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.