Cytosolic half of transmembrane domain IV of the human bile acid transporter hASBT (SLC10A2) forms part of the substrate translocation pathway.
We report the involvement of transmembrane domain 4 (TM4) of hASBT in forming the putative translocation pathway, using cysteine-scanning mutagenesis in conjunction with solvent-accessibility studies using the membrane-impermeant, sulfhydryl-specific methanethiosulfonate reagents. We individually mutated each of the 21 amino acids in TM4 to cysteine on a fully functional, MTS-resistant C270A-hASBT template. The single-cysteine mutants were expressed in COS-1 cells, and their cell surface expression levels, transport activities [uptake of the prototypical hASBT substrate taurocholic acid (TCA)], and sensitivities to MTS exposure were determined. Only P161 lacked cell-surface expression. Overall, cysteine replacement was tolerated at charged and polar residues, except for mutants I160C, Y162C, I165C, and G179C (<or=20% TCA uptake versus the control). TCA uptake was significantly inhibited by MTSES and MTSET for N164C, T167C, S168C, A171C, V173C, and P175C. Interestingly, all of these residues were clustered along one face of the putative alpha helix. TM4 mutants were not sensitive to equilibrative (12 mM) sodium concentrations, thereby ruling out a direct role of TM4 in sodium translocation. Our results demonstrate that primarily the cytosolic half of TM4 is highly solvent-accessible and plays an important role in ASBT function and substrate translocation. Consistent with the existing experimental data, a three-dimensional model for the orientation of TM4 is proposed.
Khantwal
Chandra M
CM
Department of Pharmaceutical Sciences, University of Maryland, 20 North Penn Street, Baltimore, Maryland 21201, USA.
Swaan
Peter W
PW
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D019295:Computational Biology; D003545:Cysteine / Q000737:chemistry; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D008698:Mesylates / Q000737:chemistry / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D029363:Organic Anion Transporters, Sodium-Dependent / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D017354:Point Mutation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D027981:Symporters / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics
D008698:Mesylates; D029363:Organic Anion Transporters, Sodium-Dependent; D027981:Symporters; C071908:sodium-bile acid cotransporter; C109153:(2-sulfonatoethyl)methanethiosulfonate; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; D003545:Cysteine