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Cysteine Residues in the TM9-TM11 Region of the Human Equilibrative Nucleoside Transporter Subtype 1 Play an Important Role in Inhibitor Binding and Translocation Function.
MedLine Citation:
PMID:  22837314     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
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 TM6 as the residue responsible for MMTS (membrane-permeable sulfhydryl modifier)-mediated enhancement of the binding of the ENT1 inhibitor nitrobenzylthioinosine (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, 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 [(3)H]NBMPR and the cellular uptake of [(3)H]2-chloroadenosine. These studies established that Cys378 is an extracellular-located residue modified by 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 co-mutation of Cys378, indicating that disruption of the fifth intracellular loop modifies the conformation of TM10 and its extracellular loop. 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 hENT1.
Authors:
Jamie S Park; James R Hammond
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-7-26
Journal Detail:
Title:  Molecular pharmacology     Volume:  -     ISSN:  1521-0111     ISO Abbreviation:  -     Publication Date:  2012 Jul 
Date Detail:
Created Date:  2012-7-27     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0035623     Medline TA:  Mol Pharmacol     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Affiliation:
Western University.
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