Cystic fibrosis (CF), the most common single-gene hereditary disease among people of Northern European descent, is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator, CFTR. We’ll go into the genetics of CF in another post, but here we’ll discuss the connection between the symptoms of cystic fibrosis and the effects of CF mutations on the CFTR protein, its structure, and function as a chloride channel.
CF affects multiple tissues and organs in affected patients. CF patients have hypersaline (extra salty) sweat, have trouble digesting food and gaining normal weight, and as teens and young adults suffer from respiratory symptoms and eventually die of complications of lung infections and respiratory disease. Half the patients die before they reach their late 30’s. Life expectancy for the most severe forms are shorter. CF is a life-long disease, requiring constant management:
Here’s a historical view of the changes in the median survival age for CF patients, along with developments in treatment:
Note that before 1940, the survival age is less than 1 year. In medieval times, midwives reportedly licked the foreheads of newborns, and those with extra salty sweat were abandoned to die, because they would die within a few weeks anyway of malnutrition, no matter how much breast milk they consumed. As treatments are found that prolong patients’ lives, additional complications arise in later ages.
What’s the common factor behind these multiple symptoms affecting different organs and body systems? A disorder in osmosis, caused by the failure of CFTR to conduct chloride (Cl-) ions across cell surface membranes. Cells lining the small airway passages of the lung and the pancreatic ducts have CFTR protein on their plasma membranes in position to conduct Cl- ions into or out of the airways or ducts.
CFTR is an integral membrane protein with multiple transmembrane domains, as shown in this figure from Wikipedia:
The CFTR protein is made by cells that line small airway passages of the lung, and the secretory ducts in the pancreas, and sweat glands in the skin, among others.
Q: In what compartment of these cells will the CFTR protein be synthesized?
Q: If you wanted to track the synthesis and eventual cellular location of isotopically labeled CFTR protein, what isotope would you use: 32P, 35S, or 15N?
In such labeling experiments, cells are grown in medium containing isotopically labeled subunits that are incorporated into the type of macromolecule the experimenter wants labeled.
Q: To label newly synthesized proteins, the cells should be given what type of labeled subunits (amino acids, monosaccharides, nucleotides, or fatty acids)?
The most common mutation in CF patients is called deltaF508. In this mutation, phenylalanine, the 508th amino acid in the CFTR polypeptide chain, is missing. This amino acid is normally located in the nucleotide binding domain 1 (NBD1, also sometimes called NBF1), and mediates interaction of NBD1 with another region (CL4) of the protein. The figures below show the location of NBD1 in a schematic of CFTR domains and the location of F508 (Phe508) in a model of NBD1. The deltaF508 mutant protein exhibits defects in folding. The mis-folded deltaF508 CFTR protein remains in the ER where it is eventually degraded.
Phenylalanine is a hydrophobic amino acid.
Q: What do you think would be the least likely location of of hydrophobic amino acids like phenylalanine in the structure of proteins – on the surface interacting with water molecules, in an interior pocket, or on the surface interacting with another protein?
The cell recognizes that the deltaF508 mutant CFTR protein is mis-folded, and most of it is destroyed. Less than 1% reaches the plasma membrane.
Q: In the mutant deltaF508 CFTR protein, what levels of protein structure (primary, secondary, tertiary, quaternary) must be altered, given the information thus far?
Q: Describe the route of CFTR from its site of synthesis to its final location, the plasma membrane, naming the cellular compartments or organelles in order.
At the plasma membrane, CFTR acts as a gated chloride ion channel. Once opened, chloride ions are free to diffuse through the channel down the concentration gradient.
Q: This is an example of what type of transport?
Q: If you plotted the rate of chloride transport versus the difference in chloride concentration across the membrane, what would this plot look like?
To understand why the lack of CFTR function causes problems, let’s look at the small airways in the lung. The cells lining the small airways have cilia that constantly beat to move a thin layer of protective mucus. The mucus layer floats atop a thin layer of liquid. The cilia move the mucus along with any trapped particles and bacteria up through the trachea and to the back of the throat, where it is swallowed into the digestive tract. The cilia require the thin layer of liquid to have room to beat.
To maintain the layer of water, the airway lining pumps sodium ions into the passageway. To balance the charge of the Na+ ions, the CFTR channel opens and allows exit of Cl- ions into the airway liquid. If the cells lack CFTR, or the CFTR channel doesn’t open, the charge difference severely limits the amount of Na+ that enters the airway liquid. Without enough NaCl, the airway liquid is hypotonic.
Q: In the absence of CFTR function, which way will water flow via osmosis, into the airway or out of the airway into the surrounding tissues?
See the video clip below for the answer.
Q: What are the channels that permit water molecules to cross cell membranes?
Now see what happens in the airway as a result:
Without enough water, the cilia cannot beat, the mucus accumulates and dries, and bacteria colonize and establish long-term infections.
The problems in the pancreas are similar: mucus plugs prevent pancreatic digestive enzymes from reaching the GI tract to break down proteins and lipids. In the affected tissues, lack of CFTR chloride channel function causes a problem with osmotic balance and affects secretion.
Now that you’ve explored the biochemical and physiological basis of CF, see if you can make sense of the various treatments for management of CF symptoms.
Adrian W. R. Serohijos, Tamás Hegedus, Andrei A. Aleksandrov, Lihua He, Liying Cui, Nikolay V. Dokholyan, and John R. Riordan, 2008. Phenylalanine-508 mediates a cytoplasmic–membrane domain contact in the CFTR 3D structure crucial to assembly and channel function. PNAS 105: 3256-3261.
Rabeh, W. M., Bossard, F., Xu, H., Okiyoneda, T., Bagdany, M., Mulvihill, C. M., Du, K., di Bernardo, S., Liu, Y., Konermann, L., Roldan, A., and Lukacs, G. L. (2012). Correction of both NBD1 energetics and domain interface is required to restore ΔF508 CFTR folding and function. Cell 148, 150–163.
Cystic Fibrosis http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0001167/
Cystic Fibrosis Gene http://www.ornl.gov/sci/techresources/Human_Genome/posters/chromosome/cftr.shtml
National Jewish Health page on Cystic Fibrosis http://www.nationaljewish.org/healthinfo/conditions/cysticfibrosis/