The CFTR gene codes for an ABC transporter-class ion channel protein that conducts chloride ions across epithelialcell membranes. Mutations of the CFTR gene affecting chloride ion channel function lead to dysregulation of epithelial fluid transport in the lung, pancreas and other organs, resulting in cystic fibrosis. Complications include thickened mucus in the lungs with frequent respiratory infections, and pancreatic insufficiency giving rise to malnutrition and diabetes. These conditions lead to chronic disability and reduced life expectancy. In male patients, the progressive obstruction and destruction of the developing vas deferens (spermatic cord) and epididymis appear to result from abnormal intraluminal secretions, causing congenital absence of the vas deferens and male infertility.
Nearly 1000 cystic fibrosis-causing mutations have been described. The most common mutation, DeltaF508 (ΔF508) results from a deletion (Δ) of three nucleotides which results in a loss of the amino acid phenylalanine (F) at the 508th position on the protein. As a result, the protein does not fold normally and is more quickly degraded. The vast majority of mutations are infrequent. The distribution and frequency of mutations varies among different populations which has implications for genetic screening and counseling.
Mutations consist of replacements, duplications, deletions or shortenings in the CFTR gene. This may result in proteins that may not function, work less effectively, are more quickly degraded, or are present in inadequate numbers.
It has been hypothesized that mutations in the CFTR gene may confer a selective advantage to heterozygous individuals. Cells expressing a mutant form of the CFTR protein are resistant to invasion by the Salmonella typhi bacterium, the agent of typhoid fever, and mice carrying a single copy of mutant CFTR are resistant to diarrhea caused by cholera toxin.
DeltaF508 (ΔF508), full name CFTRΔF508 or F508del-CFTR (rs113993960), is a specific mutation within the CFTR gene involving a deletion of three nucleotides spanning positions 507 and 508 of the CFTR gene on chromosome 7, which ultimately results in the loss of a single codon for the amino acidphenylalanine (F). A person with the CFTRΔF508 mutation will produce an abnormal CFTR protein that lacks this phenylalanine residue and which cannot fold properly. This protein does not escape the endoplasmic reticulum for further processing. Having two copies of this mutation (one inherited from each parent) is by far the most common cause of cystic fibrosis (CF), responsible for nearly two-thirds of cases worldwide.
The CFTR protein is largely expressed in cells of the pancreas, intestinal and respiratory epithelia, and all exocrine glands. When properly folded, it is shuttled to the cell membrane, where it becomes a transmembrane protein responsible for opening channels which release chloride ions out of cells; it also simultaneously inhibits the uptake of sodium ions by another channel protein. Both of these functions help to maintain an ion gradient that causes osmosis to draw water out of the cells. The ΔF508 mutation leads to the misfolding of CFTR and its eventual degradation in the ER. In organisms with two complements of the mutation, the protein is entirely absent from the cell membrane, and these critical ion transport functions are not performed.
Having a homozygous pair of genes with the ΔF508 mutation prevents the CFTR protein from assuming its normal position in the cell membrane. This causes increased water retention in cells, corresponding dehydration of the extracellular space, and an associated cascade of effects on various parts of the body. These effects include: thicker mucous membranes in the epithelia of afflicted organs; obstruction of narrow respiratory airways as a result of thicker mucous and inhibition of the free movement of mucocilia; congenital absence of the vas deferens due to increased mucus thickness during fetal development; pancreatic insufficiency due to blockage of the pancreatic duct with mucus; and increased risk of respiratory infection due to build-up of thick, nutrient-rich mucus where bacteria thrive. These are the symptoms of cystic fibrosis, a genetic disorder; however, ΔF508 is not the only mutation that causes this disorder.
