Residues Met89 and Ser160 in the Human Equilibrative Nucleoside Transporter 1 Affect Its Affinity for Adenosine, Guanosine, S-(4-Nitrobenzyl)-mercaptopurine Riboside, and Dipyridamole

The human equilibrative nucleoside transporter 1 (hENT1) is an important modulator of the physiological action of adenosine. We identified amino acid residues involved in adenosine transport using a yeast-based assay to rapidly screen and identify randomly generated hENT1 mutants that exhibited decreased sensitivity to inhibition of adenosine transport by various hENT1 competitive inhibitors. We identified Met89 and Ser160 as important in the affinity of hENT1 for various substrates and inhibitors. Mutation to Met89Cys or Ser160Cys significantly (p 0.05) increased the S-(4-nitrobenzyl)-mercaptopurine riboside (NBMPR) IC50 values by approximately 4and 6-fold, respectively (42 13 and 65 1.6 nM) compared with the wild-type transporter (11 0.7 nM). The double mutant Met89Cys/Ser160Cys synergistically increased the NBMPR IC50 value to approximately 19-fold of that of the wild-type transporter. In contrast, compared with wild-type hENT1, the sensitivity to dipyridamole inhibition was significantly (p 0.05) increased by only the Ser160Cys ( 2.6-fold) or the double mutant Met89Cys/Ser160Cys ( 4.7-fold) but not by the Met89Cys mutant. Mutation to Met89Cys or Ser160Cys increased the Km of adenosine ( 8and 3-fold) and the Ki of guanosine ( 6and 2-fold). The double mutant increased both the Km value of adenosine and the Ki value of guanosine by 8-fold and seemed to confer no additional reduction in adenosine or guanosine affinity than that by mutation of Met89 alone. Together, these data indicate that transmembrane domains (TMDs) 2 (Met89) and 4 (Ser160) of hENT1 interact and are important in conferring sensitivity to NBMPR. In contrast, Ser160 and Met89 of hENT1, respectively, play a dominant role in conferring sensitivity to dipyridamole and adenosine/ guanosine affinity. The endogenous nucleoside adenosine is an important regulator in autocrine and paracrine signaling through its interactions with adenosine receptors (A1, A2A, A2B, and A3) located in the plasma membrane (Mubagwa and Flameng, 2001). The extracellular adenosine concentration, and therefore its physiological and pharmacological activity, is modulated by nucleoside transporters that actively (concentrative nucleoside transporters) or facilitatively (equilibrative nucleoside transporters, ENTs) transport adenosine into the cell (Kong et al., 2004). For example, ENTs modulate extracellular adenosine concentration at the adenosine receptors in ethanol-induced ataxia (Choi et al., 2004), in adenosinemediated cardioprotection in ischemia/reperfusion injury (Chaudary et al., 2004; Taniguchi et al., 2004), and in adenosine-mediated neuromodulation (Snell et al., 2004). ENTs also transport a number of antiviral and anticancer drugs such as ribavirin (Jarvis et al., 1998) and gemcitabine (Mackey et al., 1998). Therefore, a better understanding of the molecular mechanisms by which adenosine and other nucleosides (including nucleoside drugs) bind to and are translocated by nucleoside transporters may aid in the development of new drugs that either modulate adenosine availability to adenosine receptors or are improved antiviral or anticancer drugs. hENT1, an equilibrative nucleoside transporter, is the most widely expressed member of the ENT family and is ubiquitously expressed (Pennycooke et al., 2001) including in the intestine (Chandrasena et al., 1997) and the kidney (Franco et al., 1990). hENT1 has broad nucleoside substrate selectively (Ward et al., 2000) and has an affinity for adenosine in the low micromolar range (Ward et al., 2000). hENT1 This work was supported by National Institutes of Health grant GM54447. Parts of this work were presented at the American Association of Pharmaceutical Scientists (AAPS) 2003 and 2004 National Meetings and 2003 AAPS Drug Transport Workshop. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.104.008102. ABBREVIATIONS: ENT, equilibrative nucleoside transporter; NBMPR, S-(4-nitrobenzyl)-mercaptopurine riboside; TMD, transmembrane domain. 0026-895X/05/6703-837–844$20.00 MOLECULAR PHARMACOLOGY Vol. 67, No. 3 Copyright © 2005 The American Society for Pharmacology and Experimental Therapeutics 8102/1193908 Mol Pharmacol 67:837–844, 2005 Printed in U.S.A. 837 at A PE T Jornals on D ecem er 8, 2017 m oharm .aspeurnals.org D ow nladed from is characterized by potent inhibition by NBMPR (Ward et al., 2000) and by inhibition by the non-nucleosides dipyridamole and dilazep (Visser et al., 2002). A number of hENT1 amino acid residues have been identified which, when mutated, alter the affinity of hENT1 toward its substrates or inhibitors (Fig. 1 and Table 1). Using two different yeast expression assays, our laboratory has identified a number of these amino acid residues (SenGupta et al., 2002; Endres et al., 2004). The “adenosine rescue” assay is one of these and relies on the transport of extracellular adenosine by heterologously expressed hENT1 for yeast growth and survival. This assay can identify randomly generated hENT1 mutants that are capable of transporting adenosine despite the presence of the hENT1 inhibitors such as NBMPR, dipyridamole, or dilazep. In this article, we report the use of this adenosine rescue assay to identify hENT1 amino acid residues Met89 and Ser160 which, when either singly or simultaneously mutated, selectively reduce hENT1 affinity for adenosine or guanosine (but not other natural nucleosides) and hENT1 sensitivity to inhibition by NBMPR or dipyridamole but not dilazep. In addition, we have identified for the first time that these two residues seem to act synergistically in reducing the sensitivity of the transporter to NBMPR inhibition. Materials and Methods Screening of Random Mutants by Phenotypic Complementation. We screened our library of randomly generated hENT1 expressed in yeast strain W303-1A (MATa ade2-1, can1-100, cyh2, his3-11,15, leu1-c, leu2-3,112, trp1-1, and ura3-1) for random mutants resistant to inhibition by NBMPR, dipyridamole, and dilazep in an adenosine rescue plate assay described previously (Endres et al., 2004). In brief, the yeast cells transformed with random mutants were replica-plated onto GR-Ura-Ade [2% galactose, 1% raffinose, 1% yeast nitrogen base (Difco, Detroit, MI), and 1% amino acid mix-uracil-adenine) plates containing 0 or 150 M adenosine and the presence or absence of 2 M NBMPR, 50 M dipyridamole, or 10 M dilazep (Sigma-Aldrich, St. Louis, MO). These plates were incubated at 30°C for 4 days and scored for growth. The yeast-expressing random mutants that showed resistance to inhibition of adenosine complementation were identified, and the plasmid’s contribution to the inhibitor-resistant phenotype was confirmed by rescreening. Candidate plasmids that successfully reproduced the plate assay phenotype were sequenced using the BigDye Terminator reaction kit (Applied Biosystems, Foster City, CA) and analyzed by the University of Washington DNA Sequencing and Gene Analysis Center