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J. Biol. Chem., Vol. 283, Issue 30, 20653-20663, July 25, 2008

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Conserved Aspartic Acid Residues Lining the Extracellular Loop I of Sodium-coupled Bile Acid Transporter ASBT Interact with Na⁺ and 7-OH Moieties on the Ligand Cholestane Skeleton^*

From the Department of Pharmaceutical Sciences, University of Maryland, Baltimore, Maryland 21201

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 7-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 7-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.

Received for publication, April 15, 2008 , and in revised form, May 23, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant DK61425 (to P. W. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Pfizer Global Research and Development, La Jolla, CA 92121.

² To whom correspondence should be addressed: University of Maryland, 621 HSF-II, 20 Penn St., Baltimore, MD 21201. Tel.: 410-706-0103; Fax: 410-706-5017; E-mail: pswaan{at}rx.umaryland.edu.

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