CFTR Assays

CFTR is an integral membrane protein that functions as a channel to allow passage of Cl- ions, as well as bicarbonate, across membranes. Various assays have been reported for measuring CFTR ion channel activity as well as its stability in the membrane.

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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.

Reference Material:

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.

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CFTR Functional Assays

ATPase Assays

Although CFTR’s main function is to operate as an anion channel, it also displays enzymatic activity through cleavage of ATP (an ATP-ase). The protein has a slow turnover rate for this enzymatic activity, as it is only needed to regulate the open/closed state in support of channel function.

Measuring the ATP-ase activity is sometimes done when researchers have purified CFTR or the ATP-binding subunits of CFTR, and they wish to determine that the protein is in a “native” or functional conformation.

Reference Material:

Csanády L, Vergani P, Gadsby DC. Strict coupling between CFTR's catalytic cycle and gating of its Cl- ion pore revealed by distributions of open channel burst durations. Proc Natl Acad Sci USA. 2009 Dec 4. [Epub ahead of print]

Wellhauser L, Kim Chiaw P, Pasyk S, Li C, Ramjeesingh M, Bear CE.  A small-molecule modulator interacts directly with deltaPhe508-CFTR to modify its ATPase activity and conformational stability. Mol Pharmacol, 2009. 75(6): p. 1430-8.

Electrophysiology Assays

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.

Reference Material:

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.”

Reference Material:

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, González 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.

A publication describing the discovery of a CFTR potentiator compound and its early stage clinical results demonstrates the utility of these cells.

Reference Material:

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 U S A, 2009. 106(44): p. 18825-30

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Updated: 9/12/2014