Received for publication July 10, 2006.
Revised July 30, 2006.
Accepted for publication August 9, 2006.

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 seven of the apical sodium-dependent bile acid transporter (ASBT) in substrate interaction warranted examination of its aqueous accessiblity. Consequently, cysteine substitution of 22 consecutive amino acids was performed against a methanethiosulfonate (MTS)-resistant background (C270A). Activity and susceptibility to polar MTS derivatives (MTSES, MTSET, and MTSEA) of mutants were evaluated in COS-1 cells. Q297C, F307C and Y308C represented loss-of-function mutants; whereas, 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, all functional mutants lining the entirety to TM7 were susceptible to the relatively smaller MTSEA; moreover, MTSEA sensitivity was significantly amended by co-application with substrates. This selective pattern of modification suggests that the highly conserved, extracellular half of TM7 lies within a water-filled cavity easily accessible from the apical 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. Additionally, computational analysis of solvent-accessible domains identified 5 major solvent pockets that predominantly line the hydrophilic face of TM7. Combined, our data suggest that TM7 plays a dominant role in the hASBT substrate translocation process.

Key words: Organic anion, Mutagenesis/Chimeric approaches, Cholesterol metabolism/lipoproteins

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