BIOM30002 Lecture 20: T4_L20_summary
12 Jun 2018
School
Department
Course
Professor
L20 Molecular Basis of CF
Summary
- Early example of single-gene causing disease identified though positional cloning
- CFTR is large ion channel protein – multiple domains
- Many variants, majority rare – F508del most common in northern Europeans
- Six classes of mutations/variants
- Testing typically for a specific panel (ethnic specific?) of variants
- Different types of testing technologies
History
- In 1938, CF was identified mucous plugs in pancreas in babies dying of malnutrition
- Called cystic fibrosis of the pancreas
- “Salty baby”
- Later, CF was characterised by fat and protein malabsorption, failure to thrive, lung disease
and abnormal electrolyte composition in sweat
- Significant increase in life expectancy from past to now
- 1983: abnormal cAMP mediated regulation of Cl- transport (sweat ducts)
- 1985: CF gene mapped to chromosome 7 at position q31.2 (7q31.2) by linkage in families
- 1989: positional cloning pre-human genome project method (not next generation
sequencing)
CFTR gene codes for an ion channel protein
- Gene:
1. 190 kb, 27 exons (old 24 exons, lack 6, 14 and 17 renumber)
2. Codes for large integral glycosylated membrane-spanning protein
- CF transmembrane conductance regulator = CFTR protein
1. 170 kDa, 1480 AA
CFTR protein
- Member of ABC (ATP-binding cassette) superfamily
- Gated chloride channel: different from other ABC because passage of ions by down
concentration gradient
- cAMP-dependent phosphorylation
- roles:
1. anion transport, mostly Cl- and HCO3-
2. mucociliary clearance
3. immunity/inflammation
- expressed in epithelial cells at apical side
- 5 domains
1. MSD1 (membrane-spanning domain 1): channel for passage of Cl-, transmembrane,
N-terminus
2. MSD2 (membrane-spanning domain 2)
3. NBD1 (nucleotide-binding domain 1): bind and hydrolyse ATP, intracellular
4. NBD2 (nucleotide-binding domain 2): C-terminus, to MSD2
5. R (regulatory domain): several sites phosphorylated by cAMP-dependent protein
kinase, e.g. PKA), intracellular
- Most common variant delta F508del on NBD1
Models of CFTR channel protein
- Activation of CFTR channel relies on phosphorylation (eg. PKA) on R domain
- Closed state open state
- There is one ATP permanently bound to NBDs
- Activation:
1. +PKA, second ATP binds to NBDs
2. Phosphorylation conformational change
3. Allow passage of Cl- ions down gradient
CFTR protein interact with other proteins
- C-terminal anchored to cytoskeleton close to protein that influence CFTR functions:
1. Conductance
2. Regulation of other channels (eg. ENaC = epithelial sodium channel)
3. Signal transduction
4. Localisation at apical plasma membrane
CFTR function in the airways/sweat duct – NB simplified model
Normal airway
CF airway surface
layer (ASL)
Normal
sweat duct
CF sweat duct
Cl
-
Out, down G
Build up within cell
In, down G
Cannot enter cell
Na+
In
Greater
flow in
In
Remain in duct
H2O
In
Greater flow in
In
Remain in duct
Effect
-
Dehydrated mucus
in the ASL
Sweat (salt and water)
reabsorbed
Elevated Na+ and Cl
-
in sweat
In/out of cell; down/against conc. gradient
Na+ against Cl- flow
H2O follows Na+
opposite in sweat ducts
Document Summary
Early example of single-gene causing disease identified though positional cloning. Cftr is large ion channel protein multiple domains. Many variants, majority rare f508del most common in northern europeans. Testing typically for a specific panel (ethnic specific?) of variants. In 1938, cf was identified mucous plugs in pancreas in babies dying of malnutrition. Later, cf was characterised by fat and protein malabsorption, failure to thrive, lung disease and abnormal electrolyte composition in sweat. Significant increase in life expectancy from past to now. 1983: abnormal camp mediated regulation of cl- transport (sweat ducts) 1985: cf gene mapped to chromosome 7 at position q31. 2 (7q31. 2) by linkage in families. 1989: positional cloning pre-human genome project method (not next generation sequencing) Cftr gene codes for an ion channel protein. Gene: 190 kb, 27 exons (old 24 exons, lack 6, 14 and 17 renumber, codes for large integral glycosylated membrane-spanning protein. Cf transmembrane conductance regulator = cftr protein: 170 kda, 1480 aa.
