L7 - Atypical CF Flashcards
What is classical CF?
Severe symptoms
2 mutations in CFTR
- Normally delta F508 and one other
What is atypical CF?
Mild symptoms
1 or no CFTR mutations
1-2% northern European - 2 mutations not observed
What other mutated genes could be present in atypical CF?
Might be other mutated genes - modifier genes
- Might code for proteins that regulate CFTR
- Might play a role in how the airway cell works normally
What are the two reasons the height of the ASL is lower in CF patients?
No Cl secretion
GOF mutations in ENaC
- More Na+ absorbed –> reduced ASL –> CF like symptoms
Over many ENaC gene screens in CF patients overall what was discovered about the mutations?
A range of mutations observed
- Some of these had a greater incidence than in controls
- Some polymorphisms were observed in controls
What factor did they monitor when looking at the over expression of a range of different ENaC mutations?
Amiloride sensitive Na current as a % of the control
What are the two different common variants patients can have?
A663 or T663
- Two different amino acids that people can have – different backgrounds
- Do not impact ENaC function
- Looked at if having a different background impacted ENaC if you have another mutation
Results of looking at the over expression of a range of different ENaC mutations?
In some mutations - size of current the same – mutation unlikely to be underlying symptoms in patients
In some mutations – size of current increased –> function of ENaC enhanced
In 2 mutations – size of current decreases –> function of ENaC decreased
If the size of the amiloride sensitive Na current increases when overexpressing an ENaC mutation what does this show?
Gain of function
What you would expect from symptoms
If the size of the amiloride sensitive Na current decreases when overexpressing an ENaC mutation what does this show?
Loss of function –> less Na absorption –> height of ASL increases
CF like symptoms as height of ASL too high (rather than too low like normal)
Normal individual nasal transepithelial potential difference, Cl current, amiloride current
PD = -15.2
Cl = 16.3
Amilroide - 6.5
Classic CF patient nasal transepithelial potential difference, Cl current, amiloride current
PD = 31.1 Cl = 0.08 (decreased) Amiloride = 19.4 (increased)
One CF mutation and one ENaC mutation patient nasal transepithelial potential difference, Cl current, amiloride current
PD = -25 Cl = -15 Amiloride = 2- (increased)
alphaW493R – GOF ENaC mutation
F508del – CFTR mutation
What does nasal transepithelial potential difference, Cl current, amiloride current show?
Amiloride – change in potential indicates function of ENaC
Isotprotenolol – stimulates Cl handling – indicates CFTR function
What is the alphaW493R mutation and where is it found?
Found on extracellular loop of subunit
Previous study showed the mutation increased ENaC currents
What did the results with the alphaW493R mutation and amiloride show?
Currents greater in absence of amiloride
Currents greater in W493R mutant (mutation in ENaC alpha subunit)
What two things could cause greater currents in the alphaW493R mutant?
Number of channels – not this
Open probability of channels
What does Na feedback inhibition lead to?
Works to promote endocytosis of ENaC from membrane
ENaC opens –> rise in intracellular Na –> promotes endocytosis of ENaC
from membrane
How did they test if Na feedback inhibition was inhibited in alphaW493R mutant?
Tested by activating the Na channels with low extracellular Na
If extracellular Na is low –> little Na entering cell –> low intracellular Na –> little ENaC endocytosis triggered –> then go to high extracellular Na
Certain % of ENaC channels will be removed from membrane
Compare low Na currents with high Na currents
- Get idea of how much endocytosis occur
Do this with wild-type and mutant and see if % are similar
- Should be less endocytosis in mutant
What were the results of Na feedback inhibition experiments on the alphaW493R mutant?
With high Na - more Na does enter but it quickly pulls ENaC out of membrane so currents soon drop
In mutant
- Na feedback inhibition is still occurring
- % drop is very similar between WT and mutant
- Starting surface expression unchanged
Therefore must be channel open probability affecting currents
What are the two populations of ENaC channels?
Uncleaved - Near silent - Low Po - Generate smaller ENaC currents Cleaved - Very active - High Po - Generate larger ENaC currents
How did they test if the alpha W493R mutant increased the likelihood of cleavage?
Tested the response to chymotrypsin
What were the results of the chymotrypsin experiment to investigate cleavage?
WT –> when chymotrypsin added increased cleaved channels –> increased open probability (1 –> 5)
Mutant
- In absence of chymotrypsin already higher open probability
- When chymotrypsin added no effect
What did the results of the chymotrypsin experiment to investigate cleavage show?
Chymotrypsin cannot activate near silent channels - resistant to cleavage
Not the cleavage that is increasing the open probability
- Something else has already increased the open probability
Reduced response to chymotrypsin not due to increased endogenous cleavage before chymotrypsin added
Overall what was the alpha W493R mutation doing to channel cleavage?
Mutation in ENaC was making the channels resistant to cleavage
What is Na self-inhibition?
Where Na entering the cell blocks the pore of the channel –> reduces current flow
What did the results of Na self inhibition with the alpha W493R mutation show?
WT - 1mM to --> 110 mM - Decays to a new steady state - Na self-inhibition Mutant - 1mM to --> 110 mM - No decay/dip - Na self-inhibition is not occurring - This allows a bigger increase in currents --> higher open probability
Overall, how was the alpha W493R mutation increasing open probability?
By blocking Na self inhibition
What is the beta V348M mutation?
ENaC GOF mutation
How did they look at the open probability of the beta V348M mutation?
Measured amilroide sensitive whole cell currents before adding MTSET
What is MTSET?
Sulfhydryl agent
Binds to cysteine
What does MTEST do to the ENaC channel?
Stabilises open state of channel –> ENaC will have an open probability of 1
Measure current before and after MTSET gives an estimate of Po
Example calculation of current before and after MTSET
Amiloride sensitive current before MTSET = 12 µA
Amiloride sensitive current after MTSET = 20 µA
Ratio = 12/20 = 0.6
Apparent Po of channels = 0.6
Have to make an assumption that the number of channels isn’t changing
Why did they have to modify ENaC to have a cysteine background when using MTSET to look at open probability?
Had to be used to ensure MTSET worked – it binds to cysteine
How did they confirm that there was no change in beta ENaC channel number to prove it was changes in open probability affecting Na currents?
Western blot analysis
What was the ASL like in a atypical CF mouse model (overexpression of beta ENaC subunit)?
Height of ASL reduced and cilia bent
- Mucous clearance reduced
- Leads to mucous plaques and plugs
28 days post birth - only 50% survival
What did a slice through the lung of an atypical CF mouse model look like?
Mucous plugs
When an atypical CF mouse model was given intra-tracheal injection of bacteria what happened?
Clearance of bacteria monitored after 3 days
Reduced clearance in atypical CF model
Increases chance of death
Which of the following is correct when thinking about the causes of all forms of CF?
- LOF in CFTR
- LOF in ENaC
- GOF in CFTR
- GOF in ENaC
- LOF in CFTR
- LOF in ENaC
- GOF in ENaC
