Linked Life Data

19653651

Source:http://linkedlifedata.com/resource/pubmed/id/19653651

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
36
pubmed:dateCreated
2009-9-8
pubmed:abstractText
Site-directed alkylation of consecutively introduced cysteines was employed to probe the solvent-accessible profile of highly conserved transmembrane helix 3 (TM3), spanning residues V127-T149 of the apical sodium-dependent bile acid transporter (ASBT), a key membrane protein involved in cholesterol homeostasis. Sequence alignment of SLC10 family members has previously identified a signature motif (ALGMMPL) localized to TM3 of ASBT with as yet undetermined function. Cysteine mutagenesis of this motif resulted in severe decreases in uptake activity only for mutants M141C and P142C. Additional conservative and nonconservative replacement of P142 suggests its structural and functional importance during the ASBT transport cycle. Significant decreases in transport activity were also observed for three cysteine mutants clustered along the exofacial half of the helix (M129C, T130C, S133C) and five mutants consecutively lining the cytosolic half of TM3 (L145C-T149C). Measurable surface expression was detected for all TM3 mutants. Using physicochemically different alkylating reagents, sites predominantly lining the cytosolic half of the TM3 helix were found to be solvent accessible (i.e., S128C, L143C-T149C). Analysis of substrate kinetics for select TM3 mutants demonstrates significant loss of taurocholic acid affinity for mutants S128C and L145C-T149C. Overall, we conclude (i) the functional and structural importance of P142 during the transport cycle and (ii) the presence of a large hydrophilic cleft region lining the cytosolic half of TM3 that may form portions of the substrate exit route during permeation. Our studies provide unique insight into molecular mechanisms guiding the ASBT transport cycle with respect to substrate binding and translocation events.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Sep
pubmed:issn
1520-4995
pubmed:author
pubmed:issnType
Electronic
pubmed:day
15
pubmed:volume
48
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
8528-39
pubmed:dateRevised
2011-5-13
pubmed:meshHeading
pubmed-meshheading:19653651-Alkylation, pubmed-meshheading:19653651-Amino Acid Sequence, pubmed-meshheading:19653651-Animals, pubmed-meshheading:19653651-Bile Acids and Salts, pubmed-meshheading:19653651-Cysteine, pubmed-meshheading:19653651-Cytosol, pubmed-meshheading:19653651-Humans, pubmed-meshheading:19653651-Membrane Glycoproteins, pubmed-meshheading:19653651-Molecular Sequence Data, pubmed-meshheading:19653651-Mutagenesis, Site-Directed, pubmed-meshheading:19653651-Organic Anion Transporters, Sodium-Dependent, pubmed-meshheading:19653651-Protein Structure, Secondary, pubmed-meshheading:19653651-Protein Transport, pubmed-meshheading:19653651-Signal Transduction, pubmed-meshheading:19653651-Sodium, pubmed-meshheading:19653651-Substrate Specificity, pubmed-meshheading:19653651-Symporters
pubmed:year
2009
pubmed:articleTitle
The cytosolic half of helix III forms the substrate exit route during permeation events of the sodium/bile acid cotransporter ASBT.
pubmed:affiliation
Department of Pharmaceutical Sciences, University of Maryland, Baltimore, Maryland 21201, USA.
pubmed:publicationType
Journal Article, Research Support, N.I.H., Extramural