title: Functional reconstitution and channel activity measurements of purified wildtype and mutant CFTR protein.
abstract: The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.
authors: Programme in Molecular Structure and Function, Hospital for Sick Children.
authors: Paul D W
authors: PD
authors: Eckford
authors: Programme in Molecular Structure and Function, Hospital for Sick Children.
authors: Canhui
authors: C
authors: Li
authors: Programme in Molecular Structure and Function, Hospital for Sick Children; Department of Biochemistry, University of Toronto; Department of Physiology, University of Toronto; bear@sickkids.ca.
authors: Christine E
authors: CE
authors: Bear
mesh_terms: D000818:Animals; D001692:Biological Transport; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D011510:Proteolipids / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D061987:Sf9 Cells
chemical_list: C505032:CFTR protein, human; D011510:Proteolipids; D011994:Recombinant Proteins; C020485:proteoliposomes; D019005:Cystic Fibrosis Transmembrane Conductance Regulator