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Modulation of ileal bile acid transporter (ASBT) activity by depletion of plasma membrane cholesterol: association with lipid rafts

机译:通过消耗质膜胆固醇调节回肠胆汁酸转运蛋白(ASBT)活性:与脂筏相关联。

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First published December 3, 2007; doi:10.1152/ajpgi.00237.2007.-Apical sodium-dependent bile acid transporter (ASBT) represents a highly efficient conservation mechanism of bile acids via mediation of their active transport across the luminal membrane of terminal ileum. To gain insight into the cellular regulation of ASBT, we investigated the association of ASBT with cholesterol and sphingolipid-enriched specialized plasma membrane microdomains known as lipid rafts and examined the role of membrane cholesterol in maintaining ASBT function. Human embryonic kidney (HEK)-293 cells stably transfected with human ASBT, human ileal brush-border membrane vesicles, and human intestinal epithelial Caco-2 cells were utilized for these studies. Floatation experiments on Optiprep density gradients demonstrated the association of ASBT protein with lipid rafts. Disruption of lipid rafts by depletion of membrane cholesterol with methyl-3-cyclodextrin (M(beta)CD) significantly reduced the association of ASBT with lipidrafts, which was paralleled by a decrease in ASBT activity in Caco-2 and HEK-293 cells treated with M(beta)CD. The inhibition in ASBT activity by IVCD was blocked in the cells treated with M(beta)CD-cholesterol complexes. Kinetic analysis revealed that MpCD treatment decreased the Vmax of the transporter, which was not associated with alteration in the plasma membrane expression of ASBT. Our study illustrates that cholesterol content of lipid rafts is essential for the optimal ctivity of ASBT and support the association of ASBT with lipid rafts. These findings suggest a novel mechanism by which ASBT activity may be rapidly modulated by alterations in cholesterol content of plasma membrane and thus have important implications in processes related to maintenance of bile acid and cholesterol homeostasis.
机译:首次发布于2007年12月3日; doi:10.1152 / ajpgi.00237.2007.-钠依赖性胆汁酸转运蛋白(ASBT)通过介导胆汁酸通过末端回肠腔膜的主动转运来代表一种高效的保存机制。为了深入了解ASBT的细胞调控,我们研究了ASBT与胆固醇和富含鞘脂的特殊质膜微结构域(称为脂质筏)的关系,并研究了膜胆固醇在维持ASBT功能中的作用。将稳定转染了人ASBT,人回肠刷状边界膜囊泡和人肠上皮Caco-2细胞的人胚胎肾(HEK)-293细胞用于这些研究。在Optiprep密度梯度上进行的浮选实验表明ASBT蛋白与脂质筏相关。通过用甲基-3-环糊精(MβCD)消耗膜胆固醇破坏脂筏,显着降低了ASBT与脂筏的缔合,与此同时,在处理的Caco-2和HEK-293细胞中ASBT活性降低MβCD。由IVCD对ASBT活性的抑制在用MβCD-胆固醇复合物处理的细胞中被阻断。动力学分析表明,MpCD处理降低了转运蛋白的Vmax,这与ASBT质膜表达的改变无关。我们的研究表明,脂筏的胆固醇含量对于ASBT的最佳功能性至关重要,并支持ASBT与脂筏的关联。这些发现表明,通过其质膜胆固醇含量的变化可以快速调节ASBT活性的新机制,因此在与维持胆汁酸和胆固醇稳态有关的过程中具有重要意义。

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