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Cystic fibrosis is an inherited, chronic disease which currently leads to a reduced life expectancy. CF principally affects the respiratory and digestive systems in children and young adults. The sweat glands and the reproductive system are also usually involved. Sweat is needed to cool the body; mucus is needed to lubricate the respiratory, digestive, and reproductive systems and preventing them from drying out and from becoming infected. Cystic Fibrosis causes the body to produce thick mucus that can block small ducts and tubes throughout the body, especially in the lungs and pancreas. The abnormally thick mucus causes a variety of complications including airway obstruction, bacterial infections, lung damage, digestive difficulties and a variety of other problems.
CF is caused by mutations in a large gene that encodes a large protein called the cystic fibrosis transmembrane conductance regulator (CFTR). The CFTR protein is found in cell membranes, its role to transport chloride ions out of the cell. The abnormality in the gene affects the way in which sodium and water move in and out of the body's cells. The water and sodium help the body to produce thin, slippery mucus. Mucus is a slippery substance that lubricates the linings of the airways, reproductive system, digestive system and some organs and bodily tissues.
When the CFTR gene is mutated, it either produces a CFTR protein that does not work or as in a large number of cases, there is no CFTR protein produced at all. When there is no CFTR protein present this is because part of the DNA code in the CFTR gene is missing, making the CFTR protein shorter than normal. The cell's quality control system destroys the CFTR protein, as it is too short.
Function of CFTR Protein: Ion Transport
The cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of the ATP-binding cassette transporter that plays a critical role in fluid and electrolyte transport across epithelial tissues. CFTR is composed of two membrane-spanning domains, nucleotide-binding domains linked by a regulatory domain. The membrane-spanning domains assemble to form a transmembrane pore with deep intracellular and shallow extracellular passages that guide anions towards a filter, which determines the infiltration properties of CFTR. Anion flow through the CFTR pore is powered by cycles of ATP binding and hydrolysis at two ATP-binding sites. Stable ATP binding occurs at one ATP-binding site, whereas rapid ATP yield occurs at the other. These ATP-binding sites are located at the border of the two nucleotide-binding domains. The R domain contains multiple phosphorylation sites on the surface of an unstructured domain. Phosphorylation of the R domain stimulates CFTR function by enhancing ATP-dependent channel gating at the nucleotide-binding domains. Thus, CFTR is an anion channel.
CFTR is principally expressed in the apical membrane of epithelia where it provides
a pathway for Cl and HCO3 movement and controls the rate of fluid flow through its role as an anion channel and regulating the function of ion channels and transporters in epithelial cells Thus, CFTR plays a fundamental role in transepithelial fluid and electrolyte transport.
In sweat duct epithelia, CFTR drives the reabsorption of salt, while in intestinal, pancreatic and respiratory airway epithelia CFTR powers the secretion of Cl_ and HCO3_. The importance of CFTR is dramatically highlighted by the consequences of CFTR malfunction in CF
and related disorders.
The cystic fibrosis transmembrane conductance regulator (CFTR)1 forms a Cl− channel that is an essential component of epithelial Cl− transport systems in many organs, including the intestines, pancreas, lungs, sweat glands, and kidneys. In the Cl− secretory intestinal epithelium, Cl−enters the cells through a Na+-K+-2Cl− co transporter in the basolateral membrane and exits through CFTR in the apical membrane; water follows osmotically. Absorptive epithelia use similar transporters and channels, but their polarised distribution between the apical and basolateral membranes is usually reversed. A major determinant of the transepithelial Cl− transport rate is the level of activation of CFTR which depends on the extent to which it is phosphorylated. This is determined by the relative activities of kinases and phosphatases, the activities of which are often hormonally regulated.
Structural Basis of Channel Function
Transmembrane topology of CFTR.Gray balloons on the M7-M8 loop indicateN-linked glycosylation sites. R, R-domain.
The sweat gland represents the consequences of the CF defect uncomplicated by obstruction or infection. Sweat secretion is achieved by two parallel systems. The physiologically relevant system that mediates thermal sweating operates via a cholinergically-stimulated increase in Ca2+. Small amounts of sweating can also be stimulated by elevating cAMP.
In any event, the cAMP-mediated secretory response is entirely absent in CF individuals and is reduced to half normal values in heterozygotes, indicating that CFTR Cl- channels are rate-limiting for cAMP-mediated sweat secretion.
Reduced salt reabsorption from sweat is apparent to anyone with CF. Normally, as primary sweat moves along the reabsorptive duct, most of the salt is reabsorbed. Reabsorption is driven by the large electrochemical gradient for Na+ which flows into the cell through Na+ channels in the apical membrane. The basolateral Na+, K+-ATPase then transports Na+ out of the cell and into the blood. The Na+ movement instantaneously creates a negative voltage in the lumen, which provides an electrochemical gradient that forces Cl- out of the lumen and into the ductal cells via apically located CFTR Cl- channels. The apical membrane of sweat ducts contains perhaps the most dense concentration of CFTR Cl- channels known in normal tissues.
