2.A.41 The Concentrative Nucleoside Transporter (CNT) Family
Sequenced members of the CNT family are derived from Gram-negative and Gram-positive bacteria as well as yeast and animals. They are of about 400 residues (bacterial) or about 600-700 residues (eukaryotes) with 10-14 transmembrane α-helical spanners (TMSs). The rat CNT1 has been shown to have 13 TMSs with the hydrophilic N-terminus in the cytoplasm and the C-terminus on the extracellular side of the intestinal or renal brush border membranes of these polarized epithelial cells (Hamilton et al., 2001). The C. albicans homologue is probably of the same topology. The first 3 TMSs of the mammalian CNT1 are non-essential and are, in fact, absent from the bacterial systems (Hamilton et al., 2001). Nucleoside drug analogues and inhibitors used in cancer chemotherapy include docetaxel, uridine-furane and S-(4-nitrobenzyl)-6-thioinosine (Drápela et al. 2018). Members of this family have been reported to have the CNT1 fold (Ferrada and Superti-Furga 2022). CNTs can transport NAD+ and cyclic ADP-riblse which acts on ryanodine receptors (RyRs) (Astigiano et al. 2022)
In bacteria and yeast, CNT family members are energized by H+ symport, but in mammals they are energized by Na+ symport. The different transporters exhibit differing specificities for nucleosides. Thus, the E. coli NupC permease transports all nucleosides (both ribo- and deoxyribonucleosides) except hypoxanthine and guanine nucleosides. Another system in E. coli, NupG, a member of the MFS (TC #2.A.1.10.1), transports all ribo- and deoxyribonucleosides, while a NupG homologue, XapB (TC #2.A.1.10.2), apparently transports only xanthine. Similarly, in B. subtilis, there is evidence for three distinct nucleoside permeases, one specific for hypoxanthine and guanine nucleosides, a second specific for adenine nucleosides, and a third (B. subtilis NupC) specific for pyrimidine nucleosides (cytidine and uridine and the corresponding deoxyribonucleosides).
The mammalian permease members of the CNT family also exhibit differing specificities. Thus, rats possess at least two NupC homologues, one specific for both purine and pyrimidine nucleosides (gbU10279) and one specific for purine nucleosides (gbU25055). At least three paralogues have been characterized from humans. One human homologue (CNT1) transports pyrimidine nucleosides and adenosine, but deoxyadenosine and guanosine are poor substrates of this permease. Another (CNT2) is selective for purine nucleosides. Alteration of just a few amino acyl residues in TMSs 7 and 8 interconverts their specificities. A third homologue (CNT3) transports both purine and pyrimidine nucleosides with broad specificity (Ritzel et al., 2001). All of these transporters also accumulate various nucleoside analogue drugs such as cladribrine, 2CdA. The rat CNT2 transports this drug much better than the human orthologue due to residue substitutions in the C-terminal half of the proteins (Owen et al., 2006).
The phylogenetic tree for the CNT family shows three clusters. One includes the NupC proteins of E. coli and B. subtilis, the second includes all mammalian symporters, and the third includes functionally uncharacterized bacterial homologues (Saier et al., 1999).
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).
The generalized transport reaction for permeases of the CNT family is:
Nucleoside (out) + n[H+ or Na+] (out) → Nucleoside (in) + n[H+ or Na+] (in)
Pyrimidine nucleoside:H+ symporter, NupC (Craig et al. 1994; Patching et al. 2005). Wild-type NupC had an apparent affinity for uridine of 22.2 +/- 3.7 muM and an apparent binding affinity of 1.8-2.6 mM, and various mutants with alterred properties were isolated and characterized (Sun et al. 2017). ADP-glucose is also a substrate of this system (Almagro et al. 2018).
NupC of E. coli (P0AFF2)
Nucleoside permease NupX
NupX of Escherichia coli
Concentrative nucleoside transporter, CNT, of 418 aas and 12 TMSs. A repeat-swapped model of VcCNT predicts that nucleoside transport occurs via a mechanism involving an elevator-like substrate binding domain movement across the membrane (Vergara-Jaque et al. 2015).
CNT of Vibrio cholerae
Concentrative nucleotide transporter of 590 aas and 12 TMSs in a 1 + 4 + 1 + 1 + 4 + 1 TMS arrangement, DmCnt1. Mutations in DmCnt1 alters spermatid maturation and mating behavior (Maaroufi et al. 2022).
Cnt1 of Drosophila melanogaster
Pyrimidine-preferring nucleoside:Na+ symporter, CNT1 (Na+/nucleoside = 2)(transports uridine, gemcitabine and 5'-deoxy-5'-fluorouridine) (Larráyoz et al., 2004), but in addition to pyrimidine nucelosides, it transports adenosine (Altaweraqi et al. 2020).
SLC28A1 of Homo sapiens
Purine nucleoside, uridine, and 2'3'dideoxyinosine cladribrine:Na+ symporter, CNT2 (Na+/nucleoside = 1) (Owen et al., 2006; Altaweraqi et al. 2020).
SLC28A2 of Homo sapiens
Broad-specificity nucleoside:Na+, H+ and Li+ symporter, hCNT3 (Slc28a3) transports a broad range of both purine and pyrimidine nucleosides as well as anticancer and antiviral nucleoside drugs, but guanosine, 3'azido-3-deoxythymidine and 2',3'-dideoxycytidine, which are substrates with Na+, are not substrates with H+. Both of the two cation-binding sites can apparently bind Na+, but only one can bind H+, and the Na+ and H+ forms transport different ranges of substrates. (Note: Cnt1 and Cnt2 are Na+-specific.) (Smith et al., 2005). (Na+/nucleoside = 2; Na+ + H+/nucleoside = 2; H+/nucleoside = 1). The matricellular protein, cysteine-rich angiogenic inducer 61 (CYR61), negatively regulates synthesis of the nucleoside transporters hENT1 and hCNT3, both of which transport the anti-cancer agent, gemcitabine (Hesler et al. 2016). Also probably transports gemcitabine, 3'-azido-3'-deoxythymidine (AZT), ribavirin and 3-deazauridine. Modeling revealed mobility of selected binding site and homotrimer interface residues (Latek 2017). The 3-D structure has been solved at 3.6 Å resolution by cryoEM (Zhou et al. 2020). As for its bacterial homologs, hCNT3 presents a trimeric architecture with additional N-terminal transmembrane helices to stabilize the conserved central domains. The conserved binding sites for the substrate and sodium ions unravel the selective nucleoside transport and distinct coupling mechanism (Zhou et al. 2020). A multistep elevator-like transport mechanism for nucleoside transport has been proposed (Duan et al. 2021).
CNT3 of Homo sapiens (Q9ERH8)
Broad specificity nucleoside:H+ symporter (1:1 stoichiometry). Adenosine, uridine, inosine, and guanosine are transported but not cytidine, thymidine or the nucleobase hypoxanthine (Km range: 15-65 μM). Purine and uridine nucleoside drug analogues including cordycepin (3'-deoxyadenosine) are substrates.
CNT of Candida albicans, (Q874I3)
Solute carrier family 28 member 3 (Concentrative Na+-nucleoside cotransporter 3) (CNT 3) (hCNT3). This protein is distinct from TC# 2.A.41.2.6 (78% identity) although these two proteins are called Slc28a3 and CNT3 and have the same description in UniProt (see 2.A.41.2.6 for a more complete description).
SLC28A3 of Homo sapiens
Putative pseudouridine transporter, PsuT
PsuT of Escherichia coli