Lecture 8 - CF: Molecular Basis Flashcards
In general, what does the mutation in CFTR lead to?
Abnormal cAMP-mediated regulation of chloride transport
Which gene is mutated?
CFTR gene
CF transmembrane conductance regulator
What is the CFTR protein?
Chloride channel protein
To which family does CFTR belong?
ABC superfamily of membrane transporters
ATP-binding cassette
Describe the energy requirements of the chloride channel
ATP required to open and close the channel
Ion movement through the channel is downstream (no ATP)
Describe and draw the structure of the CFTR protein
5 domains:
• 2 x MSD’s (MSD 1, MSD 2) which form the channel
• Regulatory domain; R domain
• Nucleotide binding domains: NBD 1, NBD 2
Order: • N terminus • MSD 1 • NBD1 • MSD2 • NBD 2 • R domain • C terminus
What is the important feature of NBDs?
ATP binding → channel opens
NB when ATP is hydrolysed, the channel closes again
What is the function of the R domain?
(Regulatory domain)
Phosphorylated by cAMP
Phosphorylation leads to fine tuning of the channel function
Describe the opening and closing of CFTR
ATP binds → opening of channel
ATP hydrolysis → closing of channel
What are some secondary roles of the CFTR channel?
Carboxy terminal region: • regulation of ENaC (a sodium channel) • conductance • signal transduction • cytoskeleton
Describe the role of CFTR in airways
How is this disrupted in CF?
Normally:
• Cl- ions moving out of the cell down a concentration gradient
(Na+ and water moving into cells)
In CF:
• Cl- ions maintained within the cell
• Increased Na+ and water movement into the cell
• Dehydrated mucous
Describe the role of CFTR in sweat ducts
How is this disrupted in CF?
Normally:
• Cl- ions moving into the cell down a concentration gradient
In CF:
• blockage of Cl- movement into the cells
• Na+ and water maintained outside the cell
What is the CF Mutation Database?
Collection of mutations that cause Cystic Fibrosis of varying and unknown significance
What are mutations of ‘varying clinical consequence’?
Mutations that have decreased penetrance
Difficult for clinicians, because the severity of the mutation is often not known
Which type of mutation is most common in CF?
Missense (40%)
Which exons are most commonly mutated in CF?
Membrane spanning domains: 4, 8
Regulatory domain: 14
20
What are the classes of mutation?
6 classes • Class I: no protein production • Class II: Defective processing • Class III: Defective regulation • Class IV: Reduced ion conductance • Class V: reduced protein production • Class VI: accelerated turnover from the cell surface
What happens in Class I mutation?
Premature nonsense mutation
• STOP codon before the end of the protein
• truncated protein produced
• defective protein is unstable and tagged for degradation
There are also mutations whereby the mRNA is not produced properly
What happens in Class II mutation?
Gene is transcribed and translated into the ER
However, the protein never makes it out of the ER due to e.g. mutation in folding, targeting to cell surface
→ Degradation of the protein in the ER
What happens in Class III mutation?
CLOSED CHANNEL
- Protein makes it to the cell surface
- ATP doesn’t bind properly to NBD
- Channel never opens
What happens in Class IV mutation?
REDUCED CONDUCTANCE
• Protein makes it to the cell surface
• Dysfunction of phosphorylation of the R domain
• Channel only opens somewhat, may be partially open / closed
What happens in Class V mutation?
Protein is produced, but not in the amounts required
What happens in Class VI mutation?
Protein is functional and makes it to the cell surface, but is removed too soon
What is the most common mutation in CF?
Describe it
F508del • Class II mutation • In NBD 1 • (Defective processing) • Protein missfolds • doesn't get out of ER and is targeted for degradation