Being a heterozygouscarrier (having a single copy of ΔF508) results in decreased water loss during diarrhea because malfunctioning or absent CFTR proteins cannot maintain stable ion gradients across cell membranes. Typically there is a build-up of both Cl− and Na+ ions inside affected cells, creating a hypotonic solution outside the cells and causing water to diffuse into the cells by osmosis. Several studies indicate that heterozygous carriers are at increased risk for various symptoms. For example, it has been shown that heterozygosity for cystic fibrosis is associated with increased airway reactivity, and heterozygotes may be at risk for poor pulmonary function. Heterozygotes with wheeze have been shown to be at higher risk for poor pulmonary function or development and progression of chronic obstructive lung disease. One gene for cystic fibrosis is sufficient to produce mild lung abnormalities even in the absence of infection.
The CFTR gene is located on the long arm of chromosome 7, at position q31.2, and ultimately codes for a sequence of 1,480 amino acids. Normally, the three DNAbase pairs A-T-C (paired with T-A-G on the opposite strand) at the gene's 507th position form the template for the mRNA codon A-U-C for isoleucine, while the three DNA base pairs T-T-T (paired with A-A-A) at the adjacent 508th position form the template for the codon U-U-U for phenylalanine. The ΔF508 mutation is a deletion of the C-G pair from position 507 along with the first two T-A pairs from position 508, leaving the DNA sequence A-T-T (paired with T-A-A) at position 507, which is transcribed into the mRNA codon A-U-U. Since A-U-U also codes for isoleucine, position 507's amino acid does not change, and the mutation's net effect is equivalent to a deletion ("Δ") of the sequence resulting in the codon for phenylalanine at position 508.
ΔF508 is present on at least one copy of chromosome 7 in approximately one in 30 Caucasians. Presence of the mutation on both copies causes the autosomal recessive disease cystic fibrosis. Scientists have estimated that the original mutation occurred over 52,000 years ago in Northern Europe. The young allele age may be a consequence of past selection. One hypothesis as to why the otherwise detrimental mutation has been maintained by natural selection is that a single copy may present a positive effect by reducing water loss during cholera, though the introduction of pathogenic Vibrio cholerae into Europe did not occur until the late 18th century. Another theory posits that CF carriers (heterozygotes for ΔF508) are more resistant to typhoid fever, since CFTR has been shown to act as a receptor for Salmonella typhi bacteria to enter intestinal epithelial cells.
Cystic fibrosis ΔF508 heterozygotes may be overrepresented among individuals with asthma and may have poorer lung function than non-carriers. Carriers of a single CF mutation have a higher prevalence of chronic rhinosinusitis than the general population. Approximately 50% of cystic fibrosis cases in Europe are due to homozygous ΔF508 mutations (this varies widely by region), while the allele frequency of ΔF508 is about 70%. The remaining cases are caused by over 1,500 other mutations, including R117H, 1717-1G>A, and 2789+56G>A. These mutations, when combined with each other or even a single copy of ΔF508, may cause CF symptoms. The genotype is not strongly correlated with severity of the CF, though specific symptoms have been linked to certain mutations.
The CFTR gene is approximately 189 kb in length, with 27 exons and 26 introns. CFTR is a glycoprotein with 1480 amino acids. The protein consists of five domains. There are two transmembrane domains, each with six spans of alpha helices. These are each connected to a nucleotide binding domain (NBD) in the cytoplasm. The first NBD is connected to the second transmembrane domain by a regulatory "R" domain that is a unique feature of CFTR, not present in other ABC transporters. The ion channel only opens when its R-domain has been phosphorylated by PKA and ATP is bound at the NBDs. The carboxyl terminal of the protein is anchored to the cytoskeleton by a PDZ-interacting domain.Caveat: The crystal structure included at the top is not the full CFTR channel (the cartoon version is OK). The correct PDB accession number for the channel structure is 5UAK. The structure shown (PDB# 1XMI) shows a homopentameric assembly of mutated NBD1, the first nucleotide binding domain (NBD1) of the transporter.
Location and function
The CFTR protein is a channel protein that controls the flow of H2O and Cl− ions in and out of cells inside the lungs. When the CFTR protein is working correctly, as shown in Panel 1, ions freely flow in and out of the cells. However, when the CFTR protein is malfunctioning as in Panel 2, these ions cannot flow out of the cell due to blocked CFTR channels. This occurs in cystic fibrosis, characterized by the buildup of thick mucus in the lungs.
