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 (SLC10A2). TM6 is linked exofacially to TM7 via extracellular loop 3. TM7 was identified previously as lining part of the substrate permeation path ( 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 and stability may account for lack of activity for mutant P234C. Subsequent Pro234 amino acid replacement reveals its participation in both structural and functional aspects of the transport cycle. Application of polar MTS reagents (1 mM) significantly inhibited the activity of six mutants (V235C, S239C, F242C, R246C, A248C, and 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 preincubation 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, probably 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.