L7 Atypical CF Flashcards
What are the two types of cystic fibrosis
Classical and atypical
Stats regarding atpyical CF
Mild symptoms observed with only 1 or NO CFTR mutations
In Northern Europe - small incidence of two mutations and no symptoms
Upper airway cell model
APICAl
ENaC - Na absorption
CFTR - Cl secretion
Upper airway cell model
NKCC1
Na/K ATPase
K Channel - recycle K to allow function of NKCC1
What is the effect of increased Na absorption
Bring down the height of ASL - just in the CFTR mutants
Normal subunit composition of ENaC
abg(amma)
Where can mutations occur in ENaC
In all three o the mutants
How many ENaC mutations have been screened
45 different mutations
What is a common ENaC variant
A663 - no effect on function
What is an effect of the A663 mutation
This mutation predisposes to other mutations
How were the EnaC mutants screened
OE express a number of common ENaC mutations and record the amiloride sensitive Na current
What were the results of the ENaC mutant screening
Some - size of the current is exactly the same so unlikely this mutation is having any affect on function
Some there an increase in the function - could cause symptoms
Two there is a loss of function
- height of the ASL would increase
What is the activity of ENaC in classical CF - why is this
ENaC enhanced
No longer inhibition of ENaC
Where does the aW493R occur
In the effector loop of one of the subunits
Describe the trace seen for aW493R compared to the wildtype
When amiloride removed greater current seen in the mutant compared to the wildtype
In the aW493R mutant is there a difference on the T/A backgrounds
No effect on these results
What is Na feedback inhibition
As Na moves into the cells and [Na] increases
This promotes the endocytosis of ENaC from the membrane
What is the question asked regarding Na feedback inhibition
Is there a change in this in mutants
Does the mutations prevent the endocytosis of ENaC
Describe how the question of the mutations affecting Na feedback inhibition was adressed
Use ECF of low Na - Na still able to move in but endocytosis prevented
Does aW493R affect Na feedback inhibition
Comparing low and high Na
Decreased currents with high Na outside
This is also seen in the mutant
Percentage drop is similar low to high
Describe how else ENaC channel activity may be activated
Cleavage by proteases to increase the Po
Two classes of cleavage
Uncleaved
Cleaved
Describe the uncleaved channels
Near silent
Low Po
Generate smaller currents
Describe the cleaved channels
Very active
High Po
Generate larger currents
How is cleavage investigated?
Looking at the response to chemotrypsin
Describe the response of the wildtype channels to chemotrypsin
Near silent channels now have a much increased Po
From one open to 5
Describe the response of the mutant channels to chemotrypsin
Already have a high Po which is unchaged
Conclusions drawn form the chemotrypsin expts
No activation of the near silent channels in the mutant. Reduced response to chymotrypsin not due to increase endogenous cleavage before chymotrypsin is added
Channels are resistant to cleavage but this doesnt have an effect
What is Na self inhibition
That the Na moving through the pore actuall blocks the pore reducing the size of current flow
When stepping from 1mM Na to 110 mM Na what is the effect seen in WT and the mutants
WT peaks and then quickly declines due to Na self inhibition
Mutant - peak current is much biggger and dont get the peak followed by inhibition
Higher Po because Na going through the pore doesnt block more Na moving throguh
What is a ENaC mutation found in the beta subunit
BV348M
What is the effect of BV348M on current
Steps up from 4uA to 5.5uA
Describe how the Po of the BV348M-ENaC channels was measured
Measure the amiloride sensitive whole cell currents - add MSET this stabilises the open state of ENaC by binding to cystenine the open state of ENaC
We assume this gives a Po of 1
Compare current before and after MTSET addition to work out the apparent Po
In order to measure Po using MTSET what must first be done
Have to make a background cysteine mutant - to allow MTSET to bind
What is the effect of BV348M on Po
WT Po of .24
BV348M Po of .33
So Po of this mutant channel (BV348M) has increased
Descrbe what was seen in single channel recordings of WT and BV348M mutants
BV348M Increased Po - enhanced function
What was seen in a western blot of WT and BV348M cells
No signigicant different
No change in N number
With atypical mutations more likely to affect ___ than ____
Po than N number
Describe the effects of overexpressing SCNN1B (B subunit)
Decreased height of the PCL
Mucous clearance is decreased
Mouse mutants die sooner - due to problems with mucociliary clearance - plugging the airways