CFTRs have two transmembrane domains, each linked to a nucleotide-binding domain. CFTR also contains another domain called the regulatory domain. Other members of the ABC transporter superfamily are involved in the uptake of nutrients in prokaryotes, or in the export of a variety of substrates in eukaryotes. ABC transporters have evolved to transduce the free energy of ATP hydrolysis to the uphill movement of substrates across the cell membrane. They have two main conformations, one where the cargo binding site is facing the cytosol or inward facing (ATP free), and one where it is outward facing (ATP bound). ATP binds to each nucleotide binding domain, which results in the subsequent NBD dimerization, leading to the rearrangement of the transmembrane helices. This changes the accessibility of the cargo binding site from an inward facing position to an outward facing one. ATP binding, and the hydrolysis that follows, drives the alternative exposure of the cargo binding site, ensuring a unidirectional transport of cargo against an electrochemical gradient. In CFTR, alternating between an inward-facing conformation to an outward-facing one results in channel gating. In particular, NBD dimerization (favoured by ATP binding) is coupled to transition to an outward-facing conformation in which an open transmembrane pathway for anions is formed. Subsequent hydrolysis (at the canonical active site, site 2, including Walker motifs of NBD2) destabilizes the NBD dimer and favours return to the inward-facing conformation, in which the anion permeation pathway is closed off .
In the airways of the lung, CFTR is most highly expressed by rare specialized cells called pulmonary ionocytes. In the skin CFTR is strongly expressed in the sebaceous and eccrine sweat glands. In the eccrine glands, CFTR is located on the apical membrane of the epithelial cells that make up the duct of these sweat glands.
Normally, the protein allows movement of chloride and thiocyanateions (with a negative charge) out of an epithelial cell into the Airway Surface Liquid and mucus. Positively charged sodium ions follow passively, increasing the total electrolyte concentration in the mucus, resulting in the movement of water out of the cell via osmosis.
In epithelial cells with motile cilia lining the bronchus and the oviduct, CFTR is located on the apical cell membrane but not on cilia. In contrast, ENaC (Epithelial sodium channel) is located along the entire length of the cilia.
In sweat glands, defective CFTR results in reduced transport of sodium chloride and sodium thiocyanate in the reabsorptive duct and therefore saltier sweat. This is the basis of a clinically important sweat test for cystic fibrosis often used diagnostically with genetic screening.
Cystic fibrosis transmembrane conductance regulator has been shown to interact with:
Cystic fibrosis: More than 1,800 mutations in the CFTR gene have been found but the majority of these have not been associated with cystic fibrosis. Most of these mutations either substitute one amino acid (a building block of proteins) for another amino acid in the CFTR protein or delete a small amount of DNA in the CFTR gene. The most common mutation, called ΔF508, is a deletion (Δ) of one amino acid (phenylalanine) at position 508 in the CFTR protein. This altered protein never reaches the cell membrane because it is degraded shortly after it is made. All disease-causing mutations in the CFTR gene prevent the channel from functioning properly, leading to a blockage of the movement of salt and water into and out of cells. As a result of this blockage, cells that line the passageways of the lungs, pancreas, and other organs produce abnormally thick, sticky mucus. This mucus obstructs the airways and glands, causing the characteristic signs and symptoms of cystic fibrosis. In addition, only thin mucus can be removed by cilia; thick mucus cannot, so it traps bacteria that give rise to chronic infections.
Cholera: ADP-ribosylation caused by cholera toxin results in increased production of cyclic AMP which in turn opens the CFTR channel which leads to oversecretion of Cl−. Na+ and H2O follow Cl− into the small intestine, resulting in dehydration and loss of electrolytes.
CFTR has been a drug target in efforts to find treatments for related conditions. Ivacaftor (trade name Kalydeco, developed as VX-770) is a drug approved by the FDA in 2012 for people with cystic fibrosis who have specific CFTR mutations Ivacaftor was developed by Vertex Pharmaceuticals in conjunction with the Cystic Fibrosis Foundation and is the first drug that treats the underlying cause rather than the symptoms of the disease. Called "the most important new drug of 2012", and "a wonder drug" it is one of the most expensive drugs, costing over US$300,000 per year, which has led to criticism of Vertex for the high cost.
^Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou JL (September 1989). "Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA". Science. 245 (4922): 1066–73. Bibcode:1989Sci...245.1066R. doi:10.1126/science.2475911. PMID2475911.
^Marcorelles P, Gillet D, Friocourt G, Ledé F, Samaison L, Huguen G, Ferec C (March 2012). "Cystic fibrosis transmembrane conductance regulator protein expression in the male excretory duct system during development". Human Pathology. 43 (3): 390–7. doi:10.1016/j.humpath.2011.04.031. PMID21840567.
^Araújo FG, Novaes FC, Santos NP, Martins VC, Souza SM, Santos SE, Ribeiro-dos-Santos AK (January 2005). "Prevalence of deltaF508, G551D, G542X, and R553X mutations among cystic fibrosis patients in the North of Brazil". Brazilian Journal of Medical and Biological Research = Revista Brasileira de Pesquisas Medicas e Biologicas. 38 (1): 11–5. doi:10.1590/S0100-879X2005000100003. PMID15665983.
^Bobadilla, JL; Macek Jr, M; Fine, JP; Farrell, PM (2002). "Cystic fibrosis: a worldwide analysis of CFTR mutations--correlation with incidence data and application to screening". Human Mutation. 19 (6): 575–606. doi:10.1002/humu.10041. PMID12007216.
^Verkman, A.S.; Song, Y.; Thiagarajah, J.R. (2003). "Role of airway surface liquid and submucosal glands in cystic fibrosis lung disease". American Journal of Physiology. Cell Physiology. 284 (1): C2–C15. doi:10.1152/ajpcell.00417.2002. PMID12475759.
^Pier, G.B.; Grout, M.; Zaidi, T.; Meluleni, G.; Mueschenborn, S.S.; Banting, G.; Ratcliff, R.; Evans, M.J.; Colledge, W.H. (7 May 1998). "Salmonella typhi uses CFTR to enter intestinal epithelial cells". Nature. 393 (6680): 79–82. Bibcode:1998Natur.393...79P. doi:10.1038/30006. PMID9590693.
^Dahl, Morten; Nordestgaard, Børge G.; Lange, Peter; Tybjaerg-Hansen, Anne (January 8, 2001). "Fifteen-year follow-up of pulmonary function in individuals heterozygous for the cystic fibrosis phenylalanine-508 deletion". Allergy Clin Immunol. 107 (5): 818–823. doi:10.1067/mai.2001.114117. PMID11344348.
^Dahl, M; Tybjaerg-Hansen, A; Lange, P; Nordestgaard, BG (June 27, 1998). "DeltaF508 heterozygosity in cystic fibrosis and susceptibility to asthma". Lancet. 351 (9120): 1911–3. doi:10.1016/s0140-6736(97)11419-2. PMID9654257.
^Wang, XinJing; Kim, Jean; McWilliams, Rita; Cutting, Garry R. (March 2005). "Increased prevalence of chronic rhinosinusitis in carriers of a cystic fibrosis mutation". Arch Otolaryngol Head Neck Surg. 131 (3): 237–40. doi:10.1001/archotol.131.3.237. PMID15781764.
^Morral, N; Bertranpetit, J; Estivill, X; Nunes, R; Casals, T; Gimenez, J; Angelicheva, D (1994). "The origin of the major cystic fibrosis mutation (ΔF508) in European populations". Nature Genetics. 7 (2): 169–75. doi:10.1038/ng0694-169. PMID7920636.
^ abcEnuka Y, Hanukoglu I, Edelheit O, Vaknine H, Hanukoglu A (March 2012). "Epithelial sodium channels (ENaC) are uniformly distributed on motile cilia in the oviduct and the respiratory airways". Histochemistry and Cell Biology. 137 (3): 339–53. doi:10.1007/s00418-011-0904-1. PMID22207244.
