Document Detail


Cytosolic half of transmembrane domain IV of the human bile acid transporter hASBT (SLC10A2) forms part of the substrate translocation pathway.
MedLine Citation:
PMID:  18311924     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
We report the involvement of transmembrane domain 4 (TM4) of hASBT in forming the putative translocation pathway, using cysteine-scanning mutagenesis in conjunction with solvent-accessibility studies using the membrane-impermeant, sulfhydryl-specific methanethiosulfonate reagents. We individually mutated each of the 21 amino acids in TM4 to cysteine on a fully functional, MTS-resistant C270A-hASBT template. The single-cysteine mutants were expressed in COS-1 cells, and their cell surface expression levels, transport activities [uptake of the prototypical hASBT substrate taurocholic acid (TCA)], and sensitivities to MTS exposure were determined. Only P161 lacked cell-surface expression. Overall, cysteine replacement was tolerated at charged and polar residues, except for mutants I160C, Y162C, I165C, and G179C (<or=20% TCA uptake versus the control). TCA uptake was significantly inhibited by MTSES and MTSET for N164C, T167C, S168C, A171C, V173C, and P175C. Interestingly, all of these residues were clustered along one face of the putative alpha helix. TM4 mutants were not sensitive to equilibrative (12 mM) sodium concentrations, thereby ruling out a direct role of TM4 in sodium translocation. Our results demonstrate that primarily the cytosolic half of TM4 is highly solvent-accessible and plays an important role in ASBT function and substrate translocation. Consistent with the existing experimental data, a three-dimensional model for the orientation of TM4 is proposed.
Authors:
Chandra M Khantwal; Peter W Swaan
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2008-03-01
Journal Detail:
Title:  Biochemistry     Volume:  47     ISSN:  0006-2960     ISO Abbreviation:  Biochemistry     Publication Date:  2008 Mar 
Date Detail:
Created Date:  2008-03-18     Completed Date:  2008-05-27     Revised Date:  2013-07-18    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  3606-14     Citation Subset:  IM    
Affiliation:
Department of Pharmaceutical Sciences, University of Maryland, 20 North Penn Street, Baltimore, Maryland 21201, USA.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Animals
COS Cells
Cercopithecus aethiops
Computational Biology
Cysteine / chemistry
Cytosol / metabolism
Humans
Mesylates / chemistry,  pharmacology
Models, Molecular
Molecular Sequence Data
Organic Anion Transporters, Sodium-Dependent / chemistry*,  drug effects,  genetics
Point Mutation
Protein Structure, Secondary
Protein Structure, Tertiary
Sequence Alignment
Symporters / chemistry*,  drug effects,  genetics
Grant Support
ID/Acronym/Agency:
DK061425/DK/NIDDK NIH HHS; R01 DK061425/DK/NIDDK NIH HHS
Chemical
Reg. No./Substance:
0/Mesylates; 0/Organic Anion Transporters, Sodium-Dependent; 0/Symporters; 145420-23-1/sodium-bile acid cotransporter; 155450-07-0/(2-sulfonatoethyl)methanethiosulfonate; 155450-08-1/(2-(trimethylammonium)ethyl)methanethiosulfonate; 52-90-4/Cysteine

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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