Cystic Fibrosis

• Cystic Fibrosis


  Genetically inherited disease
• Cystic Fibrosis


  • The defect is in the gene coding for a chloride ion channel, the cystic fibrosis transmembrane conductance regulator (CFTR), which is expressed in all epithelial cells
  • The gene is on chromosome 7
  • More than 2000 different mutations in the CFTR gene
  • It is inherited as an autosomal recessive disease
  • Multi organ disease affecting all organs lined within epithelial cells
• Carriers


  Have a single copy of the defective gene (g) and it is masked by the dominant normal gene (G) (heterozygous)
• Homozygous


  2 copies of the defective gene must be inherited for the child to have CF
• Processing of the Normal CFTR

  1. The CF gene is transcribed in the nucleus and the normal protein is synthesised on the ER and transported to the cell membrane in the golgi vesicles

  2. During the transport process the protein undergoes post-translational modification

  3. Protein becomes glycosylated by the addition of sugar residues

  4. The sugar residues are essential for normal trafficking of CFTR to the cell membrane

  5. The protein finally becomes an integral part of the cell surface membrane where its activity as a chloride channel is further regulated by 2 distinct mechanisms
• Regulation of CFTR Chloride Channel Activity

  • CFTR consists of 2 transmembrane domains (TM1 & TM2). 2 nucleotide binding domains (NBD1 & NBD2) and a unique intracellular hydrophilic regulatory (R) domain
  • Each membrane spanning domain has six membrane spanning segments that make up the pore through which chloride ions move and determine the ion selectivity of the channel
  • Activation of CFTR by phosphorylation is catalysed by the cyclic AMP dependant protein kinase, protein kinase A
  • The R domain is phosphorylated at 4 sites by protein kinase A which causes a conformational change in the protein and the channel to open
  • Binding of ATP on NBD1 causes NBD1 and NBD2 to dimerise and activate chloride conductance
  • ATP hydrolysis at NBD2 closes the channel and inhibits chloride conductance
  • Dephosphorylation of the R domain ensures the channel remains closed
  • The extracellular loops in TM2 contain the sites for glycosylation of the protein
• Activation of CFTR Chloride Channel Activity by 2-adrenergic Receptors

  1. Agents that increase intracellular cAMP in airway epithelial cells include the β2-agonists that act on β2-agonists

  2. Activation of β2-agonists receptors stimulates the stimulatory G-protein that activates adenyl cyclase, which is the enzyme responsible for the formation of cAMP

  3. cAMP is highly compartmentalised and the local increase in cAMP activation leads to an increase in protein kinase A activity and phosphorylation and activation of CFTR

  4. the reason behind this compartmentalised response is that CFTR and the β2-adrenergic receptors co-localise in the cell membrane of airway epithelial and both CFTR and β2-adrenergic receptors bind to the ezrin binding protein 50

  5. These proteins form a tri-molecular complex (CFTR-EBP50- β2 adrenergic receptor)

  6. Ezrin is a cytoskeletal protein that also binds protein kinase A ensuring that protein kinase A is in close proximity to CFTR

  7. Following channel activated the complex falls apart and the signal is turned off

  8. These protein-protein interactions (macromolecular complex assembly) are essential for full activation of the channel by the β2-agonist receptor pathway

  9. These interactions may be critical for a rapid and specific signal transduction from the receptor to the channel in a compartmentalised fashion

  10. It also indicates that defective forms of CFTR may lead to abnormal CFTR function in response to receptor-based signalling
• Syntaxin 1A Inhibits CFTR Activity

  • CFTR is negatively regulated by another membrane protein, syntaxin 1A, which binds at the amino (N) terminus of CFTR and negatively regulates channel opening time and also CFTR trafficking to the cell membrane
  • Therefore protein networks are important for both positive (β2-AR) and negative (syntaxin 1A) regulation of CFTR
  • Protein-protein interactions are also important in the way that CFTR regulates other ion channels such as the epithelial sodium channel
• Cytoplasmic C-Terminal Tail of CFTR Interacts with Regulatory Networks


  • The C terminus of CFTR interacts with protein that link CFTR to a complex network of regulatory proteins, through the protein binding domains (PDZ) of EBP50 to the epithelial sodium channel (ENaC)
  • In this case, CFTR normally assembled as a complex with ezrin, protein kinase A (PK-A) and the YAP and cYES proteins
  • As before the function is to tether PK--A near to CFTR and to link CFTR to the cytoskeleton and localise it to the plasma membrane
  • EBP50 interacts with YAP and this recruits cYES to the complex, which acts to inhibit ENaC activity. Since CFTR is defective in CF we can speculate that this complex is disrupted in CF, so there is both decreased chloride channel activity and increased sodium activity in CF
• Processing of the F508 CFTR

