Atypical cystic fibrosis Flashcards
Does atypical CF see mutations in CFTR
Can have one or no CFTR mutations
Do heterozygotes of atypical cystic fibrosis present any symptoms
Can present mild to severe symptoms even though they are only carriers - unlike CFTR single mutants who see no symptoms.
Which channel is mutated in atypical CF
ENaC
What kind of mutation was hypothesised to be seen in ENaC channels
GOF - enhanced sodium absorption, this depletes the ASL to give CF like symptoms
What is strange about the F61L and A334T mutant ENaC channels
LOF - height of ASL goes to high, cilia can’t function normally?
How are shifts in potential used to measure ENaC function
Addition of amiloride sees a negative shift in potential due to ENaC function
How are shifts in potential used to measure CFTR function
Change to a chloride free/ low chloride solution and the resultant shift is a measure of CFTR function
Why is ENaC function in a classical CF patient so high?
Because the mutant CFTR channel cant suppress the activity of ENaC function
What were the experimental results from the individual with the delF508/W493R mutations of CFTR/ENaC
Starting point of -25mV lies between the WT and CF patient. Addition of amiloride gives +20mV which is the same as a CF patient Response to low chloride is fairly close to normal as the patient has 50% CFTR function. Therefore it must be ENaC function causing the problem.
Which mutation is found on the extracellular loop of the ENaC alpha subunit
alphaW493R
What effect does the alphaW493R mutation have on ENaC function
GOF - increased current when amiloride is washed.
What is sodium feedback inhibition?
If ENaC opens Na floods into the cell increasing intracellular sodium. This increase triggers the endocytosis of ENaC from the membrane. Mutations can disrupt this system.
How do you test for Na feedback inhibition
Compare the ratio of currents with high and low extracellular sodium.
Outline the steps taken to investigate Na feedback inhibition in the W493R mutant compared to WT
First looked at surface expression, expression of ENaC was the same between WT and mutant channels.
Then measured the amiloride sensitive current in low extracellular Na (smaller increase in intracellular Na and less endocytosis)
Then changed to high sodium which triggers the endocytosis process. Can then compare the ratios between the two amiloride sensitive currents.
Although there is a higher current for the mutant channels in both low and high extracellular Na, the ratios between the two are roughly the same as WT, so the amount of endocytosis is occuring, Na feedback inhibition is not effected by the mutation.
What two forms does ENaC exist as and what differs between the two forms?
ENaC can be cleaved or uncleaved (cleaved by proteases)
The cleaved form has a higher Po so could be responsible for the increased current because: I = N.Po.g.(Vm-Ei)
Uncleaved ENaC is near silent with few opening events, Low Po and expect to generate low ENaC currents.
What is the role of chymotrypsin
Cleaves ENaC - taking it from low Po to high Po - increasing the current generated by ENaC
What effect does chymotrypsin have on W493R mutant currents
Looks like the response to chymotrypsin is lost becuase they are already cleaved? However this is not the case - Evidence for this is shown in the image. WT shows lots of flickering (constant opening and closing of channels)
The mutant channels don’t react to the chemotrypsin. Before the chemotrypsin is added there is already much more activity from the mutant channels which is underpinning the big currents.
What is sodium self inhibition
Sodium ions impact on the pore of ENaC to close it. Represented by the small dip in current following the peak in the image.
What happens to Na self inhibition in the W493R mutant
No sodium self inhibtion
If Na self inhibition didn’t exist in the WT then it would be around the same current seen in the mutant. The reason that the peak is smaller than the mutant is due to Na self inhibiton
Give a summary of the W493R mutation
No change in Na feedback
High currents not due to increased cleavage
There is a loss of Na self inhibition
This results in high currents, greater water reabsorption and CF like symptoms.
What is the second studied GOF mutation in ENaC
þV348M
What is the role of MTSET
Sulhydryl agent which binds to cysteine. This stabilises ENaC in an open state, essentially creating a Po of around 1. If the current is measured before and after the addition of MTSET and compared, an estimate of Po is generated.
What would be the apparent Po of ENaC if the amiloride sensitive current before MTSET was 12uA and after MTSET was 20uA
12/20 = 0.6
What was the difference in the Po between WT and V348M mutant ENaC channels
WT Po =0.24
BV348M Po = 0.33
This GOF mutant has an increase in Po, more water reabsorption etc
