Molecular cloning and functional characterization of inhibitor-sensitive (mENT1) and inhibitor-resistant (mENT2) equilibrative nucleoside transporters from mouse brain

Abstract

Mammalian cells express at least two subtypes of equilibrative nucleoside transporters, i.e. ENT1 and ENT2, which can be distinguished functionally by their sensitivity and resistance respectively to inhibition by nitrobenzylthioinosine. The ENT1 transporters exhibit distinctive species differences in their sensitivities to inhibition by dipyridamole, dilazep and draflazine (human mouse rat). A comparison of the ENT1 structures in the three species would facilitate the identification of the regions involved in the actions of these cardioprotective agents. We now report the molecular cloning and functional expression of the murine (m)ENT1 and mENT2 transporters. mENT1 and mENT2 encode proteins containing 458 and 456 residues respectively, with a predicted 11-transmembrane-domain topology. mENT1 has 88 % and 78 % amino acid identity with rat ENT1 and human ENT1 respectively ; mENT2 is more highly conserved, with 94 % and 88 % identity with rat ENT2 and human ENT2 respectively. We have also isolated two additional distinct cDNAs that encode proteins similar to mENT1 ; these probably represent distinct mENT1 isoforms or alternative splicing products. One cDNA encodes a protein with two additional amino acids

Expression of human equilibrative nucleoside transporter 1 in mouse neurons regulates adenosine levels in physiological and hypoxic‐ischemic conditions

“…This is a potent and selective inhibitor for ENT1 and no drug with similar selectivity is currently available for ENT2. Dipyridamole is used to inhibit ENT2 but it also inhibits ENT1; in addition, it has low nanomolar affinity for human ENT2 but high nanomolar or micromolar affinity for ENT2 inhibition in rat and mouse cells (Yao et al 1997;Kiss et al 2000;Parkinson et al 2005).…”

Cloned Blood–Brain Barrier Adenosine Transporter is Identical to the Rat Concentrative Na+ Nucleoside Cotransporter CNT2

“…Adenosine transporters may be either sodium-dependent concentrative nucleoside transporters (CNT) or equilibrative nucleoside transporters (ENT), and may be either insensitive or sensitive to inhibition by NBTI (Cass et al, 1998). The ENT2 isoform is expressed at the choroid plexus epithelium in brain (Anderson et al, 1999), and ENT1 and ENT2 are expressed in mouse brain (Kiss et al, 2000). However, it is not known which ENT or CNT isoform mediates BBB transport of adenosine in vivo at the brain capillary endothelium.…”

Current Progress on Equilibrative Nucleoside Transporter Function and Inhibitor Design

“…41 These observations suggest that in addition to the main high-affinity dilazep and dipyridamole binding site, hENT1 may contain a second low-affinity allosteric site for these ligands. 42,43 Hence, it is assumed that hENT1 contains a high-affinity binding site for permeants, NBMPR, and other inhibitors, such as dilazep and dipyridamole, and an allosteric low-affinity site that binds nucleosides, nucleobases, and inhibitors when present at high concentrations. It is currently not clear whether residue 33 of hENT1 and hENT2 is directly involved in permeant or inhibitor binding or whether the effects observed with M33 mutations are due to changes in the ENT tertiary structure.…”