Identification of Cysteines Involved in the Effects of Methanethiosulfonate Reagents on Human Equilibrative Nucleoside Transporter 1

Abstract

Inhibitor and substrate interactions with equilibrative nucleoside transporter 1 (ENT1; SLC29A1) are known to be affected by cysteine-modifying reagents. Given that selective ENT1 inhibitors, such as nitrobenzylmercaptopurine riboside (NBMPR), bind to the N-terminal half of the ENT1 protein, we hypothesized that one or more of the four cysteine residues in this region were contributing to the effects of the sulfhydryl modifiers. Recombinant human ENT1 (hENT1), and the four cysteine-serine ENT1 mutants, were expressed in nucleoside transport-deficient PK15 cells and probed with a series of methanethiosulfonate (MTS) 3 H]2-chloroadenosine transport. The results of this study also indicate that the hENT1-C193S mutant may be useful as a MTSET/MTSESinsensitive transporter for future cysteine substitution studies to define the extracellular domains contributing to the binding of substrates and inhibitors to this critical membrane transporter.

Cysteine Residues in the Transmembrane (TM) 9 to TM11 Region of the Human Equilibrative Nucleoside Transporter Subtype 1 Play an Important Role in Inhibitor Binding and Translocation Function

“…We showed previously that a 10-min incubation of intact cells with 1 mM MMTS enhanced the (Park et al, 2011). It was also determined that treatment with MMTS, MTSET, or MTSES decreased [ 3 H]NBMPR binding to hENT1 in broken cell preparations.…”

“…Cysteine mutants were expressed in PK15-NTD cells as described previously (Park et al, 2011). In brief, cells were cultured in modified Eagle's medium supplemented with 10% BGS, 100 units of penicillin, 100 g/ml streptomycin, 0.1 mM nonessential amino acids, and 1 mM sodium pyruvate at 37°C in 5% CO 2 and plated (90% confluent) onto 12-well plates.…”

“…Cells were treated with 0.1% DMSO (controls) or MTS reagent (1 mM MMTS, 5 mM MTSET, and 5 mM MTSES) for 10 min at room temperature We previously showed via a concentration/time course analysis that a 10-min incubation at the MTS concentrations used in this study was sufficient to achieve a maximal effect of each of the reagents on hENT1 activity. (Park et al, 2011). In some cases, 1 mM adenosine or 10 nM NBMPR was included with the MTS reagent incubation protocol to assess the ability of these ENT1 ligands to protect against MTS reagent modification of the cysteines.…”

“…Alternative techniques for advancing knowledge of ENT1 structure-function have thus been used, including cysteine mutagenesis in conjunction with cysteineselective sulfhydryl-modifying reagents (Valdés et al, 2004;Riegelhaupt et al, 2010;Park et al, 2011;Yao et al, 2011). The importance of cysteines in hENT1 function has been shown in multiple studies using N-ethylmaleimide (NEM) and pchloromercuribenzene sulfonate (pCMBS) as sulfhydryl modifiers.…”

“…We have recently described modification of hENT1 ligand binding and transport function using a series of methanethiosulfonate (MTS) reagents (Park et al, 2011 ]2-chloroadenosine uptake by hENT1. The negatively charged, membrane-impermeable (2-sulfonatoethyl)-methanethiosulfonate (MTSES), like pCMBS (also negatively charged), was found to inhibit NBMPR binding only in broken cell preparations, again implicating an intracellular cysteine.…”