Chimeric Constructs between Human and Rat Equilibrative Nucleoside Transporters (hENT1 and rENT1) Reveal hENT1 Structural Domains Interacting with Coronary Vasoactive Drugs

Abstract

We have recently isolated cDNAs from human placenta and rat jejunum encoding the prototypic human and rat equilibrative nitrobenzylthioinosine (NBMPR)-sensitive nucleoside transporters hENT1 and rENT1. The two proteins (456 and 457 residues, M r 50,000) are 78% identical in amino acid sequence and contain 11 potential transmembrane segments (TMs) with a large putative extracellular loop between TMs 1 and 2 and a large cytoplasmic loop between TMs 6 and 7. When expressed in Xenopus oocytes, recombinant hENT1 and rENT1 transport both purine and pyrimidine nucleosides, including adenosine, and are inhibited by nanomolar concentrations of NBMPR. hENT1 is also potently inhibited by coronary vasodilator drugs (dipyridamole, dilazep, and draflazine), whereas rENT1 is insensitive to inhibition by these compounds (dipyridamole IC 50 values 190 nM (hENT1) and >10 M (rENT1) at 10 M uridine). In the present study, we have generated reciprocal chimeras between hENT1 and rENT1, using splice sites at residues 99 (end of TM 2) and 231 (end of TM 6), to identify structural domains of hENT1 responsible for transport inhibition by vasoactive compounds. Transplanting the amino-terminal half of hENT1 into rENT1 converted rENT1 into a dipyridamole/dilazep-sensitive transporter, whereas the amino-terminal half of rENT1 rendered hENT1 dipyridamole/dilazep-insensitive. Domain swaps within the amino-terminal halves of hENT1 and rENT1 identified residues 100 -231 (incorporating TMs 3-6) of hENT1 as the major site of vasodilator interaction. Since these drugs function as competitive inhibitors of nucleoside transport and NBMPR binding, TMs 3-6 are likely to form part of the substrate-binding site.

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

“…Comparative genomic analysis of ENT proteins across species also indicated that transmembrane domains 3-6 (exons 4-6) are highly conserved and are involved in binding and transport of nucleosides (Sankar et al, 2002). Experimental studies with chimeras from ENTs of rats and humans have demonstrated the involvement of transmembrane domains 3-6 in interactions with inhibitors and/or permeants (Sundaram et al, 1998(Sundaram et al, , 2001bYao et al, 2001Yao et al, , 2002.…”

“…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).…”

Ligand-gated Ion Channels

“…The affinities of draflazine, dilazep, KF24345 and dipyridamole at ENT1 transporters are species dependent, exhibiting lower affinity at rat transporters than at human transporters (Sundaram et al, 1998;Hammond and Archer, 2004).…”