Transmembrane Domain II of the Human Bile Acid Transporter SLC10A2 Coordinates Sodium Translocation

Human apical sodium-dependent bile acid transporter (hASBT, SLC10A2) is responsible for intestinal reabsorption of bile acids and plays a key role in cholesterol homeostasis. We used a targeted and systematic approach to delineate the role of highly conserved transmembrane helix 2 on the expression and function of hASBT. Cysteine mutation significantly depressed transport activity for >60% of mutants without affecting cell surface localization of the transporter. All mutants were inaccessible toward chemical modification by membrane-impermeant MTSET reagent, strongly suggesting that transmembrane 2 (TM2) plays an indirect role in bile acid substrate translocation. Both bile acid uptake and sodium dependence of TM2 mutants revealed a distinct α-helical periodicity. Kinetic studies with conservative and non-conservative mutants of sodium sensitive residues further underscored the importance of Gln 75 , Phe 76 , Met 79 , Gly 83 , Leu 86 , Phe 90 , and Asp 91 in hASBT function. Computational analysis indicated that Asp 91 may coordinate with sodium during the transport cycle. Combined, our data propose that a consortium of sodium-sensitive residues along with previously reported residues (Thr 134 , Leu 138 , and Thr 149 ) from TM3 may form the sodium binding and translocation pathway. Notably, residues Gln 75 , Met 79 , Thr 82 , and Leu 86 from TM2 are highly conserved in TM3 of a putative remote bacterial homologue (ASBT NM ), suggesting a universal mechanism for the SLC10A transporter family. Background: Transmembrane domains are critical to the structure and function of bile acid transporters. Results: Sodium sensitive residues follow distinct α-helical periodicity along TM2. Conclusion: TM2 forms part of the sodium translocation pathway, which is highly conserved in its putative remote bacterial homologue ASBT NM . Significance: The proposed sodium translocation mechanism may be universal to other SLC10A family members.