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Molecular Pharmacology

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Research ArticleArticle

Transmembrane Domain VII of the Human Apical Sodium-Dependent Bile Acid Transporter ASBT (SLC10A2) Lines the Substrate Translocation Pathway

Naissan Hussainzada, Antara Banerjee and Peter W. Swaan
Molecular Pharmacology November 2006, 70 (5) 1565-1574; DOI: https://doi.org/10.1124/mol.106.028647
Naissan Hussainzada
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Antara Banerjee
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Peter W. Swaan
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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 (≤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.

Footnotes

  • This research was supported by grant DK061425 (to P.W.S.) from the National Institutes of Health, National Institute for Digestive Diseases and Kidney.

  • A.B. and N.H. contributed equally to this work.

  • ABBREVIATIONS: SLC10, solute carrier 10; ASBT, apical sodium-dependent bile acid transporter; hASBT, human apical sodium-dependent bile acid transporter; TM, transmembrane; SCAM, substituted cysteine accessibility method; TCA, taurocholic acid; GDCA, glychodeoxycholic acid; CA, cholic acid; sulfo-NHS-SS-biotin, (sulfosuccinimidyl 2-(biotinamido)-ethyl-1,3-dithiopropionate; MTSEA-biotin, N-biotinaminoethyl methanethiosulfonate; wt, wild type; PBS, phosphate-buffered saline.

  • ↵1 Current affiliation: Schering-Plough Biopharma, Palo Alto, California.

    • Received July 7, 2006.
    • Accepted August 9, 2006.
  • The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 70 (5)
Molecular Pharmacology
Vol. 70, Issue 5
1 Nov 2006
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Research ArticleArticle

Transmembrane Domain VII of the Human Apical Sodium-Dependent Bile Acid Transporter ASBT (SLC10A2) Lines the Substrate Translocation Pathway

Naissan Hussainzada, Antara Banerjee and Peter W. Swaan
Molecular Pharmacology November 1, 2006, 70 (5) 1565-1574; DOI: https://doi.org/10.1124/mol.106.028647

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Research ArticleArticle

Transmembrane Domain VII of the Human Apical Sodium-Dependent Bile Acid Transporter ASBT (SLC10A2) Lines the Substrate Translocation Pathway

Naissan Hussainzada, Antara Banerjee and Peter W. Swaan
Molecular Pharmacology November 1, 2006, 70 (5) 1565-1574; DOI: https://doi.org/10.1124/mol.106.028647
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