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
What happens in S1255P?
- Class III mutation
- In NBD 2
- Defective regulation
NB don’t need to memorise this
What happens in Q1412X?
- Class VI mutation
- Instability at the cell surface
NB don’t need to memorise this
What happens in Splice mutation intron 4 donor site?
- Class I mutation
- In MSD 1
- Absent protein
NB don’t need to memorise this
Why is important to know the mutations?
Therapies can be targeted to the particular mutation for better results
Which therapies are used for Class I mutation?
Aminoglycoside antibiotics:
• allow ‘read through’ of mRNA despite nonsense mutations
Which therapies are used for Class II mutation?
‘Correctors’
• improve processing
Which therapies are used for Class III mutation?
‘Potentiators’
active proteins, keep the channel open
Which therapies are used for Class IV mutation?
‘Flavanoid’
Augment channel function
Which therapies are used for Class V mutation?
Increase levels of correctly spliced RNA
What is happening in F508del?
Phenylalanine n° 508 deletion
• three bases are deleted
• NB only two bases from the F508 codon deleted, and one for Isoleucine
How is CFTR mutation tested for?
Earlier: 1. RFLP analysis S23 2. Multiplex PCR; SNP analysis 3. MALDI-TOF Mass Spectrometry
How many mutations are screened for in newborns?
12 mutations
More being added all the time
Describe the different frequencies of F508del in people of different ethnicities
Different ethnicities are affected to greater and lesser degrees by certain mutations.
For example Northern Europeans (esp. Danes) are particularly affected by the F508del mutation
How is Mass Spectrometry used for screening?
(MALDI-TOF MS)
• Large number of samples can be tested simultaneously
• Looking for a medium n° of SNPs
What is an RFLP?
Restriction fragment length polymorphism
• When DNA from different individuals is digested with restriction enzymes, one gets fragments with different lengths.
Describe RFLP analysis
- PCR + Restriction enzymes
- Run on an acrylamide gel
- Analysis of RFLPs
Describe Multiplex PCR
• Fluorscent tagging to see SNPs
Where is CFTR expressed?
Apical membrane of epithelium
In a wide range of tissues
What is the function of the MSD’s?
Form the ion channel through the membrane
Draw a rough diagram of the CFTR gene
S 18 • 27 exons • 2 x MSD coding regions • 2 x NBD coding regions • R domain coding region
What is the R117H mutation?
- Class IV mutation
- In MSD 1
- Defective conduction through the channel
Describe the difference between class III and IV mutation. Which mutation is more severe?
Class III: ATP doesn’t bind, hence channel doesn’t open
Class IV: dysfunctional phosphorylation of R domain, channel has reduced conductance
Class III mutation is more severe
What is the importance of the external projections of the CFTR?
These are loops of the MBD that are located on the outside of the cell
These can interact with other, external molecules, altering the function of the CFTR channel
Which class of mutation is F508del?
Class II
How and when was the CFTR gene identified?
1989
Identified by positional cloning
How does the CFTR regulate ENaC?
The TRL (Threonine-Arg-Leu) sequence on the cytoplasmic C-terminus interacts with it
(Inhibition of ENaC)
