title: Transmembrane domain VII of the human apical sodium-dependent bile acid transporter ASBT (SLC10A2) lines the substrate translocation pathway.
abstract: Recent evidence implicating transmembrane (TM) segment 7 of the apical sodium-dependent bile acid transporter (ASBT) in substrate interaction warranted examination of its aqueous accessibility. Therefore, cysteine substitution of 22 consecutive amino acids was performed against a methanethiosulfonate (MTS)-resistant background (C270A). Activity and susceptibility to polar MTS derivatives [(2-aminoethyl)-methanethiosulfonate (MTSEA), [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET), and methanethiosulfonate ethylsulfonate (MTSES)] of mutants were evaluated in COS-1 cells. Thr289, Tyr293, Gln297, Ala301, Phe307, and Tyr308 represented loss-of-function mutants; furthermore, the measurable residual activities for T289C, Y293C, and A301C (<or=20% control) proved insensitive to MTS treatment. MTSES and MTSET inhibition was confined to residues lining the extracellular half of TM7; amino acids situated deeper within the membrane were unaffected. In contrast, the entire length of TM7 was susceptible to the relatively smaller MTSEA; moreover, MTSEA sensitivity was significantly amended by coapplication with substrates. This selective pattern of modification suggests that the highly conserved lower half of TM7 lies within a water-filled cavity easily accessible from the extracellular milieu, whereas residues approaching the cytosolic/membrane interface reside in pores for which accessibility is modulated by molecular volume. Functionally inactive and MTS-inaccessible residues (T289C, Y293C, Q297C, and A301C) within TM7 may play a structural role critical to transporter function; conversely, MTS-sensitive residues are spatially distinct and may demarcate a face of the TM involved in substrate translocation. In addition, computational analysis of solvent-accessible domains identified five key solvent pockets that predominantly line the hydrophilic face of TM7. Combined, our data suggest that TM7 plays a dominant role in the hASBT translocation process.
authors.affiliation: Department of Pharmaceutical Sciences, University of Maryland, Baltimore, 21201, USA.
authors.forename: Naissan
authors.initials: N
authors.lastname: Hussainzada
authors.forename: Antara
authors.initials: A
authors.lastname: Banerjee
authors.forename: Peter W
authors.initials: PW
authors.lastname: Swaan
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000187:drug effects; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D019295:Computational Biology; D003545:Cysteine / Q000378:metabolism; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D008698:Mesylates / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D050505:Mutant Proteins / Q000378:metabolism; D029363:Organic Anion Transporters, Sodium-Dependent / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000187:drug effects; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism; D012997:Solvents; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000187:drug effects; D027981:Symporters / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D008698:Mesylates; D050505:Mutant Proteins; D029363:Organic Anion Transporters, Sodium-Dependent; D012997:Solvents; D027981:Symporters; C093171:methanethiosulfonate ethylammonium; C071908:sodium-bile acid cotransporter; C074502:methanethiosulfonate; D005020:Ethyl Methanesulfonate; D012964:Sodium; D003545:Cysteine