Lecture 8 - Molecular Basis of Cystic Fibrosis Flashcards
On which chromosome is the CFTR gene?
Cr 7
Length of CFTR gene
70kB
Number of exons in CFTR gene
27
What is the CFTR protein?
A large, integral, glycosylated, membrane-spanning protein
Weight of CFTR protein
170kD
Is CFTR responsible for active transport?
No.
Despite using ATP, CFTR moves Cl- with concentration gradient
Which family does CFTR belong to?
ATP-Binding Cassette (ABC) superfamily of membrane transporters
How is CFTR channel regulated?
By cAMP-dependent phosphorylation
Which cells express CFTR?
Epithelial cells, normally on apical surface
What are the domains of the CFTR protein? 1) 2) 3) 4) 5)
1) Membrane-spanning domain 1
2) MSD 2
3) Nucleotide-binding domain 1
4) NBD 2
5) Regulatory domain
Function of membrane-spanning domains
Form pore through which Cl- move
Function of nucleotide-binding domains
Bind and hydrolyse ATD
Function of regulatory domain
Several sites that can be phosphorylated by cAMP-dependent kinases
EG: Protein kinase A
Examples of different direction of Cl- flow through CFTR
Lungs: Cl- flows out of cell
Sweat duct epithelial cells: Cl- flows into cells
How does CFTR interact with other proteins?
Largely through C-terminal, which is anchored to cytoskeleton
Example of protein with which CFTR interacts
ENaC sodium channel
Domain on C-terminal of CFTR which interacts with other proteins
TRL - Threonine - Arginine - Leucine
Normal function of CFTR in cell in lung
1)
2)
1) Cl- moves out of cell through CFTR
2) Na+, H2O move into cell down concentration gradient
Defective function of CFTR in cell in lung
1)
2)
3)
1) Cl- ions can’t escape cell through CFTR
2) Buildup of Cl- in cell results in a greater concentration gradient.
3) ENaC is not inhibited by CFTR. Unregulated uptake of Na+ into cells, leading to water osmotically being absorbed into cells. Dehydration of ASL
Normal function of CFTR in a sweat duct
1)
2)
1) Cl-, Na+ and H2O enter cell down concentration gradient
2) Sweat is secreted, but ions are largely reabsorbed by cells
Defective function of CFTR in a sweat duct
1)
2)
1) Cl- can’t enter cell, so Na+ ions don’t either
2) Buildup of Cl- and Na+ ions in sweat, leading to abnormally salty sweat
Number of known mutations in CFTR
Over 1900
Where are most mutations found in CFTR?
Exons 4, 8, 14, 20
Most frequent mutation in CFTR
Missense
Class I CFTR defect
No protein production
Class II CFTR defect
Defective processing
(maturation, premature degradation)
Protein can’t leave ER, Golgi
Class III CFTR defect
Defective regulation (defective ATP binding, hydrolysis)
Channels don’t open
Class IV CFTR defect
Defective or reduced opening of ion channel, ion conductance
Channels can open, but not much
Class V CFTR defect
Reduced protein production
promoter or splicing abnormality
Class VI CFTR defect
Accelerated turnover from cell surface
Quite a rare type of defect
Common mutations leading to a class I defect
Nonsense, missense, frameshift mutations
Do mutations in CFTR gene have to be pathogenic?
No.
Some are non-pathogenic, some have reduced penetrance
What could lead to a class VI defect?
Nonsense mutation placing a stop codon near the C-terminal
Protein instability at cell surface
What could lead to a class III defect?
Mutation in nucleotide-binding domain 2.
ATP can’t be hydrolysed
Or mutation in R domain
What could lead to a class II defect?
Mutation in nuclear binding domain 1.
Defective cell processing
This is where F508del occurs.
What could lead to a class IV defect?
Mutation in membrane-spanning domain 1.
Cl- have more trouble moving through pore
What could lead to a class I defect?
Mutation in membrane-spanning domain 1.
Normal levels of mRNA, absent protein
What could lead to a class V defect?
Mutation in membrane-spanning domain 1.
Reduced number of transcripts
Most common mutation
F508del
Which class of defect results from F508del?
Class II
Protein is misfolded, retained in ER, degraded
Way to correct class I defects
Aminoglycosides
Allow ‘read through’ of mRNA
Way to correct class II defects
‘Correctors’ to improve protein processing
Way to correct class III defects
‘Potentiators’ to activate protein
Way to correct class IV defects
Flavinoid compounds to improve channel conductance
channel more likely to be open
Way to correct class V defects
Improve number of correctly-spliced mRNA molecules
How does F508del occur?
1)
2)
3)
1) C from end of isoleucine and TT from phenylalanine are deleted.
2) Last amino acid of phenylalanine codon (T) combined with first two amino acids of isoleucine codon (AT) leads to isoleucine codon.
3) 3-base, out of frame deletion, but only phenylalanine has been removed.
Frequency of F508del mutation
~50% CF sufferers homozygous for F508del
F508del accounts for ~75% of CFTR mutations in northern Europe
How was CFTR first discovered to be the protein involved in CF?
Positional cloning
Least common place in CFTR gene for a mutation to occur.
Promoter
