Mareux Elodie, Lapalus Martine, Amzal Rachida, Almes Marion, Aït-Slimane Tounsia, Delaunay Jean-Louis, Adnot Pauline, Collado-Hilly Mauricette, Davit-Spraul Anne, Falguières Thomas, Callebaut Isabelle, Gonzales Emmanuel, Jacquemin Emmanuel
Inserm, Physiopathogénèse et traitement des maladies du foie, UMR_S 1193, Hepatinov, Université Paris-Saclay, Orsay, France.
Paediatric Hepatology & Paediatric Liver Transplant Department, Reference Center for Rare Paediatric Liver Diseases, FILFOIE, ERN RARE LIVER, Assistance Publique-Hôpitaux de Paris, Faculty of Medicine Paris-Saclay, CHU Bicêtre, Le Kremlin-Bicêtre, France.
Liver Int. 2020 Aug;40(8):1917-1925. doi: 10.1111/liv.14518. Epub 2020 Jun 8.
BACKGROUND & AIM: The canalicular bile salt export pump (BSEP/ABCB11) of hepatocytes is the main adenosine triphosphate (ATP)-binding cassette (ABC) transporter responsible for bile acid secretion. Mutations in ABCB11 cause several cholestatic diseases, including progressive familial intrahepatic cholestasis type 2 (PFIC2) often lethal in absence of liver transplantation. We investigated in vitro the effect and potential rescue of a BSEP mutation by ivacaftor, a clinically approved cystic fibrosis transmembrane conductance regulator (CFTR/ABCC7) potentiator.
The p.T463I mutation, identified in a PFIC2 patient and located in a highly conserved ABC transporter motif, was studied by 3D structure modelling. The mutation was reproduced in a plasmid encoding a rat Bsep-green fluorescent protein. After transfection, mutant expression was studied in Can 10 cells. Taurocholate transport activity and ivacaftor effect were studied in Madin-Darby canine kidney (MDCK) clones co-expressing the rat sodium-taurocholate co-transporting polypeptide (Ntcp/Slc10A1).
As the wild-type protein, Bsep was normally targeted to the canalicular membrane of Can 10 cells. As predicted by 3D structure modelling, taurocholate transport activity was dramatically low in MDCK clones expressing Bsep . Ivacaftor treatment increased by 1.7-fold taurocholate transport activity of Bsep (P < .0001), reaching 95% of Bsep activity. These data suggest that the p.T463I mutation impairs ATP-binding, resulting in Bsep dysfunction that can be rescued by ivacaftor.
These results provide experimental evidence of ivacaftor therapeutic potential for selected patients with PFIC2 caused by ABCB11 missense mutations affecting BSEP function. This could represent a significant step forward for the care of patients with BSEP deficiency.
肝细胞胆小管胆汁盐输出泵（BSEP/ABCB11）是负责胆汁酸分泌的主要三磷酸腺苷（ATP）结合盒（ABC）转运蛋白。ABCB11基因突变会引发多种胆汁淤积性疾病，包括2型进行性家族性肝内胆汁淤积症（PFIC2），若无肝移植，通常会致命。我们在体外研究了临床批准的囊性纤维化跨膜电导调节因子（CFTR/ABCC7）增强剂依伐卡托对BSEP突变的影响及潜在的挽救作用。
通过三维结构建模研究了在一名PFIC2患者中鉴定出的位于高度保守的ABC转运蛋白基序中的p.T463I突变。该突变在编码大鼠Bsep-绿色荧光蛋白的质粒中重现。转染后，在Can 10细胞中研究突变体表达。在共表达大鼠牛磺胆酸钠共转运多肽（Ntcp/Slc10A1）的Madin-Darby犬肾（MDCK）克隆中研究牛磺胆酸盐转运活性和依伐卡托的作用。
与野生型蛋白一样，Bsep正常靶向Can 10细胞的胆小管膜。如三维结构建模所预测，在表达Bsep的MDCK克隆中，牛磺胆酸盐转运活性极低。依伐卡托处理使Bsep的牛磺胆酸盐转运活性提高了1.7倍（P <.0001），达到Bsep活性的95%。这些数据表明p.T463I突变损害了ATP结合，导致Bsep功能障碍，而依伐卡托可挽救该功能障碍。
这些结果为依伐卡托对由影响BSEP功能的ABCB11错义突变引起的特定PFIC2患者的治疗潜力提供了实验证据。这可能是BSEP缺乏症患者护理方面向前迈出的重要一步。
