Aqueous Accessibility of Transmembrane Domain 6 of the Human Apical Na+‐dependent Bile Acid Transporter is Modulated by Substrate Binding

Abstract

Along the terminal ileum, the human apical Na+-dependent bile acid transporter (hASBT) couples internalization of two sodium ions to one bile acid molecule. Our previous study (Hussainzada et al. (2006) Mol. Pharmacol. 70:) defined transmembrane segment (TM) 7 in forming part of the putative substrate translocation pore using cysteine mutagenesis and thiol modification (SCAM). In the present study, SCAM analysis was extended to TM6, since it lies adjacent to TM7 according to a previously developed model (Zhang et al., (2004) Biochemistry 43:) and may therefore line another face of the translocation pore. Most cysteine substitutions in TM6 were well tolerated and all mutants were expressed at the plasma membrane. Two mutants, I229C and G237C were functionally inactive, suggesting a role for these residues during the transport cycle. Seven residues were sensitive to chemical labeling by various MTS reagents (Val235, Ser239, Leu240, Phe242, Arg246, Ala248 and Tyr253), of which four (Val235, Ser239, Phe242, Arg246) had altered MTS accessibility upon binding of Na+ and/or bile acid substrates. Most TM6 residues were significantly Na+-sensitive, suggesting involvement of this TM in Na+ interactions. In conclusion, TM6 may play a critical role in hASBT transport function by either directly interacting with Na+ ions or by transducing protein conformational changes occurring after Na+ binding. Supported by NIH RO1 DK61425.