Pharmacological chaperone Proteostasis modulator -- modulate proteins that control folding of CFTR in ER (lumacaftor)

1. Describe the clinical phenotype (ducts and tubes are blocked by sticky mucus) 2. Investigate biochemical abnormality (defective chloride transport across epithelia) 3. Identify defective gene (CFTR chloride channel identification and cloning) 4. Characterise normal function of gene product (Epithelia chloride channel with abnormal regulation) 5. Understand how disruption interrupts normal function (defective ion transport caused by protein biosynthesis and channel synthesis defects) 6. Drug therapies based on rational understanding of disease

Chest massage to dislodge mucus and antibiotic IV to fight lung infections

- meconium ileus at birth - distal intestinal obstructive syndrome (DIOS) as an adult - loss of exocrine pancreas function - congenital bilateral absence of vas deferens - Air passage mucus accumulation

Infection by Pseudomonas aeruginosa. The free swimming form is susceptible to antibiotics, but once a colony forms, it it protected by a biofilm. Once the infection becomes mucoid it is very hard to eradicate. Many patients are treated with prophylactic antibiotics.

Polar cell, has apical face and basolateral face -- separated by tight junctions Na/K-ATPase needed to create a gradient for NKCC1 function - pump K+ in, Na+ out - K+ passive movement out via ion channel NKCC1 actively takes up Cl- from basolateral membrane -- Na+/K+/2Cl- all taken in Cl- passive exit through apical membrane Na+ movement across tight junction into lumen Water movement follows

ABC transporter family (ATP binding cassette) CFTR is the only ABC to form the ion channel, not just regulate one

- phosphorylation of the R domain stimulates channel gating by facilitating the interaction between ATP and NBDs - phosphorylation by PKA is required for channel opening (cAMP mediated) - ATP binding and hydrolysis gates channel opening and closing - the higher the concentration of ATP, the less time there is between channel openings - NBD1 is predicted to tightly bind ATP, whereas NBD2 hydrolyses ATP - the binding of ATP opens the channel, hydrolysis closes channel - this acts as a timing mechanism for closing

Walker A: lysine interacts with either alpha or gamma phosphate of ATP and is essential for hydrolysis Walker B: aspartate coordinates Mg+ in MgATP and is required for ATP binding LSGGQ: conserved glutamine may control the rate of ATP hydrolsis

the surface of NBD1 -- the key site of NBD and MSD communication

Pancreatic insufficient or sufficient. Several studies suggest a relationship between genotype and phenotype. ie PI phenotype in patients with two severe mutations (eg homozygous F508del), PS phenotype in patients with one severe one mild, or two mild mutations

1. defective protein production 2. defective processing 3. defective channel regulation 4. defective channel conduction 5. reduced cell surface expression 6. instability at cell surface

Defective processing. Immunoprecipitation studies found that there was no maturation in F508del CFTR -- the channel was not present in the Golgi, had not been fully glycosylated. Confocal microscopy looking at slices of intestinal epithelia showed CFTR pattern resembling apical membrane (stained with biotin), in homoF508del CFTR there was staining throughout the cell (not the same as basolateral). The protein cannot leave the ER and is tagged with ubiquitin and trafficked to the proteosome

NBD mutations such as F508del, G1244E, G1349D reduce the open channel probability, but don't affect chloride current. Loss of function is due to the loss of cAMP activated Cl- channel opening at the apical membrane

MSD R347P decreases Cl- flow. It has been suggested that this is due to it changing the channel from a multi-ion pore to a single-ion pore, thus disrupting ion flow