Defining the substrate translocation pathway of the human Apical Sodium‐Dependent Bile Acid Transporter ASBT (SLC10A2): Role of Transmembrane Domain IV

Abstract

ASBT, comprises of seven transmembrane helices based on homology modeling and mutagenesis studies with extracellular loop 3 as the bile acid binding site. In the current study participation of transmembrane helix 4 in the formation of sodium or/and bile acid translocation pathway was investigated using cysteine-scanning mutagenesis in conjunction with the membrane-impermeant, sulfhydryl-specific reagents, sodium methanethiosulfonate (MTSES) and methanethiosulfonate bromide (MTSET). All twenty-one ASBT mutants were created from a fully functional, MTS-resistant C270A-ASBT template by successively changing each residue along TM4 to a cysteine. These single cysteine mutants were then expressed in COS-1 cells and their expression levels, transport activities, sensitivities to MTS reagents were determined. Overall, cysteine replacement was tolerated at charged and polar residue except for mutants I160C, P161C, Y162C, I165C and G179C which showed ¡Ü 20% TCA uptake. P161 in this group showed no cell surface expression. TCA uptake was significantly inhibited by MTSES and MTSET for N164C, T167C, S168C, A171C, V173C and P175C. Interestingly, all of these residues along with residues showing sodium dependency and reduced TCA uptake were clustered along one face of the putative alpha-helix. Our results strongly suggest that TM4 is relatively solvent accessible and plays an important role in ASBT function. Supported by NIH RO1 DK061425.