L7 - atypical CF Flashcards
give some features of atypical CF?
- milder symptoms than classical CF
- 1 or NO CFTR mutations
- carriers usually asymptomatic
- must be other genes mutated in these patients
what kind of mutations were proposed to be in ENaC to cause CF?
GOF mutations in ENaC proposed to cause atypical CF - mimic CF patients - as when there is no CFTR, there cannot be the inhibitory effect on ENaC- ENaC increases in function anyway
- more Na absorbed for PCL - depletion of ASL
where were mutations in ENaC discovered in CF patients?
- mutations discovered in all 3 subunits
- A - 11 mutations
- B - 7 mutations
- Y - 8 mutations
- in individuals who didnt fit into normal CFTR mutations - ENaC mutations were observed
what effect do mutations have on ENaC function?
- 9 different mutations tested
- with 4 mutations there were bigger amiloride sensitive currents - increased function GOF of ENaC
- with 3 mutations there was no difference to control
- with 2 mutations tested there was a LOF in ENaC
- shows variation in ENaC mutations
what does the a-W493R mutations show?
- is on the EC loop of subunit a
- a previous study showed increased ENaC currents
- current study confirmed this - GOF in ENaC
- the amiloride sensitive current in a-W493R cells was increased, and the response when amiloride was washed out was greater
what is NA+ feedback inhibition?
- when ENaC opens, Na+ floods into the cell
- high IC [Na+] triggers endocytosis of ENaC from the membrane
What is proposed that happens to ENaC endocytosis in GOF mutations?
propose that endocytosis of ENaC doesn’t occur during GOF ENaC mutations - removal of ENaC from membrane not triggered
- high numbers of ENaC remain in membrane
what did a-W493R mutations show about feedback inhibition?
- take difference in I amil between low and high EC [Na} currents - at low EC Na feedback inhibition is prevented - take differences between 2 conditions - gives degree of endocytosis of ENaC
- same degree/ratio of endocytosis between mutant and WT channels - feedback inhibition is unaffected
- FEEDBACK INHIBITION/ ENDOCYTOSIS IS NOT WHY WE GET GOF IN ENaC
how does cleaving channels effect the Po?
cleaved ENaC channels have a higher Po (GOF)- larger ENaC currents
what did experiments using chymotrypsin on a-W493R suggest?
- chymotrypsin can cleave ENaC channels
- in ENaC a-W493R mutant repsonse to chymotrysin is lost
- suggests that channels could already be cleaved?
How was it found that ENaC channels are not already cleaved and what did this show?
- looked at single channel currents
- when chymotrypsin added there was no activation of near silent channels
- can see mutant channels open a lot more even before the addiitons of chymotrypsin
- Po is a lot higher in mutant ENaC - sustained openings but this is not due to cleavage
are Na+ feedback inhibition and Na+ self-inhibition different?
Yes
what happens in Na+ self-inhibition?
Na+ impacts on the channel of the pore to close it.
- in the a-W493R mutant - self-inhibition doesn’t occur? - larger currents in mutant
- WT peaks sooner as Na self-inhibition kicks in - doesn’t occur in mutant
why is the ENaC mutation a-W493R GOF?
there is a loss in NA+ SELF-INHIBITION
- get larger currents
Is the B-V348M mutaiton GOF or LOF and how can this be shown?
B-V348M mutation is a GOF mutation in ENaC
- The is a bigger response/current when amiloride is washed out compared to WT - ENaC has increased function with this mutation
