From the Department of Pharmaceutical Sciences, University of Maryland, Baltimore, Maryland 21201.
From the Department of Pharmaceutical Sciences, University of Maryland, Baltimore, Maryland 21201.
J Biol Chem. 2013 Nov 8;288(45):32394-32404. doi: 10.1074/jbc.M113.518555. Epub 2013 Sep 17.
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.
人顶膜钠依赖性胆汁酸转运蛋白（hASBT，SLC10A2）负责胆汁酸的肠吸收，在胆固醇稳态中发挥关键作用。我们采用靶向和系统的方法来描绘高度保守的跨膜螺旋 2 对 hASBT 表达和功能的作用。半胱氨酸突变显著降低了超过 60%的突变体的转运活性，而不影响转运体的细胞表面定位。所有突变体都无法被不透膜的 MTSET 试剂进行化学修饰，这强烈表明跨膜 2（TM2）在胆汁酸底物转运中起间接作用。TM2 突变体的胆汁酸摄取和钠离子依赖性均显示出明显的α-螺旋周期性。对保守和非保守的钠离子敏感残基突变体的动力学研究进一步强调了 Gln（75）、Phe（76）、Met（79）、Gly（83）、Leu（86）、Phe（90）和 Asp（91）在 hASBT 功能中的重要性。计算分析表明，Asp（91）可能在转运循环中与钠离子协同作用。综合来看，我们的数据表明，一组钠离子敏感残基与之前报道的 TM3 中的残基（Thr（134）、Leu（138）和 Thr（149））可能形成钠离子结合和转运途径。值得注意的是，TM2 中的残基 Gln（75）、Met（79）、Thr（82）和 Leu（86）在假定的远程细菌同源物（ASBTNM）的 TM3 中高度保守，这表明 SLC10A 转运蛋白家族具有普遍的机制。
