RT Journal Article SR Electronic T1 Transmembrane Domain VII of the Human Apical Sodium-Dependent Bile Acid Transporter ASBT (SLC10A2) Lines the Substrate Translocation Pathway JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 1565 OP 1574 DO 10.1124/mol.106.028647 VO 70 IS 5 A1 Naissan Hussainzada A1 Antara Banerjee A1 Peter W. Swaan YR 2006 UL http://molpharm.aspetjournals.org/content/70/5/1565.abstract AB 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. The American Society for Pharmacology and Experimental Therapeutics