Activity of the Bile Salt Export Pump (ABCB11) Is Critically Dependent on Canalicular Membrane Cholesterol Content

Abstract

Mutations in ATP8B1 cause severe inherited liver disease. The disease is characterized by impaired biliary bile salt excretion (cholestasis), but the mechanism whereby impaired ATP8B1 function results in cholestasis is poorly understood. ATP8B1 is a type 4 P-type ATPase and is a flippase for phosphatidylserine. Atp8b1-deficient mice display a dramatic increase in the biliary extraction of cholesterol from the canalicular (apical) membrane of the hepatocyte. Here we studied the hypothesis that disproportionate cholesterol extraction from the canalicular membrane impairs the activity of the bile salt transporter, ABCB11, and as a consequence causes cholestasis. Using single pass liver perfusions, we show that not only ABCB11-mediated transport but also Abcc2-mediated transport were reduced at least 4-fold in Atp8b1 deficiency. We show that canalicular membranes of cholestatic Atp8b1-deficient mice have a dramatically reduced cholesterol to phospholipid ratio, i.e. 0.75 ؎ 0.24 versus 2.03 ؎ 0.71 for wild type. In vitro depletion of cholesterol from mouse liver plasma membranes using methyl-␤-cyclodextrin demonstrated a near linear relation between cholesterol content of the membranes and ATPdependent taurocholate transport. Abcc2-mediated transport activity was not affected up to 30% of membrane cholesterol depletion but declined to negligible levels at 70% of membrane cholesterol depletion. These effects were reversible as cholesterol repletion of the liver membranes completely restored Abcb11-and Abcc2-mediated transport. Our data demonstrate that membrane cholesterol content is a critical determinant of ABCB11/ABCC2 transport activity, provide an explanation for the etiology of ATP8B1 disease, and suggest a novel mechanism protecting the canalicular membrane against luminal bile salt overload.

A Flippase-Independent Function of Atp8b1, the Protein Affected in Familial Intrahepatic Cholestasis Type 1, Is Required for Apical Protein Expression and Microvillus Formation in Polarized Epithelial Cells

“…This observation challenges a recently proposed model in which the morphological aberrancies and loss of ectoenzymes from the canalicular membrane in liver biopsies of Atp8b1 G308V/G308V mutant mice are primarily ascribed to a disruption of APL asymmetry, which would render the canalicular membrane hypersensitive to extraction of membrane sterols and proteins by hydrophobic bile salts. 21,33 Our current findings therefore imply that ATP8B1 serves a dual role. Besides catalyzing APLT activity, we propose that ATP8B1 may provide a molecular scaffold in the apical membrane to recruit structural components or modulators of the actin cytoskeleton involved in microvilli formation.…”

Interaction of bile salts with rat canalicular membrane vesicles: Evidence for bile salt resistant microdomains

“…Fourth, cholesterol is a critical component of lipid microdomains. Feeding mice lacking Atp8b1 with a cholate diet renders them cholestatic and markedly reduces the cholesterol to phospholipid ratio in cLPM [29]. These and other authors have shown that the activity of Bsep depends on membrane cholesterol content [29,30].…”

“…BSRMs contain the transporters required for canalicular bile formation and are enriched in cholesterol. The function of the ABCtransporters is modulated by membrane cholesterol, since a positive correlation between membrane cholesterol content and transporter activity for Bsep [30], BCRP [32] and MRP2 (Guyot and Stieger unpublished) expressed in Sf9 cells and for Bsep in cLPM [29] has been demonstrated. Stimulation of Mrp2 will increase BS independent bile flow and consequently lower BS concentration in the canaliculus.…”

Inner workings and biological impact of phospholipid flippases

“…ATP8B1 mutant mice display a dramatic increase in biliary output of canalicular cholesterol. Subsequent studies have shown that the activity of the bile salt pump is crucially dependent on the cholesterol content of the canalicular membrane (Paulusma et al, 2009). The exoplasmic leaflet of this membrane is rich in sphingolipids that are tightly packed with cholesterol to provide maximum resistance against the detergent action of hydrophobic bile salts.…”