ABCMdb

PMID: 25867140

Eckford PD, Li C, Bear CE
Functional reconstitution and channel activity measurements of purified wildtype and mutant CFTR protein.
J Vis Exp. 2015 Mar 9;(97). doi: 10.3791/52427., [PubMed]

Sentences

No. Mutations Sentence Comment

9 ABCC7 p.Gly551Asp 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. Login to comment 28 ABCC7 p.Gly551Asp G551D-CFTR, a less common mutation, is thought to be properly folded yet is dysfunctional as a chloride channel at the cell surface 6 . Login to comment 29 ABCC7 p.Gly551Asp The development of small molecule correctors and potentiators has the goal of correcting folding and/or trafficking of mutants such as F508del- CFTR to the cell surface, and potentiating or increasing the channel activity of mutations such as G551D when present on the cell surface, respectively. Login to comment 30 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1244Glu ABCC7 p.Gly1349Asp ABCC7 p.Ser1255Pro ABCC7 p.Ser1251Asn ABCC7 p.Ser549Arg ABCC7 p.Ser549Asn ABCC7 p.Gly178Arg While the correctors VX-809 and VX-661 (are not yet approved for use in patients, the potentiator Kalydeco (ivacaftor; VX-770) is being used at 150 mg every 12 hr in CF patients >6 years with at least one G551D-CFTR mutation, and more recently for patients with one of G178R, S549N, S549R, G551S, G1244E, S1251N, S1255P and G1349D. Login to comment 35 ABCC7 p.Gly551Asp The system was used to interrogate the effects of the potentiator VX-770/Kalydeco on Wt- (wild-type), F508del- and G551D-CFTR and it was shown for the first time that the drug interacts directly with the CFTR protein to potentiate its channel activity in an ATP-independent manner, demonstrating the utility and applicability of these methods to the study of the interaction of CFTR and mutants with nucleotides and small molecules from a population perspective to answer clinically relevant questions about the protein. Login to comment 52 ABCC7 p.Gly551Asp Crude membrane preparation 1. Obtain a fresh or thaw a frozen Sf9 cell pellet from a 500 ml culture of cells over-expressing wildtype-, F508del-, or G551D-CFTR protein with a C-terminal His10-tag, grown by standard cell culture methods, as described previously 17,28 . Login to comment 115 ABCC7 p.Gly551Asp For G551D- and F508del-CFTR, use a protein:lipid ratio of approximately 1:200-1:1,200 (w/w) in order to detect efflux activity. Login to comment 271 ABCC7 p.Gly551Asp This rapid purification protocol yields Wt-CFTR from Sf9 cells at near homogeneity, however for F508del- and G551D-CFTR, the method is better described as an enrichment procedure. Login to comment 274 ABCC7 p.Gly551Asp As shown in Figure 3D, significant iodide efflux is measured for both F508del- (protein:lipid mass ratio of approximately 1:600) and G551D-CFTR (protein:lipid ratio of approximately 1:300). Login to comment 276 ABCC7 p.Gly551Asp However both F508del-CFTR and G551D purified by these methods, reconstituted and subjected to efflux measurements respond as expected to small molecule potentiators (Figure 4), and show similar channel function to protein purified by the more rigorous PFO purification method 19 . Login to comment 281 ABCC7 p.Gly551Asp As shown in Figure 4, all three CFTR genotypes tested are significantly potentiated by active small molecule potentiators such as Kalydeco/VX-770 or VRT-532 (Figure 4C; shown for G551D), while an inactive analog has no effect on the iodide efflux activity of CFTR (shown for F508del-CFTR; Figure 4B). Login to comment 298 ABCC7 p.Gly551Asp (D) Multiple genotypes produce regulated CFTR signals in the iodide efflux assay, including Wt-, F508del- and G551D-CFTR versus control. Login to comment 300 ABCC7 p.Gly551Asp (c)The American Society for Biochemistry and Molecular Biology. Figure 4: The phosphorylation-dependent potentiation of CFTR by small molecules can be interrogated using the iodide efflux system for Wt-, F508del- and G551D-CFTR. Login to comment 305 ABCC7 p.Gly551Asp (c) Potentiation of G551D-CFTR by 10 &#b5;M VRT-532 or VX-770. Login to comment 310 ABCC7 p.Gly551Asp The method described here is advantageous as it allows rapid purification of Wt-CFTR or high enrichment of F508del- and G551D-CFTR in moderate quantities that is highly functional in assays including ATPase and direct measurements of channel function, including single channel measurements in planar bilayer systems and demonstrated measures of CFTR population channel function that are highly relevant to the study of small molecules 19-21 . Login to comment