Erythrocyte nucleoside transport: asymmetrical binding of nitrobenzylthioinosine to nucleoside permeation sites

Abstract

1. Nitrobenzylthioinosine is a potent and specific inhibitor of nucleoside translocation in animal cells. Kinetic and inhibitor binding studies were undertaken to clarify how this inhibitor interacts with the nucleoside transporter from human and nucleoside‐permeable type sheep erythrocytes. 2. [3H]nitrobenzylthioinosine inhibition of zero‐trans [U‐14C]uridine influx into nucleoside‐permeable type sheep cells was consistent with simple competitive inhibition (apparent Ki 1 nmol/l). Analysis of results using total inhibitor levels instead of cell‐free inhibitor concentrations did not affect the inhibition pattern, but increased the apparent Ki value by 5‐fold. 3. In contrast, [3H]nitrobenzylthioinosine was a non‐competitive inhibitor of zero‐trans [U‐14C]uridine efflux (apparent Ki 1·5 nmol/l). Dipyridamole, another potent inhibitor of nucleoside translocation, also inhibited zero‐trans [U‐14C]uridine influx in a competitive manner (apparent Ki 20‐40 nmol/l). 4. [3H]nitrobenzylthioinosine bound to high‐affinity sites on cell membranes from human and nucleoside‐permeable type sheep cells (apparent KD values ≃ 1 nmol/l). Binding of inhibitor to these sites was competitively blocked by uridine, a well characterized substrate for the nucleoside transporter (apparent Ki 1·25 and 0·9 mmol/l, respectively). These apparent Ki values are close to the apparent Km for uridine equilibrium exchange in human erythrocytes. 5. Similarly, deoxycytidine was found to be a competitive inhibitor of high‐affinity [3H]nitrobenzylthioinosine binding activity (apparent Ki 1·0 and 1·2 mmol/l for human and nucleoside‐permeable type sheep cell membranes, respectively). This contrasts with a previous report that this nucleoside had no effect on inhibitor binding activity. Transport studies confirmed that deoxycytidine is a substrate for the erythrocyte nucleoside transporter. Apparent Km and Vmax values for [U‐14C]‐deoxycytidine zero‐trans influx into human and nucleoside‐permeable type sheep cells were comparable to those obtained for [U‐14C]uridine. 6. It is suggested from these results that nitrobenzylthioinosine competes directly with nucleosides for the permeation site of the nucleoside transporter, but that inhibitor binds preferentially to the external membrane surface.

Identification and Mutational Analysis of Amino Acid Residues Involved in Dipyridamole Interactions with Human and Caenorhabditis elegans Equilibrative Nucleoside Transporters

“…This common mechanism of inhibition suggested that the residues involved in dipyridamole binding by hENT1 and CeENT1 may be conserved and was consistent with the results of previous studies that had suggested that the dipyridamole-binding site overlaps with the exofacial permeant binding site of ENT transporters (16,18,39).…”

Demonstration of Equilibrative Nucleoside Transporters (hENT1 and hENT2) in Nuclear Envelopes of Cultured Human Choriocarcinoma (BeWo) Cells by Functional Reconstitution in Proteoliposomes

“…NBMPR Binding to BeWo Membrane Fractions-Because [ 3 H]NBMPR binds specifically and with high affinity to functional nucleoside transporters, equilibrium binding analysis has been used extensively as a surrogate marker for the presence of the es transporter protein (1,8,32,33). BeWo membrane fractions collected at different sucrose concentrations were first assayed for NBMPR binding activity at a single concentration (5 nM) under conditions previously shown in studies with intact cells (19) to be sufficient to label over 40% of the high affinity sites.…”

“…, which has been used extensively in the characterization of equilibrative nucleoside transport proteins (1,8,32,33), was performed as follows. Membrane preparations, proteoliposomes (ϳ10 g of protein/ml final concentration), and intact nuclei and nuclear membranes (at ϳ40 g of protein/ml) were incubated for 45 min at room temperature with a range of concentrations (0.24 -24 nM) of [ 3 H]NBMPR in either the absence (total binding) or presence (nonspecific binding) of 10 M NBMPR (1-ml final assay volume).…”

Selective Inhibition of Human Equilibrative and Concentrative Nucleoside Transporters by BCR-ABL Kinase Inhibitors

“…inhibitor) binding site overlaps with the uridine (i.e. permeant) binding site of hENT1 (33)(34)(35). Dipyridamole resistance was seen when Met 33 was changed to Ala in TM1 of hENT1, thereby identifying it as a key residue for dipyridamole interaction with hENT1.…”