Abstract
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 (ASBTNM), suggesting a universal mechanism for the SLC10A transporter family.
Highlights
Transmembrane domains are critical to the structure and function of bile acid transporters
High sequence conservation in transmembrane 2 (TM2) was observed among various species (Fig. 1B)
The mutations were generated using the hASBT C270A scaffold, which is largely insensitive to thiol-specific chemical reagents
Summary
Transmembrane domains are critical to the structure and function of bile acid transporters. 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 ␣-helical periodicity. Functional flexibility enabled by residues Pro234, Gly237, and Gly241 allow TM6 to form a “conformational switch” for substrate turnover [16] Both experimental data and computational modeling suggest a pivotal role for TM7 in substrate translocation [17, 18]. Conservative and non-conservative mutations were generated on functionally impaired and sodium-sensitive residues to further elucidate their functional importance/requirement
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