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Am J Physiol Gastrointest Liver Physiol 294: G489-G497, 2008. First published December 6, 2007; doi:10.1152/ajpgi.00237.2007
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MUCOSAL BIOLOGY

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

Fadi Annaba,1 Zaheer Sarwar,1 Pradeep Kumar,1 Seema Saksena,1 Jerrold R. Turner,3 Pradeep K. Dudeja,1,2 Ravinder K. Gill,1 and Waddah A. Alrefai1

1Section of Digestive Diseases and Nutrition, Department of Medicine, University of Illinois at Chicago, and 2Jesse Brown Veterans Affairs Medical Center; and 3Department of Pathology, University of Chicago, Chicago, Illinois

Submitted 24 May 2007 ; accepted in final form 30 November 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-β-cyclodextrin (MβCD) significantly reduced the association of ASBT with lipid rafts, which was paralleled by a decrease in ASBT activity in Caco-2 and HEK-293 cells treated with MβCD. The inhibition in ASBT activity by MβCD was blocked in the cells treated with MβCD-cholesterol complexes. Kinetic analysis revealed that MβCD 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 activity 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.

detergent-insoluble microdomains; floatation on Optiprep density gradient; bile acid absorption


Address for reprint requests and other correspondence: W. A. Alrefai, Univ. of Illinois at Chicago, Jesse Brown VA Medical Center. Medical Research Service (600/151), 820 South Damen Ave., Chicago, IL 60612 (e-mail: walrefai{at}uic.edu)






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