenosine uptake porter, NT4 or ENT4, of 434 aas and 11 TMSs in a 6 + 5 TMS arrangement.
Nucleobase/nucleoside porter of Plasmodium falciparum
Vacuolar protein sorting-associated protein 51, putative (VPS51) protein with two transmembrane domains of 6 and 5 TMSs, respectively, homologous to the transmembrane domains of 2.A.57.4.4 (NT4).
NT5 of Plasmodium ovale curtisi
Battenin (BTN) or ceroid lipofuscinosis neuronal-3 (CLN3), with 6 TMSs and the N- and C-termini in the cytoplasm (Nugent et al. 2008). May function in trafficking from the Golgi to the plasma membrane (Tecedor et al. 2013). Mutations give rise to the disease, juvenile neuronal ceroid lipofuscinosis (JNCL) or Batten disease in humans, a fatal childhood-onset neurodegenerative disorder caused by mutations in CLN3. May indirectly regulate activity of the Na+,K+-ATPase (Uusi-Rauva et al. 2008).
Cln3 of Mus musculus
10 TMS homologue
10 TMS homologue of Trichomonas vaginalis
Cln3 family protein of 513 aas and 11 putative TMSs.
Cln3 family protein of Oxytricha trifallax
Uncharacterized protein of 5432 aas
UP of Trypanosoma cruzi
Battenin homologue of 439 aas and 10 or 11 TMSs.
Battenin of Acanthamoeba castellanii
Battenin or Cln3, of 438 aas and 11 TMSs, is phosphorylated on both serine and threonine residues by three protein kinases (Michalewski et al. 1998). The N-terminus of Battenin is a protein interaction domain (Moroziewicz et al. 2006). Cln3 is involved in microtubule-dependent, anterograde transport of late endosomes and lysosomes. CLN3 interacts directly with active, guanosine-5'-triphosphate (GTP)-bound Rab7 and with the Rab7-interacting lysosomal protein (RILP) that anchors the dynein motor (Uusi-Rauva et al. 2012). Loss-of-function mutations in CLN3 are responsible for juvenile-onset neuronal ceroid lipofuscinosis (JNCL), or Batten disease, which is an incurable lysosomal disease that manifests with vision loss, followed by seizures and progressive neurodegeneration, robbing children of motor skills, speech and cognition, and eventually leading to death in the second or third decade of life. A current understanding of CLN3 structure, function and dysfunction in JNCL can be found in (Cotman and Staropoli 2012). Mutational variability in CLN3 gives rise to JNCL (Sher et al. 2019). The N-terminus of Battenin is a protein interaction domain (Moroziewicz et al. 2006). Dysregulation of intracellular calcium homeostasis results from the absence of a functional CLN3 protein. and has been linked to synaptic dysfunction and accelerated apoptosis in vulnerable neuronal cells. Prolonged increased intracellular calcium may be a trigger for neuronal apoptosis and cellular loss in JNCL (An Haack et al. 2011).
Cln3 of Homo sapiens
Uncharacterized protein of 418 aas and 10 - 11 TMSs
UP of Mycosphaerella pini (Red band needle blight fungus) (Dothistroma septosporum)
Uncharacterized protein of 467 aas and 10 TMSs.
UP of Phialocephala scopiformis