^Sharma S, Hanukoglu A, Hanukoglu I (2018). "Localization of epithelial sodium channel (ENaC) and CFTR in the germinal epithelium of the testis, Sertoli cells, and spermatozoa". Journal of Molecular Histology. 49 (2): 195–208. doi:10.1007/s10735-018-9759-2. PMID29453757.CS1 maint: multiple names: authors list (link)
^Sharma S, Hanukoglu I (2019). "Mapping the sites of localization of epithelial sodium channel (ENaC) and CFTR in segments of the mammalian epididymis". Journal of Molecular Histology. 50 (2): 141–154. doi:10.1007/s10735-019-09813-3. PMID30659401.
^Yonei Y, Tanaka M, Ozawa Y, Miyazaki K, Tsukada N, Inada S, Inagaki Y, Miyamoto K, Suzuki O, Okawa H (April 1992). "Primary hepatocellular carcinoma with severe hypoglycemia: involvement of insulin-like growth factors". Liver. 12 (2): 90–3. doi:10.1111/j.1600-0676.1992.tb00563.x. PMID1320177.
^Zhang H, Peters KW, Sun F, Marino CR, Lang J, Burgoyne RD, Frizzell RA (August 2002). "Cysteine string protein interacts with and modulates the maturation of the cystic fibrosis transmembrane conductance regulator". The Journal of Biological Chemistry. 277 (32): 28948–58. doi:10.1074/jbc.M111706200. PMID12039948.
^Cheng J, Moyer BD, Milewski M, Loffing J, Ikeda M, Mickle JE, Cutting GR, Li M, Stanton BA, Guggino WB (February 2002). "A Golgi-associated PDZ domain protein modulates cystic fibrosis transmembrane regulator plasma membrane expression". The Journal of Biological Chemistry. 277 (5): 3520–9. doi:10.1074/jbc.M110177200. PMID11707463.
^ abcGentzsch M, Cui L, Mengos A, Chang XB, Chen JH, Riordan JR (February 2003). "The PDZ-binding chloride channel ClC-3B localizes to the Golgi and associates with cystic fibrosis transmembrane conductance regulator-interacting PDZ proteins". The Journal of Biological Chemistry. 278 (8): 6440–9. doi:10.1074/jbc.M211050200. PMID12471024.
^Wang S, Yue H, Derin RB, Guggino WB, Li M (September 2000). "Accessory protein facilitated CFTR-CFTR interaction, a molecular mechanism to potentiate the chloride channel activity". Cell. 103 (1): 169–79. doi:10.1016/S0092-8674(00)00096-9. PMID11051556.
^Liedtke CM, Yun CH, Kyle N, Wang D (June 2002). "Protein kinase C epsilon-dependent regulation of cystic fibrosis transmembrane regulator involves binding to a receptor for activated C kinase (RACK1) and RACK1 binding to Na+/H+ exchange regulatory factor". The Journal of Biological Chemistry. 277 (25): 22925–33. doi:10.1074/jbc.M201917200. PMID11956211.
^ abPark M, Ko SB, Choi JY, Muallem G, Thomas PJ, Pushkin A, Lee MS, Kim JY, Lee MG, Muallem S, Kurtz I (December 2002). "The cystic fibrosis transmembrane conductance regulator interacts with and regulates the activity of the HCO3- salvage transporter human Na+-HCO3- cotransport isoform 3". The Journal of Biological Chemistry. 277 (52): 50503–9. doi:10.1074/jbc.M201862200. PMID12403779.
^Hegedüs T, Sessler T, Scott R, Thelin W, Bakos E, Váradi A, Szabó K, Homolya L, Milgram SL, Sarkadi B (March 2003). "C-terminal phosphorylation of MRP2 modulates its interaction with PDZ proteins". Biochemical and Biophysical Research Communications. 302 (3): 454–61. doi:10.1016/S0006-291X(03)00196-7. PMID12615054.
