Received for publication September 7, 2007.
Revised October 22, 2007.
Accepted for publication October 29, 2007.

Conformational Flexibility of Helix VI is Essential for Substrate Permeation of the Human Apical Sodium-dependent Bile Acid Transporter (ASBT)

Abstract

The present study characterizes the methanethiosulfonate (MTS) inhibition profiles of 26 consecutive cysteine-substituted mutants comprising transmembrane (TM) helix 6 of the human apical Na+-dependent bile acid transporter (hASBT, SLC10A2). TM6 is linked exofacially to TM7 via extracellular loop (EL) 3. TM7 was previously identified as lining part of the substrate permeation path (Hussainzada et al. Mol Pharmacol 70:1565, 2006). Most TM6 cysteine replacements were well-tolerated, except for five residues with either severely hampered (I229C, G249C) or abolished (P234C, G237C, G241C) activity. Disruption of protein synthesis or folding accounted for lack of activity for mutant P234C. Subsequent amino acid replacements at Pro234 demonstrate its essential nature in both structural and functional aspects of the transport cycle. Application of polar MTS reagents (1 mM) significantly inhibited activity of six mutants (V235C, S239C, F242C, R246C, A248C, Y253C), for which rates of modification were almost fully reversed (except Y253C) upon inclusion of bile acid substrates or removal of Na ⁺ from the MTS pre-incubation medium. Activity assessments at equilibrative [Na⁺] revealed numerous Na⁺ sensitive residues, suggesting their proximity in or around Na+ interaction sites. In silico modeling reveals the intimate and potentially cooperative orientation of MTS-accessible TM6 residues toward functionally important TM7 amino acids, substantiating TM6 participation during the transport cycle. We conclude a functional requirement for helical flexibility imparted by Pro234, Gly237 and Gly241, likely forming a "conformational switch" requisite for substrate turnover; meanwhile, MTS-accessible residues, which line a helical face spatially distinct from this switch, may participate during substrate permeation.

Key words: Organic anion, Structure-activity relationships and modeling, Mutagenesis/Chimeric approaches, Cholesterol metabolism/lipoproteins

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