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

Abstract

Inhibitor and substrate interactions with equilibrative nucleoside transporter 1 (ENT1; SLC29A1) are known to be affected by cysteine-modifying reagents. A previous study from our laboratory established Cys222 in transmembrane (TM) 6 as the residue responsible for methyl methanethiosulfonate (a membrane-permeable sulfhydryl modifier)-mediated enhancement of the binding of the ENT1 inhibitor nitrobenzylmercaptopurine riboside (NBMPR) in intact cells. However, the capacity of charged sulfhydryl reagents to inhibit the binding of NBMPR in broken cell preparations (allowing cytoplasmic access) was not affected by mutation of any of the cysteines (Cys87, 193, 213, or 222) in the N-terminal half of the protein. We thus hypothesized that the inhibitory effects of the modifiers were due to the one or more of the six cysteine residues in the C-terminal half of ENT1, particularly one or both of those in the fifth intracellular loop (Cys414 and Cys416). Each of the cysteines were mutated to serine or alanine and expressed in nucleoside transportdeficient PK15 cells and probed with a series of methanethiosulfonate sulfhydryl-modifying reagents. Transporter function was assessed by the site-specific binding of [ 3 H]NBMPR and the cellular uptake of [ 3 H]2-chloroadenosine. These studies established that Cys378 is an extracellular-located residue modified by [2-(trimethylammonium)ethyl] methane-thiosulfonate (MTSET) to inhibit the binding of NBMPR to intact cells. Mutation of Cys414 led to an enhancement of the ability of MTSET to inhibit NBMPR binding, and this enhancement was eliminated by the comutation of Cys378, indicating that disruption of the fifth intracellular loop modifies the conformation of TM10 and its extracellular extension. Mutation of Cys416 led to the loss of the ability of the charged sulfhydryl reagents to inhibit NBMPR binding in isolated membranes and also led to the loss of transport function. This finding further supports an allosteric interaction between the fifth intracellular loop and the extracellular NBMPR binding domain and implicates this region in the translocation function of human ENT1.

Identification of Key Residues for Urate Specific Transport in Human Glucose Transporter 9 (hSLC2A9)

“…This supports our homology model of hSLC2A9 (Figs 4A and 5A) which also indicates C181 faces the translocation pore. These findings are comparable with other transporters, like the equilibrative nucleoside transporters (ENTs) and concentrative nucleoside transporter (CNT) proteins232425. For instance, a single cysteine, C140, was located within the substrate translocation channel in rat ENT2, where it reacted with pCMBS23.…”

Substrate Dependent Regulation of the Human Equilibrative Nucleoside Transporter 1 (hENT1) in HEK293 Cells

“…There is a large extracellular loop between TM domains 1 and 2 of ENT1, which contains N-linked glycosylation sites (Vickers et al, 1999). TM domains 2, 4, 5 and 9-11 are reported to be critical for nucleoside transport (Endres and Unadkat 2005;SenGupta et al, 2002;Park and Hammond, 2012) and there is a large intracellular loop between TM domains 6 and 7, which serves as a regulatory hub for the transporter (details in following sections). An important feature of ENT1 is sensitivity to inhibition by the nucleoside analog, NBTI (S-(4-Nitrobenzyl)-6-thioinosine).…”

Visualizing nucleic acid metabolism using non-natural nucleosides and nucleotide analogs

“…There are numerous literature reports of using radiolabeled substrates to define the kinetic parameters for natural and modified nucleosides (41)(42)(43)(44)(45)(46)(47)(48). One insightful report is from the Cass laboratory that has extensively studied the mechanism of nucleoside transport using different model systems (41).…”

“…Collectively, the application of radiolabeled substrates to measure the kinetics of nucleoside uptake has provided useful information into the mechanism and regulation of nucleoside homeostasis. In addition, this methodology has been applied in studies using sitedirected mutants of various nucleoside transporters to generate structure-activity relationships (SAR) that define structural features required for efficient nucleoside transport (43)(44)(45). These kinetic studies have also found utility in other research areas.…”