  • Delta F 508 is derived from D for deletion, F is from the one letter code for amino acids and stands for phenyl-alanine and 508 is in the position in the amino sequence of the CFTR protein
  • The deletion of 3 base pairs in the CF gene, coding for a single amino acid in the final product, is responsible for the CF phenotype
  • The missing phenylalanine is critically responsible for post-translational defects in the CFTR protein. Immature forms of the protein are synthesised, but they do not become glycosylated, do not traffick through the golgi system and are not inserted into the cell membrane
  • Because of the missing amino acid in the tertiary structure the protein does not fold properly and it is taken up quickly destroyed in the proteosomes
  • The NET defect is that the CFTR is not delivered to the cell membrane and this chloride channel is missing leading to a wide range on physiological defects in epithelial cells
• Classes of CFTR Mutation in Patients with CF

  • Class 1: Premature termination of the translation of the CFTR mRNA caused by base substitutions that create stop codons or insertions that shift the reading frame. No protein synthesis.
  • Class 2: Trafficking defects, mis-folded CFTR protein is degraded in proteosomes and fails to reach the plasma membrane, (DF508)
  • Class 3: Regulatory mutants which reach the surface of the cell, but do not respond normally to activation signals (G551D).
  • Class 4: Mutant proteins reach the plasma membrane, but have altered channel properties and chloride conductance defects
  • Class 5: Unstable mRNA, reduced synthesis of functional CFTR
  • Class 6: Reduced stability of CFTR protein in the plasma membrane
• The Genetic Defect is in the CFTR Protein that acts as Chloride Channel in Cell Membranes


  Chloride ions move through a pore that opens when CFTR is activated by ATP and by phosphorylation of a regulatory domain
• Organs Affected by Cystic Fibrosis

  • CFTR is expressed by all epithelia (sheets of cells that form a barrier between different compartments of the body and line the intestines, airways and many ducts)
  • The epithelia of patients with CF are relatively impermeable to chloride and CF is therefore a multi-organ disease
  • In the sweat glands, sweat is produced by cells at the base of the sweat gland and sodium and chloride ions are normally reabsorbed by epithelial cells lining the duct, so that only water reaches the surface to cool the skin
  • In CF, the epithelial cells lining the duct fail to absorb chloride ions from the secreted sweat, and as a result sodium is also poorly absorbed
  • As a result of a defect in chloride absorbance, people with CF have unusually salty sweat
  • In the pancreas; defective chloride secretion results in defective transport of HCO3- and water into the lumen of the pancreatic ducts, and the ducts become obstructed
  • The digestive enzymes, amylase, lipase, and trypsinogen, are not delivered into the gut, resulting in a nutritional insufficiency
  • Patients require supplementation of their diet with the missing enzymes, given in capsule form before meals
  • Because lipids are not digested and absorbed patients are deficient in the uptake of the fat soluble vitamins, vitamin A, D and E, which are important anti-oxidants
  • In the lungs; Defective CFTR results in defective chloride secretion, and increased sodium and water absorption
  • Clogging and infection of the airways impedes breathing. Infection leads to inflammation and lung damage
• Diagnosis of Cystic Fibrosis

  • Excessively salty sweat is the result of defective chloride absorption in the sweat glands. A sweat test is the mainstay of diagnosis
  • Failure to thrive and gain weight (malnourishment) occurs as the defect leads to thick mucus that blocks the pancreatic duct and prevents digestive enzymes from being delivered to the gut. Thus, despite apparently good nutrition, babies with CF fail to thrive and gain weight
  • Finally, in the first few years of life, babies with CF are repeatedly admitted to hospital with chest infections. Persistent chest infection is due to defective chloride secretion and increased sodium and water absorption in the airways, leading to thick dessicated secretions that invite infection and inflammation. In 95% of cases lung disease and respiratory failure is the cause of death
  • Defective CFTR makes epithelia relatively impermeable to chloride. But chloride ions can move through CFTR in either direction, and in different organs the defect can result in defective absorption (as in sweat) or defective secretion (as in the lungs and pancreas)