In CF sweat ducts, the Cl- conductance is virtually abolished, and the duct behaves as though it were permeable only to Na+. Thus when Na+ attempts to flow out of a CF duct unaccompanied by Cl- , it creates a large excess of negative charge in the duct which sets up an opposing electrical gradient for Na+ and so greatly retards its movement. The net result is that both Na+ and Cl- are poorly reabsorbed by the CF duct, leading to the high salt content of CF sweat.
The thin duct that conveys sperm from the testes is also a Cl--based fluid secreting organ. It is perhaps more vulnerable than any other organ to the destructive effects of CF. The loss of the vas deferens in CF males is caused by degeneration secondary to obstruction. Normal genital tract structures are present in many young CF males, but that after a certain age the vas is absent, with variable sparing of the epididymus.
The pancreatic acinar cells secretes digestive enzymes and some fluid that then mixes with a bicarbonate-rich fluid secreted by the duct cells, which elevates cAMP in the usual way. The pancreatic juice flows along the pancreatic duct into the duodenum, where it participates in digestion, especially of fats. CFTR is the only significant Cl- channel in the apical membrane of ductal cells, and it functions there in conjunction with an anion exchanger to effect bicarbonate secretion. When defective there is a reduction in fluid secretion, which eventually leads to blockage of smaller ducts because of enzyme precipitation or mucus accumulation. The pancreas is unusual in that the acini entirely lack myoepithelial cells as supporting structures, and hence are extremely sensitive to damage by even minor increases in intraductal pressure. Once damage and inflammation start, a rapid loss of pancreatic function ensues.
Of the hundreds of CF mutations that have been identified, the great majority cause pancreatic insufficiency. These include all mutations that cause biosynthetic arrest of CFTR, or that are missense or nonsense mutations that result in no mature protein, and also includes normally processed proteins that are incapable of opening. However, a small set of CFTR mutations that allow some residual CFTR channel conductance lead to milder forms of CF in which pancreatic function remains sufficient for digestion. It is now widely appreciated that slight residual function can have dramatic consequences at the organ system level.
CFTR expression in the intestine is most dense in the cells that migrate across the villi border. Because the cells are the site of fluid secretion by the intestine, loss of CFTR function would be predicted to lead to under hydration of the intestines. Tissues from CF individuals were shown to lack Cl- secretion, regardless of whether they activated cAMP or Ca2+. Intestinal cells lack a Ca2+-activated Cl- channel.
The reduction of fluid secretion and salt absorption across epithelia, caused by loss of CFTR can cause CF disease. However, persistent lung infection is the most life-threatening symptom of CF. Loss of CFTR leads to increased Na+ reabsorption, probably because of increased activity of sodium channels. CFTR also influences another Cl- channel.
CFTR Cl- channels are strategically located in the lung to play a key role in fighting infections and in keeping the airway ducts free of mucus accumulation. This idea is given credence by the finding of a highly heterogeneous distribution of CFTR within the airway mucosa, with the most dense staining occurring in serous cells of the submucosal glands.
Submucosal glands are complex glands that have, via their many tubules, a greatly expanded surface area. In humans they provide most of the mucin secretion in the upper airways. Most of the gland cell volume is made up of serous cells, which form the secretory endpieces of the glands, while mucus cells line the tubules. CFTR is not found in the mucous cells. Serous cells are the primary source of fluid secretion in the glands and are essential to the formation of properly hydrated mucus. In addition, serous cells secrete a host of antibiotic compounds and tissue-protecting protease inhibitors.
It seems apparent that if the antibiotic rich fluid secretion of airway sub mucosal glands were lost, the lungs should be more vulnerable to infections. Nevertheless, a role for gland malfunction in CF was ignored until recently because the glands normally secrete to agents they raise cellular Ca2+ levels, and, as we have seen, Ca2-mediated Cl- secretion is typically spared in CF. However, intestinal crypt cells are an exception: they normally secrete to both cAMP and Ca2+, and as we saw both forms of secretion are lost from CF subjects. The same finding applies to submucosal gland serous cells.
The mechanism for this has been explored in a human cell line, called Calu-3, that shares a great many features with native serous cells. In Calu-3 cells, CFTR channels in the apical membrane are constitutively active and sustain a low level of constitutive Cl- secretion. Elevation of cystosolic Ca2+ causes a large increase in Cl- secretion by opening basolateral K+ channels to increase the driving force for Cl- exit. Thus, airway serous cells are another example of an epithelium in which Ca2+ -mediated fluid secretion should be abolished by CFTR mutations, and that has been observed.
