Atypical CF

Question 1

Classical CF - what are the most common features?

Answer 1

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-]

Question 2

Atypical CF - what are the most common features?

Answer 2

1 or 0 mutations in CFTR but similar symptoms are seen!! Mild symptoms, modifier genes may be involved

Question 3

Enac - why relevant? What do GOF mutations cause ?

Answer 3

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?

Question 4

Enac - how many sub units?

what was shown wrt CF patients screened for mutations in Enac??

Answer 4

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!!

Question 5

Overexpression study of mutant Enac – used to observe currents
What was seen

Answer 5

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

Question 6

Experimental validation of Enac GOF mutation experiments –> why do this? How?

Answer 6

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

Question 7

Enac GOF validation - shifts in potential with and without Amiloride?

Answer 7

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)

Question 8

Common mutation in atypical CF patients

Answer 8

GOF in Enac protein!

Question 9

aW493R mutation - what part of Enac does it impact? How is this confirmed?

Answer 9

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)

Question 10

What is Na+ feedback inhibition?

Answer 10

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

Question 11

What can be observed regarding Enac currents in WT cells by the mechanism of Na+ feedback inhibition?

Answer 11

(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

Question 12

What can be observed regarding Enac currents in aW493R cells by the mechanism of Na+ feedback inhibition?

Answer 12

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

Question 13

ENac cleavage - how does this impact Po??

Answer 13

ENac is cleaved which shifts the channel to have a higher Po. Hypothesis therefore that in atypical CF, more cleaved ENac therefore larger currents?

Question 14

Impact of Chymotrypsin on WT and mutant aW493R cells?

Answer 14

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!!

Question 15

Self inhibition of ENac –> what is it and how could it be causing symptoms in aW493R muts?

Answer 15

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

Question 16

BV348M mutation - what does it impact and how can we measure its impact using MTSET?

Answer 16

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

Question 17

Measuring PO using MTSET

Answer 17

Amiloride sensitive currents without MTSET = 12ma
Currents with MTSET = 20ma
Ratio = 12:20 = 0.6 = channels open 60% of time!!!

Question 18

MTSET ratios of BV348M vs WT cell channels?

Answer 18

WT cells = ratio is 0.24 so channels open 24% of the time!

BV348M - ratio is 0.33 = channels open 33% of the time

Question 19

Mice Mutant ENac overexpression - observations made?

Answer 19

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

Question 20

Impact of B sub unit on ENac as a whole?

Answer 20

B sub units are rate limiting –> increase in number of B sub units for ENac increases the functionality of the channel!!!