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Molecular Pharmacology

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Research ArticleArticle

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

Jamie S. Park and James R. Hammond
Molecular Pharmacology November 2012, 82 (5) 784-794; DOI: https://doi.org/10.1124/mol.112.079616
Jamie S. Park
Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada
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James R. Hammond
Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada
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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. 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 transport-deficient PK15 cells and probed with a series of methanethiosulfonate sulfhydryl-modifying reagents. Transporter function was assessed by the site-specific binding of [3H]NBMPR and the cellular uptake of [3H]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.

Footnotes

  • This work was supported by the Natural Sciences and Engineering Research Council of Canada. Support (grant to J.R.H. and postgraduate scholarship to J.S.P.). The graduate training of J.S.P. was also provided by the Schulich School of Medicine and Dentistry, Western University.

  • Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

    http://dx.doi.org/10.1124/mol.112.079616.

  • ABBREVIATIONS:

    hENT
    human equilibrative nucleoside transporter
    NBMPR
    nitrobenzylmercaptopurine riboside
    TM
    transmembrane
    ENT
    equilibrative nucleoside transporter
    NEM
    N-ethylmaleimide
    pCMBS
    p-chloromercuribenzene sulfonate
    MTS
    methanethiosulfonate
    MMTS
    methyl methanethiosulfonate
    MTSET
    [2-(trimethylammonium)ethyl] methane-thiosulfonate
    MTSES
    (2-sulfonatoethyl)-methanethiosulfonate
    PBS
    phosphate-buffered saline
    NBTGR
    S-(4-nitrobenzyl)-6-thioguanosine riboside
    2-Br
    2-bromohexadecanoic acid
    PK15-NTD
    nucleoside transport-deficient pig kidney epithelial cells derived from the PK15 cell line
    FTH-SAENTA
    5′-S-{2-(1-[(fluorescein-5-yl)thioureido]-hexanamido)ethyl}-6-N-(4-nitrobenzyl)-5′-thioadenosine
    BGS
    bovine growth serum
    WT
    wild-type
    NMG
    N-methylglucamine
    DMSO
    dimethyl sulfoxide
    TBS
    Tris-buffered saline
    PDVF
    polyvinylidene difluoride
    IL
    intracellular loop.

  • Received April 25, 2012.
  • Accepted July 26, 2012.
  • Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 82 (5)
Molecular Pharmacology
Vol. 82, Issue 5
1 Nov 2012
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Research ArticleArticle

Role of Cysteines in hENT1 Function

Jamie S. Park and James R. Hammond
Molecular Pharmacology November 1, 2012, 82 (5) 784-794; DOI: https://doi.org/10.1124/mol.112.079616

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Research ArticleArticle

Role of Cysteines in hENT1 Function

Jamie S. Park and James R. Hammond
Molecular Pharmacology November 1, 2012, 82 (5) 784-794; DOI: https://doi.org/10.1124/mol.112.079616
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