Functional and Molecular Characterization of Nucleobase Transport by Recombinant Human and Rat Equilibrative Nucleoside Transporters 1 and 2

Abstract

The human (h) and rat (r) equilibrative (Na ؉ -independent) nucleoside transporters (ENTs) hENT1, rENT1, hENT2, and rENT2 belong to a family of integral membrane proteins with 11 transmembrane domains (TMs) and are distinguished functionally by differences in sensitivity to inhibition by nitrobenzylthioinosine and coronary vasoactive drugs. Structurally, the proteins have a large glycosylated loop between TMs 1 and 2 and a large cytoplasmic loop between TMs 6 and 7. In the present study, hENT1, rENT1, hENT2, and rENT2 were produced in Xenopus laevis oocytes and investigated for their ability to transport pyrimidine and purine nucleobases. hENT2 and rENT2 efficiently transported radiolabeled hypoxanthine, adenine, guanine, uracil, and thymine (apparent K m values 0.7-2.6 mM), and hENT2, but not rENT2, also transported cytosine. These findings were independently confirmed by hypoxanthine transport experiments with recombinant hENT2 produced in purine-cytosine permease (FCY2)-deficient Saccharomyces cerevisiae and provide the first direct demonstration that the ENT2 isoform is a dual mechanism for the cellular uptake of nucleosides and nucleobases, both of which are physiologically important salvage metabolites. In contrast, recombinant hENT1 and rENT1 mediated negligible oocyte fluxes of hypoxanthine relative to hENT2 and rENT2. Chimeric experiments between rENT1 and rENT2 using splice sites at rENT1 residues 99 (end of TM 2), 171 (between TMs 4 and 5), and 231 (end of TM 6) identified TMs 5-6 of rENT2 (amino acid residues 172-231) as a determinant of nucleobase transport activity, suggesting that this domain forms part(s) of the ENT2 substrate translocation channel.Plasma membrane transport processes for nucleosides and nucleobases play key roles in many aspects of mammalian physiology and pharmacology (1-5). In particular, uptake of exogenous nucleosides and nucleobases is the first step of nucleotide synthesis in tissues such as bone marrow and intestinal epithelium (and certain parasitic organisms) that lack de novo pathways for purine biosynthesis (5-7). The same transport processes also mediate cellular uptake of many synthetic nucleoside and nucleobase analogs used in cancer, viral, and parasite chemotherapy (3)(4)(5)8). Independent transport processes specific for nucleosides or nucleobases as well as shared mechanisms of nucleoside and nucleobase transport have been described (2, 4).In human and other mammalian cells and tissues, uptake of nucleosides is brought about by members of the concentrative (Na ϩ -dependent) nucleoside transporter (CNT) 1 and equilibrative (Na ϩ -independent) nucleoside transporter (ENT) families (3, 5). CNTs have been described primarily in specialized epithelia, whereas ENTs occur in most, possibly all, cell types and tissues. Three CNT and two ENT isoforms have been identified. Human (h) and rat (r) CNT1 and CNT2 both transport uridine, but are otherwise selective for pyrimidine (hCNT1 and rCNT1) and purine (hCNT2 and rCNT2) nucleosides (9 -14). In contrast, hCNT3 and its mous...

Molecular Determinants of Substrate Selectivity of a Novel Organic Cation Transporter (PMAT) in the SLC29 Family

“…The importance of this region is further supported by our mutagenesis data, which identified that key residues for substrate recognition are located in TM5. Our data are consistent with results from chimeric studies of the ENTs, which also showed that the N-terminal half (TM1-6) of ENT1 and -2 contains critical domains for substrate/inhibitor recognition (21,22). Together, these results indicate that despite the distinct substrate selectivity and low sequence homology, PMATs and the ENTs share a similar protein modular organization.…”

“…Based on these data, we hypothesized that despite the difference in substrate selectivity, PMAT and ENTs may share a similar tertiary structure and a similar arrangement of functional domains for substrate recognition and translocation. Earlier studies have suggested that the N-terminal half (TM1-6) of ENT1 and ENT2 contains critical domains for substrate/inhibitor recognition and translocation (21,22). To test our hypothesis, we first constructed two chimeric transporters, chimeras T1-6 and T7-11, between human PMAT and the prototype human equilibrative nucleoside transporter, hENT1 (Fig.…”

Nucleoside anticancer drugs: the role of nucleoside transporters in resistance to cancer chemotherapy

“…hENT1 and hENT2 mediate the well-characterized equilibrative-sensitive (es) and equilibrative-insensitive (ei) processes. Although both hENT1 and hENT2 have similar selectivities for nucleosides, hENT2 also recognizes and transports hypoxanthine and other purine nucleobases (Osses et al, 1996;Baldwin et al, 1999;Yao et al, 2002). The role of hENT3 and hENT4 in nucleoside transport processes has not yet been established (Baldwin et al, 2003).…”

“…Studies of the properties of chimeras between human and rat ENTs have established that transmembrane domains 3-6 contain residues responsible for sensitivity or resistance to coronary vasodilators (Sundaram et al, 1998) and to NBMPR (Sundaram et al, 2001b). Transmembrane domains 1-6 of ENT2 appear to be responsible for transport of 3 0 -deoxynucleosides (Yao et al, 2001) while transmembrane domains 5-6 appear to be involved in its transport of nucleobases (Yao et al, 2002). Using site-directed mutagenesis, binding of NBMPR to recombinant hENT1 produced in Saccharomyces cerevisiae has been investigated.…”

Functional characterization of a H+/nucleoside co‐transporter (CaCNT) from Candida albicans, a fungal member of the concentrative nucleoside transporter (CNT) family of membrane proteins

“…CNT1 and CNT2 both transport uridine and adenosine, but are otherwise selective for pyrimidine (hCNT1 and rCNT1) and purine (hCNT2 and rCNT2) nucleosides (Huang et al, 1994;Che et al, 1995, Yao et al, 1996aWang et al, 1997;Ritzel et al, 1997Ritzel et al, , 1998. hCNT3, its mouse (m) orthologue mCNT3 and a close relative from Eptatretus stouti, an ancient marine prevertebrate, transport both pyrimidine and purine nucleosides (Ritzel et al, 2001;Yao et al, 2002b). The relationships of these proteins to transport processes defined by functional studies are: ENT1 (es), ENT2 (ei ), CNT1 (cit), CNT2 (cif ) and CNT3 (cib).…”