Figure 1. Hypothesized Structure of CFTR.
The protein contains 1480 amino acids and a number of discrete globular and transmembrane domains. Activation of CFTR relies on phosphorylation, particularly through protein kinase A but probably involving other kinases as well. Channel activity is governed by the two nucleotide-binding domains, which regulate channel gating. The carboxyl terminal (consisting of threonine, arginine, and leucine [TRL]) of CFTR is anchored through a PDZ-type-binding interaction with the cytoskeleton and is kept in close approximation (dashed arrows) to a number of important proteins. These associated proteins influence CFTR functions, including conductance, regulation of other channels, signal transduction, and localization at the apical plasma membrane. Each membrane-spanning domain contains six membrane-spanning alpha helixes, portions of which form a chloride-conductance pore. The regulatory domain is a site of protein kinase A phosphorylation. The common ΔF508 mutation occurs on the surface of nucleotide-binding domain 1.
Figure 2. Extrusion of Mucus Secretion onto the Epithelial Surface of Airways in Cystic Fibrosis.
Panel A shows a schematic of the surface epithelium and supporting glandular structure of the human airway. In Panel B, the submucosal glands of a patient with cystic fibrosis are filled with mucus, and mucopurulent debris overlies the airway surfaces, essentially burying the epithelium. Panel C is a higher-magnification view of a mucus plug tightly adhering to the airway surface, with arrows indicating the interface between infected and inflamed secretions and the underlying epithelium to which the secretions adhere. (Both Panels B and C were stained with hematoxylin and eosin, with the colors modified to highlight structures.) Infected secretions obstruct airways and, over time, dramatically disrupt the normal architecture of the lung. In Panel D, CFTR is expressed in surface epithelium and serous cells at the base of submucosal glands in a porcine lung sample, as shown by the dark staining, signifying binding by CFTR antibodies to epithelial structures (aminoethylcarbazole detection of horseradish peroxidase with hematoxylin counterstain).
Figure 3. Mechanism Underlying Elevated Sodium Chloride Levels in the Sweat of Patients with Cystic Fibrosis.
Sweat ducts (Panel A) in patients with cystic fibrosis differ from those in people without the disease in the ability to reabsorb chloride before the emergence of sweat on the surface of the skin. A major pathway for Cl absorption is through CFTR, situated within luminal plasma membranes of cells lining the duct (i.e., on the apical, or mucosal, cell surface) (Panel B). Diminished chloride reabsorption in the setting of continued sodium uptake leads to an elevated transepithelial potential difference across the wall of the sweat duct, and the lumen becomes more negatively charged because of a failure to reabsorb chloride (Panel C). The result is that total sodium chloride flux is markedly decreased, leading to increased salt content. The thickness of the arrows corresponds to the degree of movement of ions.
Figure 4. Models Explaining the Transepithelial Potential Difference across the Airway Epithelium in Cystic Fibrosis.
Under normal conditions, sodium chloride is absorbed from the airways (Panel A). The first step of this process uses sodium and chloride absorptive pathways present in the luminal (apical) membranes of airway-surface epithelial cells, designated as the mucosal surface (Panel B). In a bioelectric assay (a measurement of the transepithelial potential difference), the lumen is negative in part because of the relative impermeability of chloride as compared with sodium. The relative contribution of CFTR and other non-CFTR Cl permeability pathways is not known. The transepithelial potential difference is markedly hyperpolarized (i.e., the lumen is much more negatively charged) in cystic fibrosis. Two models have been proposed to explain this difference. In the high-salt model (Panel C), the situation resembles that of the sweat duct, in which the absence of CFTR leads to the inability to reabsorb chloride ion from airway-surface liquid. Because of the continued activity of sodium ion reabsorption, which is dependent on epithelial sodium channels, the airway surface negativity is increased (lumen-negative). According to this model, although a large charge separation is observed (with positively charged sodium ions moving across the airway wall and negatively charged chloride ions remaining behind), the net sodium chloride reabsorption decreases because of the inability to reabsorb chloride counter-ions. In the low-volume model (Panel D), both sodium and chloride are hyperabsorbed. The airways of patients with cystic fibrosis are slightly less permeable to chloride ions than they are to sodium ions, a process that leads to an increased transepithelial potential difference. This model predicts a depletion in the volume of airway-surface liquid (shown in blue). The thickness of the arrows corresponds to the degree of movement of ions.
Figure 5. Categories of CFTR Mutations.
Classes of defects in the CFTR gene include the absence of synthesis (class I); defective protein maturation and premature degradation (class II); disordered regulation, such as diminished ATP binding and hydrolysis (class III); defective chloride conductance or channel gating (class IV); a reduced number of CFTR transcripts due to a promoter or splicing abnormality (class V); and accelerated turnover from the cell surface (class VI)