title: Transmembrane helix 1 contributes to substrate translocation and protein stability of bile acid transporter SLC10A2.
abstract: The human apical sodium-dependent bile acid transporter (hASBT, SLC10A2) plays a critical role in the enterohepatic circulation of bile acids, as well as in cholesterol homeostasis. ASBT reclaims bile acids from the distal ileum via active sodium co-transport, in a multistep process, orchestrated by key residues in exofacial loop regions, as well as in membrane-spanning helices. Here, we unravel the functional contribution of highly conserved transmembrane helix 1 (TM1) on the hASBT transport cycle. Consecutive cysteine substitution of individual residues along the TM1 helix (Ile(29)-Gly(50)), as well as exofacial Asn(27) and Asn(28), resulted in functional impairment of ∼70% of mutants, despite appreciable cell surface expression for all but G50C. Cell surface expression of G50C and G50A was rescued upon MG132 treatment as well as cyclosporine A, but not by FK506 or bile acids, suggesting that Gly(50) is involved in hASBT folding. TM1 accessibility to membrane-impermeant MTSET remains confined to the exofacial half of the helix along a single, discrete face. Substrate protection from MTSET labeling was temperature-dependent for L34C, T36C, and L38C, consistent with conformational changes playing a role in solvent accessibility for these mutants. Residue Leu(30) was shown to be critical for both bile acid and sodium affinity, while Asn(27), Leu(38), Thr(39), and Met(46) participate in sodium co-transport. Combined, our data demonstrate that TM1 plays a pivotal role in ASBT function and stability, thereby providing further insight in its dynamic transport mechanism.
authors.affiliation: Department of Pharmaceutical Sciences, University of Maryland, Baltimore, Maryland 21201, USA.
authors.forename: Tatiana Claro
authors.initials: TC
authors.lastname: da Silva
authors.forename: Naissan
authors.initials: N
authors.lastname: Hussainzada
authors.forename: Chandra M
authors.initials: CM
authors.lastname: Khantwal
authors.forename: James E
authors.initials: JE
authors.lastname: Polli
authors.forename: Peter W
authors.initials: PW
authors.lastname: Swaan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D006801:Humans; D007700:Kinetics; D008247:Lysosomes / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D029363:Organic Anion Transporters, Sodium-Dependent / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D021381:Protein Transport; D017386:Sequence Homology, Amino Acid; D027981:Symporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D029363:Organic Anion Transporters, Sodium-Dependent; D027981:Symporters; C071908:sodium-bile acid cotransporter; D046988:Proteasome Endopeptidase Complex