title: Effects of cellular, chemical, and pharmacological chaperones on the rescue of a trafficking-defective mutant of the ATP-binding cassette transporter proteins ABCB1/ABCB4.
abstract: The ATP-binding cassette transporter ABCB4 is a phosphatidylcholine translocator specifically expressed at the bile canalicular membrane in hepatocytes, highly homologous to the multidrug transporter ABCB1. Variations in the ABCB4 gene sequence cause progressive familial intrahepatic cholestasis type 3. We have shown previously that the I541F mutation, when reproduced either in ABCB1 or in ABCB4, led to retention in the endoplasmic reticulum (ER)/Golgi. Here, Madin-Darby canine kidney cells expressing ABCB1-GFP were used as a model to investigate this mutant. We show that ABCB1-I541F is not properly folded and is more susceptible to in situ protease degradation. It colocalizes and coprecipitates with the ER chaperone calnexin and coprecipitates with the cytosolic chaperone Hsc/Hsp70. Silencing of calnexin or overexpression of Hsp70 have no effect on maturation of the mutant. We also tested potential rescue by chemical and pharmacological chaperones. Thapsigargin and sodium 4-phenyl butyrate were inefficient. Glycerol improved maturation and exit of the mutant from the ER. Cyclosporin A, a competitive substrate for ABCB1, restored maturation, plasma membrane expression, and activity of ABCB1-I541F. Cyclosporin A also improved maturation of ABCB4-I541F in Madin-Darby canine kidney cells. In HepG(2) cells transfected with ABCB4-I541F cDNA, cyclosporin A allowed a significant amount of the mutant protein to reach the membrane of bile canaliculi. These results show that the best strategy to rescue conformation-defective ABCB4 mutants is provided by pharmacological chaperones that specifically target the protein. They identify cyclosporin A as a potential novel therapeutic tool for progressive familial intrahepatic cholestasis type 3 patients.
authors.affiliation: INSERM and UPMC Université Paris 06, UMR_S 938, CdR Saint-Antoine, 75012 Paris, France.
authors.forename: Julien
authors.initials: J
authors.lastname: Gautherot
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Durand-Schneider
authors.forename: Danièle
authors.initials: D
authors.lastname: Delautier
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Delaunay
authors.forename: Alegna
authors.initials: A
authors.lastname: Rada
authors.forename: Julie
authors.initials: J
authors.lastname: Gabillet
authors.forename: Chantal
authors.initials: C
authors.lastname: Housset
authors.forename: Michèle
authors.initials: M
authors.lastname: Maurice
authors.forename: Tounsia
authors.initials: T
authors.lastname: Aït-Slimane
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000235:genetics / Q000378:metabolism*; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D037281:Calnexin / Q000235:genetics / Q000378:metabolism*; D002779:Cholestasis / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D003451:Cryoprotective Agents / Q000494:pharmacology*; D016572:Cyclosporine / Q000494:pharmacology*; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D020868:Gene Silencing; D005990:Glycerol / Q000494:pharmacology*; D050883:HSC70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D056945:Hep G2 Cells; D006801:Humans; D020125:Mutation, Missense; D010654:Phenylbutyrates / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects / Q000235:genetics; D019284:Thapsigargin / Q000494:pharmacology
chemical_list: C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D003451:Cryoprotective Agents; D004791:Enzyme Inhibitors; D050883:HSC70 Heat-Shock Proteins; C494371:HSPA8 protein, human; D010654:Phenylbutyrates; D037281:Calnexin; D019284:Thapsigargin; C075773:4-phenylbutyric acid; D016572:Cyclosporine; C095122:multidrug resistance protein 3; D005990:Glycerol