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

Abstract

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-beta-cyclodextrin (MbetaCD) 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 MbetaCD. The inhibition in ASBT activity by MbetaCD was blocked in the cells treated with MbetaCD-cholesterol complexes. Kinetic analysis revealed that MbetaCD treatment decreased the V(max) 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.

Digoxin Is Not a Substrate for Organic Anion-Transporting Polypeptide Transporters OATP1A2, OATP1B1, OATP1B3, and OATP2B1 but Is a Substrate for a Sodium-Dependent Transporter Expressed in HEK293 Cells

“…It is also notable that mRNA for SLC5A6, a sodium-dependent transporter that has been reported to demonstrate broad substrate selectivity (de Carvalho and Quick, 2011), is highly expressed in HEK293 cells, at 1634 MAS5 units. In contrast, the apical sodium-dependent bile acid transporter (ASBT; gene SLC10A2) is a sodium-dependent bile acid transporter localized to the apical surface of the terminal ileal enterocytes; however, consistent with the relatively low level of mRNA expression determined from gene chip analysis (56 MAS5 units), ASBT is not endogenously expressed in HEK293 cells (Annaba et al, 2008). Likewise, but not surprisingly, mRNA for the sodium taurocholate transporting polypeptide (gene SLC10A1), a sodium-dependent bile acid transporter expressed on the sinusoidal membrane of hepatocytes, was also essentially absent in HEK293 cells (22 MAS5 units; Table 3).…”

“…Because it is known that the ASBT (SLC10A2) is expressed on the apical surface of terminal ileal enterocytes, cholangiocytes, and renal proximal tubular cells (Annaba et al, 2008), it was considered a possibility that ASBT may be responsible for the sodium-dependent uptake of digoxin into HEK293 cells. However, ASBT is not expressed in HEK293 cells (Annaba et al, 2008), so this was ruled out as a potential explanation.…”

“…However, ASBT is not expressed in HEK293 cells (Annaba et al, 2008), so this was ruled out as a potential explanation. Another transporter, SMVT (SLC5A6), has recently been described as a sodium-dependent SLC family transporter that demonstrates "surprising substrate versatility" (de Carvalho and Quick, 2011).…”

Xenobiotic, Bile Acid, and Cholesterol Transporters: Function and Regulation

“…Depletion of membrane cholesterol enhances rat Ntcp uptake activity in transfected HEK293 cells (Molina et al, 2008). Moreover, reduced membrane cholesterol lowers BCRP (Storch et al, 2007), ASBT (Annaba et al, 2008), and Pgp (Dos Fenyvesi et al, 2008) transporter activity in overexpressing cell lines. In the cases of BCRP and ASBT, lower cholesterol levels did not alter cell viability or transporter subcellular localization or expression (Storch et al, 2007;Annaba et al, 2008).…”

Bile acid transporters

“…The ASBT protein resides on the plasma membrane in lipid rafts and depleting membrane cholesterol using methyl-␤ -cyclodextrin signifi cantly reduced ASBT's association with rafts and taurocholate transport ( 230 ). The cholesterol-depleted cells exhibited no change in ASBT protein expression at the plasma membrane, suggesting that the decreased taurocholate uptake was due to reduced ASBT activity.…”