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

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The cholestatic disorders 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 generally more disturbing than BRIC1 mutations with respect to expression, structural stability and/or function. Although recent data have indicated 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 have investigated known PFIC1 and BRIC1 mutations using our well-functioning methodology for expression, affinity purification and assay of the partial reactions of ATP8A2.
Mutations I91P in ATP8A2 (L127P in ATP8B1) and L308F (I344F) are located in TM1 and TM3, respectively, and E897K (E891K) is located in the exoplasmic loop between TM5 and TM6. Our experiments are focusing on the ATPase activity, rate of phosphorylation and dephosphorylation, and affinities for vanadate and the lipid substrate of the mutant proteins. L308F and E897K provide clues to where the lipid substrate might bind and enter the protein. The charge reversal from negative to positive in the E897K increases the affinity for the negatively charged PS significantly, whereas the neutral PE binds with wild type-like affinity in this mutant. The BRIC1 mutant L308F has reduced affinity for both PS and PE, but almost wild type-like maximal rate and cellular expression, in contrast to the PFIC1 mutants I91P and E897K, which display much lower 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 year2015
Number of pages1
Publication statusPublished - 2015
Event4th International Workshop on Expression, Structure and Function of Membrane Proteins - Firenze, Italy
Duration: 28 Jun 20152 Jul 2015

Workshop

Workshop4th International Workshop on Expression, Structure and Function of Membrane Proteins
CountryItaly
CityFirenze
Period28/06/201502/07/2015

    Research areas

  • Flippase, flippase mechanism, flippase structure, P-type ATPase, Cholestasis

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