The lipid flippase heterodimer ATP8B1–CDC50A is essential for surface expression of the apical sodium-dependent bile acid transporter (SLC10A2/ASBT) in intestinal Caco-2 cells

Abstract

Deficiency of the phospholipid flippase ATPase, aminophospholipid transporter, class I, type 8B, member 1 (ATP8B1) causes progressive familial intrahepatic cholestasis type 1 (PFIC1) and benign recurrent intrahepatic cholestasis type 1 (BRIC1). Apart from cholestasis, many patients also suffer from diarrhea of yet unknown etiology. Here we have studied the hypothesis that intestinal ATP8B1 deficiency results in bile salt malabsorption as a possible cause of PFIC1/BRIC1 diarrhea. Bile salt transport was studied in ATP8B1-depleted intestinal Caco-2 cells. Apical membrane localization was studied by a biotinylation approach. Fecal bile salt and electrolyte contents were analyzed in stool samples of PFIC1 patients, of whom some had undergone biliary diversion or liver transplantation. Bile salt uptake by the apical sodium-dependent bile salt transporter solute carrier family 10 (sodium/bile acid cotransporter), member 2 (SLC10A2) was strongly impaired in ATP8B1-depleted Caco-2 cells. The reduced SLC10A2 activity coincided with strongly reduced apical membrane localization, which was caused by impaired apical membrane insertion of SLC10A2. Moreover, we show that endogenous ATP8B1 exists in a functional heterodimer with transmembrane protein 30A (CDC50A) in Caco-2 cells. Analyses of stool samples of post-transplant PFIC1 patients demonstrated that bile salt content was not changed, whereas sodium and chloride concentrations were elevated and potassium levels were decreased. The ATP8B1-CDC50A heterodimer is essential for the apical localization of SLC10A2 in Caco-2 cells. Diarrhea in PFIC1/BRIC1 patients has a secretory origin to which SLC10A2 deficiency may contribute. This results in elevated luminal bile salt concentrations and consequent enhanced electrolyte secretion and/or reduced electrolyte resorption.

Human mini-guts: new insights into intestinal physiology and host–pathogen interactions

“…For example, the functional role of cyclic GMP-dependent protein kinase II (cGKII) in ion transport was contradictory between T84 cells (no cGKII involvement) 22 and mouse intestine (cGKII is involved in fluid homeostasis) 23 . Another marked difference has been reported for the bile salt transporter ASBT between Caco-2 cells and mouse intestine 24 . Although not fully understood, ASBT might be regulated by differing mechanisms or differing P-type ATPases in Caco-2 cells and mouse intestine.…”

ATP8B1 and ATP11C: Two Lipid Flippases Important for Hepatocyte Function

“…Furthermore and in contrast to Chen and colleagues, Cai et al [17] and Van der Mark et al [23] have shown that ATP8B1 depletion in Caco-2 cells and human and rat hepatocytes leaves FXR activity unaffected.…”

“…Van der Velden et al [24] observed that knockdown of ATP8B1 in Caco-2 cells led to a loss in microvilli, an unorganized apical actin cytoskeleton and a posttranscriptional defect in apical protein expression. Van der Mark et al [23], on the other hand, showed that ATP8B1 depletion in Caco-2 cells resulted in impaired membrane insertion and consequent absence of the apical sodium-dependent bile acid transporter (ASBT/SLC10A2) from the apical membrane, without a change in cellular morphology. These discrepant findings may be attributed to artifacts introduced by clonal selection of cells, use of different Caco-2 cell clones, variation in culture conditions or combinations of these factors.…”

Substrates of P4‐ATPases: beyond aminophospholipids (phosphatidylserine and phosphatidylethanolamine)

“…This finding may explain some of the extrahepatic symptoms of patients with PFIC1, such as hearing loss and diarrhea (91,95,99). Verhulst et al (76) reported that knockdown of ATP8B1 in Caco-2 cells led to loss of microvilli, and van der Mark et al (100) showed that ATP8B1 depletion did not affect the morphology of Caco-2 cells. Thus, it remains to be elucidated whether ATP8B1 is required for the formation of microvilli.…”