Mutation of Residue 33 of Human Equilibrative Nucleoside Transporters 1 and 2 Alters Sensitivity to Inhibition of Transport by Dilazep and Dipyridamole

Abstract

Human equilibrative nucleoside transporters (hENT) 1 and 2 differ in that hENT1 is inhibited by nanomolar concentrations of dipyridamole and dilazep, whereas hENT2 is 2 and 3 orders of magnitude less sensitive, respectively. When a yeast expression plasmid containing the hENT1 cDNA was randomly mutated and screened by phenotypic complementation in Saccharomyces cerevisiae to identify mutants with reduced sensitivity to dilazep, clones with a point mutation that converted Met 33 to Ile (hENT1-M33I) were obtained. Characterization of the mutant protein in S. cerevisiae and Xenopus laevis oocytes revealed that the mutant had less than one-tenth the sensitivity to dilazep and dipyridamole than wild type hENT1, with no change in nitrobenzylmercaptopurine ribonucleoside (NBMPR) sensitivity or apparent uridine affinity. To determine whether the reciprocal mutation in hENT2 (Ile 33 to Met) also altered sensitivity to dilazep and dipyridamole, hENT2-I33M was created by site-directed mutagenesis. Although the resulting mutant (hENT2-I33M) displayed >10-fold higher dilazep and dipyridamole sensitivity and >8-fold higher uridine affinity compared with wild type hENT2, it retained insensitivity to NBMPR. These data established that mutation of residue 33 (Met versus Ile) of hENT1 and hENT2 altered the dilazep and dipyridamole sensitivities in both proteins, suggesting that a common region of inhibitor interaction has been identified.

Second-site Suppression of a Nonfunctional Mutation within the Leishmania donovani Inosine-Guanosine Transporter

“…Conserved Gly residues within transmembrane domains (TM) 4 and 5 of human ENT1 (hENT1) and TM5 of LdNT1 have been shown to be critical for transport function and ligand selectivity (4,5), and a Leu within TM2 of hENT1 has also been revealed as a ligand specificity determinant (6). Additional studies have supported a role for TMs 3-6 (7) and Met 33 within TM1 of hENT1 in the binding of dipyridamole and dilazep (8). Dipyridamole and dilazep are potent inhibitors of hENT1 but do not significantly affect the majority of parasite nucleoside transporters (9).…”

Adenosine as a signaling molecule in the retina: biochemical and developmental aspects

“…The presence of nucleoside transporters was described in several tissues and central nervous system areas (Cass et al 1998, Parkinson 1999) and subdivided in two major subclasses, concentrative (CNTs, sodium-dependent) and equilibrative (ENTs, sodium-independent) (Visser et al 2002). The equilibrative transporters are ubiquitously distributed and further classified as sensitive (es or ENT1) or insensitive (ei or ENT2) to the inhibitor Nitrobenzylthioinosine (NBI).…”

Molecular Basis of Nucleobase Transport Systems in Mammals

“…The most notable characteristic of ENT1-mediated transport is that it can be inhibited by extremely low concentrations of NBMPR and dipyridamole; for example, their respective K i values for human ENT1 (hENT1) are 6 and 48 nM, respectively. 29) (Detailed information on the physiology and function of ENTs as nucleoside transporters can be found elsewhere. 30,31) ) Recently, Yao et al have demonstrated that, in the Xenopus laevis oocyte expression system, ENT1 has the capability to transport not only nucleosides but also nucleobases such as adenine, hypoxanthine, guanine, thymine, and uracil 8) (Table 1).…”