Conserved aspartic acid residues lining the extracellular loop 1 of sodium-coupled bile acid transporter ASBT Interact with Na+ and 7alpha-OH moieties on the ligand cholestane skeleton.
Functional contributions of residues Val-99-Ser-126 lining extracellular loop (EL) 1 of the apical sodium-dependent bile acid transporter were determined via cysteine-scanning mutagenesis, thiol modification, and in silico interpretation. Despite membrane expression for all but three constructs (S112C, Y117C, S126C), most EL1 mutants (64%) were inactivated by cysteine mutation, suggesting a functional role during sodium/bile acid co-transport. A negative charge at conserved residues Asp-120 and Asp-122 is required for transport function, whereas neutralization of charge at Asp-124 yields a functionally active transporter. D124A exerts low affinity for common bile acids except deoxycholic acid, which uniquely lacks a 7alpha-hydroxyl (OH) group. Overall, we conclude that (i) Asp-122 functions as a Na(+) sensor, binding one of two co-transported Na(+) ions, (ii) Asp-124 interacts with 7alpha-OH groups of bile acids, and (iii) apolar EL1 residues map to hydrophobic ligand pharmacophore features. Based on these data, we propose a comprehensive mechanistic model involving dynamic salt bridge pairs and hydrogen bonding involving multiple residues to describe sodium-dependent bile acid transporter-mediated bile acid and cation translocation.
Hussainzada
Naissan
N
Department of Pharmaceutical Sciences, University of Maryland, Baltimore, MD 21201, USA.
Da Silva
Tatiana Claro
TC
Zhang
Eric Y
EY
Swaan
Peter W
PW
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry*; D019556:COS Cells; D002522:Chlorocebus aethiops; D002776:Cholestanes / Q000737:chemistry*; D017124:Conserved Sequence; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D009154:Mutation; D029363:Organic Anion Transporters, Sodium-Dependent / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000737:chemistry; D027981:Symporters / Q000737:chemistry*
D002776:Cholestanes; D008024:Ligands; D029363:Organic Anion Transporters, Sodium-Dependent; D027981:Symporters; C071908:sodium-bile acid cotransporter; D001224:Aspartic Acid; D012964:Sodium