Atypical CF Flashcards
Classical CF - what are the most common features?
2 copies of mutated CFTR gene, class 1-3 mutations most severe and involve pancreatic insufficiency Mutation in CFTR determines the severity in CF, the amount of protein determines the sweat [Cl-]
Atypical CF - what are the most common features?
1 or 0 mutations in CFTR but similar symptoms are seen!! Mild symptoms, modifier genes may be involved
Enac - why relevant? What do GOF mutations cause ?
Increase absorption of Na+, H20 follows, height of PCL depletes, cilia bend over –> cannot clear mucus!!
GOF mutation in Enac may mirror those symptoms seen in CF?
Enac - how many sub units?
what was shown wrt CF patients screened for mutations in Enac??
31 CF patients screened for mutations in Enac (4 sub units - a, B and y, d in airway only)
11 mutations in Enac!!!
45 atypical CF patients screened, even more mutations in Enac identified!!
Overexpression study of mutant Enac – used to observe currents
What was seen
Overexpression of ENac in presence of particular mutations and observed Na+ currents
Larger Na+ currents were seen!!
In patients where no Enac enhancement was seen, patient symptoms were due to differing Cl-/Na+ handling
Experimental validation of Enac GOF mutation experiments –> why do this? How?
DId this to show that the Enac GOF mutations but Cl- handling was the same – added low Cl- solution to the upper airway of CF patients and normal patients and measured transmembrane potential shift in currents
Enac GOF validation - shifts in potential with and without Amiloride?
Classical CF – no shift in potential in response to Cl- solution (CFTR mutated so can’t work!) + Amiloride = 19mV shift (response to blocked Enac is enhanced in CF!!)
CF carrier (one copy of dF508) - Cl- shift similar to WT (change in response to low Cl- solution) but 20mV shift in response to ENac – ENac function enhanced but CFTR function unchanged!
Highlights how GOF in Enac mirrors CF-like symptoms (and not due to changes in Cl- handling)
Common mutation in atypical CF patients
GOF in Enac protein!
aW493R mutation - what part of Enac does it impact? How is this confirmed?
EC loop of the alpha sub unit
Overexpress mutation in 2 different cell backgrounds
Measure current when Amiloride added –> large change in current through cells seen (mutation-sensitive currents produced)
What is Na+ feedback inhibition?
When WT Enac opens, Na+ flood into the cell causing an increase in IC [Na+]. When this happens, Enac is retrieved from the membrane by endocytosis in a negative feedback mechanism
What can be observed regarding Enac currents in WT cells by the mechanism of Na+ feedback inhibition?
(note - the starting number of ENac channels in the membrane for both WT and mutant cells was NORMAL)
In WT cells where there in high Na+ conditions, a drop in current is observed as ENac is endocytosed out of the membrane
What can be observed regarding Enac currents in aW493R cells by the mechanism of Na+ feedback inhibition?
Inhbition of Na+ current is still seen in the presence of the mutation in ENac (we would EXPECT) the Na+ currents to remain high in high Na+ as ENac not removed from membrane –> GOF in Enac in atypical CF not to do with Na+ feedback inhibition
ENac cleavage - how does this impact Po??
ENac is cleaved which shifts the channel to have a higher Po. Hypothesis therefore that in atypical CF, more cleaved ENac therefore larger currents?
Impact of Chymotrypsin on WT and mutant aW493R cells?
Chymotrypsin is a protease that cleaves ENac (would therefore cause in increase in Po of channels in WT cells)
Response to Chym is lost in mutants –> prolonged channel opening irrespective of chymotrypsin – differences in gating!!
Self inhibition of ENac –> what is it and how could it be causing symptoms in aW493R muts?
Amount of Na+ moving through channels regulates the channel Po. Loss of S.I like in ENac GOF mutants = higher currents = greater H20 influx = CF-like thick mucus
BV348M mutation - what does it impact and how can we measure its impact using MTSET?
GOF mutation in B sub unit of ENac
MTSET stablises the ENac channel in an open state (Po therefore = 1). In the presence of MTSET Amiloride sensitive ENac currents are therefore LARGER (as channel always open and producing larger currents!!!!)
Can measure currents in absence of MTSET and compare the 2 values to calculate the % open probability
Measuring PO using MTSET
Amiloride sensitive currents without MTSET = 12ma
Currents with MTSET = 20ma
Ratio = 12:20 = 0.6 = channels open 60% of time!!!
MTSET ratios of BV348M vs WT cell channels?
WT cells = ratio is 0.24 so channels open 24% of the time!
BV348M - ratio is 0.33 = channels open 33% of the time
Mice Mutant ENac overexpression - observations made?
Increased Po of channels Thick mucus and less clearance of mucus Increased H20 reabsorption Lower height of PCL Mucus plaque and plug formation Inflammation Poor clearance of bacteria Post natal mortality
Impact of B sub unit on ENac as a whole?
B sub units are rate limiting –> increase in number of B sub units for ENac increases the functionality of the channel!!!