Identification of cysteines involved in the effects of methanethiosulfonate reagents on human equilibrative nucleoside transporter 1.

Jamie S Park, Scott J Hughes, Frances K M Cunningham, James R Hammond

Index: Mol. Pharmacol. 80(4) , 735-46, (2011)

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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) sulfhydryl-modifying reagents. Transporter function was assessed by the binding of [(3)H]NBMPR and the cellular uptake of [(3)H]2-chloroadenosine. The membrane-permeable reagent methyl methanethiosulfonate (MMTS) enhanced [(3)H]NBMPR binding in a pH-dependent manner, but decreased [(3)H]2-chloroadenosine uptake. [2-(Trimethylammonium)ethyl] methane-thiosulfonate (MTSET) (positively charged, membrane-impermeable), but not sodium (2-sulfonatoethyl)-methanethiosulfonate (MTSES) (negatively charged), inhibited [(3)H]NBMPR binding and enhanced [(3)H]2-chloroadenosine uptake. Mutation of Cys222 in transmembrane (TM) 6 eliminated the effect of MMTS on NBMPR binding. Mutation of Cys193 in TM5 enhanced the ability of MMTS to increase [(3)H]NBMPR binding and attenuated the effects of MMTS and MTSET on [(3)H]2-chloroadenosine uptake. Taken together, these data suggest that Cys222 contributes to the effects of MTS reagents on [(3)H]NBMPR binding, and Cys193 is involved in the effects of these reagents on [(3)H]2-chloroadenosine transport. The results of this study also indicate that the hENT1-C193S mutant may be useful as a MTSET/MTSES-insensitive transporter for future cysteine substitution studies to define the extracellular domains contributing to the binding of substrates and inhibitors to this critical membrane transporter.