^Wang S, Raab RW, Schatz PJ, Guggino WB, Li M (May 1998). "Peptide binding consensus of the NHE-RF-PDZ1 domain matches the C-terminal sequence of cystic fibrosis transmembrane conductance regulator (CFTR)". FEBS Letters. 427 (1): 103–8. doi:10.1016/S0014-5793(98)00402-5. PMID9613608.
^Moyer BD, Duhaime M, Shaw C, Denton J, Reynolds D, Karlson KH, Pfeiffer J, Wang S, Mickle JE, Milewski M, Cutting GR, Guggino WB, Li M, Stanton BA (September 2000). "The PDZ-interacting domain of cystic fibrosis transmembrane conductance regulator is required for functional expression in the apical plasma membrane". The Journal of Biological Chemistry. 275 (35): 27069–74. doi:10.1074/jbc.M004951200. PMID10852925.
^Sun F, Hug MJ, Lewarchik CM, Yun CH, Bradbury NA, Frizzell RA (September 2000). "E3KARP mediates the association of ezrin and protein kinase A with the cystic fibrosis transmembrane conductance regulator in airway cells". The Journal of Biological Chemistry. 275 (38): 29539–46. doi:10.1074/jbc.M004961200. PMID10893422.
Kulczycki LL, Kostuch M, Bellanti JA (January 2003). "A clinical perspective of cystic fibrosis and new genetic findings: relationship of CFTR mutations to genotype-phenotype manifestations". American Journal of Medical Genetics. Part A. 116A (3): 262–7. doi:10.1002/ajmg.a.10886. PMID12503104.
Vankeerberghen A, Cuppens H, Cassiman JJ (March 2002). "The cystic fibrosis transmembrane conductance regulator: an intriguing protein with pleiotropic functions". Journal of Cystic Fibrosis. 1 (1): 13–29. doi:10.1016/S1569-1993(01)00003-0. PMID15463806.
Tsui LC (1992). "Mutations and sequence variations detected in the cystic fibrosis transmembrane conductance regulator (CFTR) gene: a report from the Cystic Fibrosis Genetic Analysis Consortium". Human Mutation. 1 (3): 197–203. doi:10.1002/humu.1380010304. PMID1284534.
McIntosh I, Cutting GR (July 1992). "Cystic fibrosis transmembrane conductance regulator and the etiology and pathogenesis of cystic fibrosis". FASEB Journal. 6 (10): 2775–82. doi:10.1096/fasebj.6.10.1378801. PMID1378801.
Nagel G (December 1999). "Differential function of the two nucleotide binding domains on cystic fibrosis transmembrane conductance regulator". Biochimica et Biophysica Acta. 1461 (2): 263–74. doi:10.1016/S0005-2736(99)00162-5. PMID10581360.
Greger R, Schreiber R, Mall M, Wissner A, Hopf A, Briel M, Bleich M, Warth R, Kunzelmann K (2001). "Cystic fibrosis and CFTR". Pflügers Archiv. 443 Suppl 1: S3–7. doi:10.1007/s004240100635. PMID11845294.
Dahan D, Evagelidis A, Hanrahan JW, Hinkson DA, Jia Y, Luo J, Zhu T (2001). "Regulation of the CFTR channel by phosphorylation". Pflügers Archiv. 443 Suppl 1: S92–6. doi:10.1007/s004240100652. PMID11845311.
Cohn JA, Noone PG, Jowell PS (September 2002). "Idiopathic pancreatitis related to CFTR: complex inheritance and identification of a modifier gene". Journal of Investigative Medicine. 50 (5): 247S–255S. doi:10.1136/jim-50-suppl5-01. PMID12227654.
Schwartz M (February 2003). "[Cystic fibrosis transmembrane conductance regulator (CFTR) gene: mutations and clinical phenotypes]". Ugeskrift for Laeger. 165 (9): 912–6. PMID12661515.
Wong LJ, Alper OM, Wang BT, Lee MH, Lo SY (July 2003). "Two novel null mutations in a Taiwanese cystic fibrosis patient and a survey of East Asian CFTR mutations". American Journal of Medical Genetics. Part A. 120A (2): 296–8. doi:10.1002/ajmg.a.20039. PMID12833420.