Biochemical Characterization of P4-ATPase Mutations Associated with Intrahepatic Cholestatic Disease

Research output: Contribution to conferencePosterResearch

The cholestatic disorders progressive familial intrahepatic cholestasis type 1 (PFIC1, also referred to as Byler’s disease) and benign recurrent intrahepatic cholestasis type 1 (BRIC1) are caused by mutation of the P4-ATPase ATP8B1. The substrate of ATP8B1 is very likely to be phosphatidylserine (PS), and ATP-driven flipping of PS from the exoplasmic leaflet to the cytoplasmic leaflet of canalicular membranes of hepatocytes is essential to maintain the bile acid transporter ABCB11 in a functional state, and, thus, to prevent development of cholestasis [1]. Approximately 200 PFIC1 and BRIC1 families have been investigated, and more than 50 distinct disease mutations have been identified, with roughly half being missense mutations.
In this project we try to answer the question whether PFIC1 mutations are generally more disturbing than BRIC1 mutations with respect to expression, structural stability and function. We investigate the mutations in our well functioning system of ATP8A2, being expressed in mammalian HEK293T cells, affinity-purified, and reconstituted in lipid vesicles. Well-known mutations from both groups of patients have been selected for study.
I91P in ATP8A2 (L127P in ATP8B1) and L308F (I344F) are located in the first and third transmembrane segment, respectively, and E897K (E891K) is located in the exoplasmic loop between the fifth and sixth transmembrane segment. The first experiments were focusing on the ATPase activity of the mutant proteins and their dependence on PS concentration. The BRIC1 mutant L308F has an almost wild type-like maximal rate, in contrast to the PFIC1 mutants I91P and E897K, which display much lower maximal rates. Both L308F and I91P show markedly reduced affinities for PS, whereas E897K exhibits a markedly increased affinity compared with the wild type. Furthermore, the cellular expression of the PFIC1 mutants is dramatically lower than wild type flippase.
These and further results offer a potential explanation as to why the PFIC1 mutations are generally more disturbing than BRIC1 mutations. Additionally, these functional studies of residues in the transmembrane domain will contribute with important information in elucidation of the phospholipid transport mechanism of P4-type ATPases.

1. Folmer, D.E., R.P.J. Oude Elferink, and C.C. Paulusma, Biochim. Biophys. Acta (2009) 1791. 628-635.
Original languageEnglish
Publication year2014
Number of pages1
Publication statusPublished - 2014
EventNa,K-ATPase and related transport ATPases, structure, mechanism, cell biology, health and disease - Conference Center De Werelt, Lunteren, Netherlands
Duration: 30 Aug 20145 Sep 2014
Conference number: 14th P-type ATPase meeting


ConferenceNa,K-ATPase and related transport ATPases, structure, mechanism, cell biology, health and disease
Number14th P-type ATPase meeting
LocationConference Center De Werelt

    Research areas

  • Flippase, P-type ATPase, Cholestasis

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