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. Video LinkThe video component of this article can be found at

Attenuation of Phosphorylation-dependent Activation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Disease-causing Mutations at the Transmission Interface

“…1 A ). In a subset of our experiments, we confirmed that the purified full-length WT-CFTR is functional as a phosphorylation and ATP-regulated anion channel upon reconstitution in proteoliposomes using our previously published iodide efflux protocols ( 12 , 14 ).…”

Cholesterol Interaction Directly Enhances Intrinsic Activity of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

“…Each preparation was separately reconstituted into pre-formed 1,2-palmitoyl-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes. The anion electrodiffusion from liposomes normalized for the amount of CFTR inserted into liposomes were measured (Refer to CFTR quantification section in Materials and Methods for more details on the method) in order to determine the relative proportion of channel competent CFTR molecules from the two preparations [40,41]. Briefly, in this assay, CFTR protein in amphipols or LMNG micelles was reconstituted into POPC proteoliposomes with equal concentrations of potassium iodide inside and potassium glutamate outside of the liposomes to provide a driving force for iodide efflux and to maintain the osmolarity [40,41].…”

“…The anion electrodiffusion from liposomes normalized for the amount of CFTR inserted into liposomes were measured (Refer to CFTR quantification section in Materials and Methods for more details on the method) in order to determine the relative proportion of channel competent CFTR molecules from the two preparations [40,41]. Briefly, in this assay, CFTR protein in amphipols or LMNG micelles was reconstituted into POPC proteoliposomes with equal concentrations of potassium iodide inside and potassium glutamate outside of the liposomes to provide a driving force for iodide efflux and to maintain the osmolarity [40,41]. The addition of valinomycin, an ionophore that selectively facilitates potassium ion flux out of the proteoliposomes, alleviates the charge build up in the proteoliposomes and allows for iodide efflux through activated CFTR [40,41].…”

“…Briefly, in this assay, CFTR protein in amphipols or LMNG micelles was reconstituted into POPC proteoliposomes with equal concentrations of potassium iodide inside and potassium glutamate outside of the liposomes to provide a driving force for iodide efflux and to maintain the osmolarity [40,41]. The addition of valinomycin, an ionophore that selectively facilitates potassium ion flux out of the proteoliposomes, alleviates the charge build up in the proteoliposomes and allows for iodide efflux through activated CFTR [40,41]. A schematic of this assay is shown in Figure 4A.…”

Characterizing diverse orthologues of the cystic fibrosis transmembrane conductance regulator protein for structural studies

“…In this spirit, a recent report described the extraction of CFTR from insect cell membranes using a zwitterionic detergent foscholine-14 followed by rapid exchange into the non-ionic detergent dodecylmaltoside (DDM) [41]. Following this protocol, the authors measured channel flux and reconstituted ATPase activity.…”