QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: mol.106.028647v1 70/5/1565    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hussainzada, N. Articles by Swaan, P. W. Search for Related Content PubMed PubMed Citation Articles by Hussainzada, N. Articles by Swaan, P. W.

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

Department of Pharmaceutical Sciences, University of Maryland, Baltimore, Maryland.

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.

Address correspondence to: Peter W Swaan. Ph.D, University of Maryland, HSF-II, 20 Penn Street, Baltimore, MD 21201. E-mail: pswaan{at}rx.umaryland.edu

This article has been cited by other articles:

				N. Hussainzada, T. C. Da Silva, E. Y. Zhang, and P. W. Swaan Conserved Aspartic Acid Residues Lining the Extracellular Loop I of Sodium-coupled Bile Acid Transporter ASBT Interact with Na+ and 7{alpha}-OH Moieties on the Ligand Cholestane Skeleton J. Biol. Chem., July 25, 2008; 283(30): 20653 - 20663. [Abstract] [Full Text] [PDF]

				N. Hussainzada, A. Khandewal, and P. W. Swaan Conformational Flexibility of Helix VI Is Essential for Substrate Permeation of the Human Apical Sodium-Dependent Bile Acid Transporter Mol. Pharmacol., February 1, 2008; 73(2): 305 - 313. [Abstract] [Full Text] [PDF]

				N. Hussainzada and P. W Swaan Aqueous Accessibility of Transmembrane Domain 6 of the Human Apical Na+-dependent Bile Acid Transporter is Modulated by Substrate Binding FASEB J, April 1, 2007; 21(6): A1336 - A1336.