Western Blot Assays
As a membrane bound protein, CFTR's biogenesis carries it through the endoplasmic reticulum (ER) and Golgi apparatus. Within the ER the CFTR polypeptide is core glycosylated at two sites and then within the Golgi apparatus it receives complex glycosylation that is maintained at the level of the plasma membrane.
When evaluated on a Western blot the core glycosylated immature form of CFTR migrates further and is designated the "B-band." The complex glycosylated form of CFTR, representing transit through the Golgi, but not necessarily plasma membrane expression, migrates slower during gel electrophoresis due to its greater molecular weight and is termed "C-band."
Complex glycosylation appears to play a role in prolonging membrane stability. The F508del CFTR protein shows a marked drop in the level of "C-band" observed in Western blot assays.
- Chang XB, Mengos A, Hou YX, Cui L, Jensen TJ, Aleksandrov A, Riordan JR, Gentzsch M. Role of N-linked oligosaccharides in the biosynthetic processing of the cystic fibrosis transmembrane conductance regulator. J Cell Sci., 2008. 121(Pt 17): p. 2814-23
CFTR Functional Assays
The function of different CFTR mutations and their response to CFTR modulators (potentiators, correctors, and inhibitors) is commonly studied in CF. A number of assays have been developed to evaluate CFTR function; however, some of the more commonly used assays for basic research and drug discovery efforts are highlighted below.
There are two main kinds of assays that are done on cells expressing CFTR in the apical membrane:
- Ussing chamber assays use electrodes to measure ion movement across the membranes of cells grown into a monolayer with tight junctions.
- "Patch clamp" assays measure the opening and closing rates of single channels, in which patches of the cell membrane are isolated using a micropipette tip and these patches are hooked up to microelectrodes.
- Devor DC, Bridges RJ, Pilewski JM. Pharmacological modulation of ion transport across wild-type and DeltaF508 CFTR-expressing human bronchial epithelia. Am J Physiol Cell Physiol, 2000. 279(2): C461-79
- Dousmanis, AG, Nairn AC, Gadsby DC. Distinct Mg(2+)-dependent steps rate limit opening and closing of a single CFTR Cl(-) channel. J Gen Physiol, 2002. 119(6): p. 545-59
CFTR Modulator Discovery Assays
A number of high-throughput assays have been configured to screen for small molecules that repair the dysfunction of mutant CFTR. The cells chosen for the screening may have an influence on what kind of modulators are found.
F508del CFTR cells can be used in assays configured such that small molecules that correct the trafficking dysfunction are termed corrector hits. Alternatively, these assays can also be configured to find small molecules that improve the open probability of CFTR, and these small molecules are referred to as potentiator hits.
High-throughput assays to detect restored CFTR function have been published by a number of investigators including Dr. Alan Verkman (University of California, San Francisco), Dr. Luis Gallietta (Istituto Giannina Gaslini, Genova, Italy), and Vertex Pharmaceuticals, Inc. (San Diego, CA).
- Pedemonte N, Lukacs GL, Du K, Caci E, Zegarra-Moran O, Galietta LJ, Verkman AS. Small-molecule correctors of defective DeltaF508-CFTR cellular processing identified by high-throughput screening. J Clin Invest, 2005. 115(9): p. 2564-71
- Pedemonte N, Diena T, Caci E, Nieddu E, Mazzei M, Ravazzolo R, Zegarra-Moran O, Galietta LJ. Antihypertensive 1,4-dihydropyridines as correctors of the cystic fibrosis transmembrane conductance regulator channel gating defect caused by cystic fibrosis mutations. Mol Pharmacol, 2005. 68: p. 1736-1746
- Van Goor F, Straley KS, Cao D, Gonzlez J, Hadida S, Hazlewood A, Joubran J, Knapp T, Makings LR, Miller M, Neuberger T, Olson E, Panchenko V, Rader J, Singh A, Stack JH, Tung R, Grootenhuis PD, Negulescu P. Rescue of DeltaF508-CFTR trafficking and gating in human cystic fibrosis airway primary cultures by small molecules. Am J Physiol Lung Cell Mol Physiol, 2006. 290(6): p. L1117-30
A Preclinical Model for CFTR Modulator Studies
While most known CFTR modulator compounds have been discovered through screening assays using immortalized cell lines, an important pre-clinical cell-based model system used to confirm the activity of hits is primary human epithelial cells derived from lung transplant tissue.
The publications below describe the discovery of CFTR potentiator and corrector compounds and their early stage clinical results that demonstrate the utility of these cells.
- Van Goor F, Hadida S, Grootenhuis PD, Burton B, Cao D, Neuberger T, Turnbull A, Singh A, Joubran J, Hazlewood A, Zhou J, McCartney J, Arumugam V, Decker C, Yang J, Young C, Olson ER, Wine JJ, Frizzell RA, Ashlock M, Negulescu P. Rescue of CF airway epithelial cell function in vitro by a CFTR potentiator, VX-770. Proc Natl Acad Sci USA, 2009. 106(44): p. 18825-30
- Van Goor F, Hadida S, Grootenhuis PD, Burton B, Stack JH, Straley KS, Decker CJ, Miller M, McCartney J, Olson ER, Wine JJ, Frizzell RA, Ashlock M, Negulescu PA. Correction of the F508del-CFTR protein processing defect in vitro by the investigational drug VX-809. Proc Natl Acad Sci U S A. 2011 Nov 15;108(46):18843-8.