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 Gln⁷⁵, Phe⁷⁶, Met⁷⁹, Gly⁸³, Leu⁸⁶, Phe⁹⁰, and Asp⁹¹ in hASBT function. Computational analysis indicated that Asp⁹¹ may coordinate with sodium during the transport cycle. Combined, our data propose that a consortium of sodium-sensitive residues along with previously reported residues (Thr¹³⁴, Leu¹³⁸, and Thr¹⁴⁹) from TM3 may form the sodium binding and translocation pathway. Notably, residues Gln⁷⁵, Met⁷⁹, Thr⁸², and Leu⁸⁶ from TM2 are highly conserved in TM3 of a putative remote bacterial homologue (ASBT_NM), suggesting a universal mechanism for the SLC10A transporter family.