Biochemical characterization of P4-ATPase mutations associated with Intrahepatic Cholestatic Disease

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Progressive familial intrahepatic cholestasis type 1 (PFIC1) and benign recurrent intrahepatic cholestasis type 1 (BRIC1) are caused by mutation of the P4-ATPase ATP8B1 that flips phospholipid from the exoplasmic leaflet to the cytoplasmic leaflet of canalicular membranes. It is hypothesized that PFIC1 mutations are the most disturbing with respect to expression, structural stability and/or function. Although recent data indicates that the specific phospholipid substrate of ATP8B1 is phosphatidylcholine (PC) [1] whereas ATP8A2 flips phosphatidylserine (PS) and phosphatidylethanolamine (PE), there may be several mechanistic similarities between ATP8B1 and ATP8A2, and here we investigate known disease mutations using our well-functioning methodology for expression, affinity purification and assay of the partial reactions of ATP8A2.
Mutations I91P (L127P in ATP8B1) and L308F (I344F) are located in TM1 and TM3, respectively, and E897K (E981K) is located in the exoplasmic TM5-TM6 loop. Our experiments are focusing on the ATPase activity, phosphorylation rate and affinities for vanadate and the lipid substrates. L308F and E897K provide clues to where the lipid substrate might bind and enter the protein. The charge reversal from negative to positive in E897K increases the affinity for the negatively charged PS significantly, whereas the neutral PE binds with wild type-like affinity. The BRIC1 mutant L308F has reduced affinity for both lipids, but almost wild type-like maximal rate and cellular expression, in contrast to the PFIC1 mutants I91P and E897K, which display decreased expressions and maximal rates. These and further results offer a potential explanation as to why PFIC1 is generally more disturbing than BRIC1.

1. Takatsu, H. et al. (2014) J. Biol. Chem. 289, 33543-33556
Original languageEnglish
Publication year20 Sep 2015
Publication statusPublished - 20 Sep 2015
EventScandinavian Physiological Society Annual Meeting - Aarhus University, Aarhus, Denmark
Duration: 17 Sep 201520 Sep 2015


ConferenceScandinavian Physiological Society Annual Meeting
LocationAarhus University

    Research areas

  • Flippase, flippase mechanism, flippase structure, Cholestasis, Disease

See relations at Aarhus University Citationformats

ID: 93278940