Transmembrane domain II of the human bile acid transporter SLC10A2 coordinates sodium translocation.

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TitleTransmembrane domain II of the human bile acid transporter SLC10A2 coordinates sodium translocation.
Publication TypeJournal Article
Year of Publication2013
AuthorsSabit, H, Mallajosyula, SS, Mackerell, AD, Swaan, PW
JournalJ Biol Chem
Volume288
Issue45
Pagination32394-404
Date Published2013 Nov 08
ISSN1083-351X
KeywordsAmino Acid Substitution, Animals, Cercopithecus aethiops, COS Cells, Humans, Ion Transport, Kinetics, Mutation, Missense, Organic Anion Transporters, Sodium-Dependent, Protein Structure, Secondary, Protein Structure, Tertiary, Sodium, Symporters
Abstract

Human apical sodium-dependent bile acid transporter (hASBT, SLC10A2) is responsible for intestinal reabsorption of bile acids and plays a key role in cholesterol homeostasis. We used a targeted and systematic approach to delineate the role of highly conserved transmembrane helix 2 on the expression and function of hASBT. Cysteine mutation significantly depressed transport activity for >60% of mutants without affecting cell surface localization of the transporter. All mutants were inaccessible toward chemical modification by membrane-impermeant MTSET reagent, strongly suggesting that transmembrane 2 (TM2) plays an indirect role in bile acid substrate translocation. Both bile acid uptake and sodium dependence of TM2 mutants revealed a distinct α-helical periodicity. Kinetic studies with conservative and non-conservative mutants of sodium sensitive residues further underscored the importance of Gln(75), Phe(76), Met(79), Gly(83), Leu(86), Phe(90), and Asp(91) in hASBT function. Computational analysis indicated that Asp(91) may coordinate with sodium during the transport cycle. Combined, our data propose that a consortium of sodium-sensitive residues along with previously reported residues (Thr(134), Leu(138), and Thr(149)) from TM3 may form the sodium binding and translocation pathway. Notably, residues Gln(75), Met(79), Thr(82), and Leu(86) from TM2 are highly conserved in TM3 of a putative remote bacterial homologue (ASBTNM), suggesting a universal mechanism for the SLC10A transporter family.

DOI10.1074/jbc.M113.518555
Alternate JournalJ. Biol. Chem.
PubMed ID24045943
PubMed Central IDPMC3820874
Grant ListR01 DK061425 / DK / NIDDK NIH HHS / United States
DK061425 / DK / NIDDK NIH HHS / United States