Lecture 08 Atypical Cystic Fibrosis

Question 1

Compare atypical and classical cystic fibrosis

Answer 1

classical CF:
severe symptoms (some mild with 2 faulty copies)
majority 2 mutations in CFTR
normally heterozygous = carrier with no symptoms
1-2% N. Europe population have 1 mutation and have CF symptoms

atypical CF:
mild symptoms
1 or no CFTR mutations

Question 2

What could account for the differences seen in mutation severity?

Answer 2

modifier genes

Question 3

If not CFTR, what was hypothesised to cause atypical CF?

What sort of mutation?

Answer 3

ENaC - the disruption of sodium channels in the airway could result in the disruption of the PCL
Gain-of-Function (GoF) mutation in ENaC - would enhance the sodium reabsorption and therefore water reabsorption leading to reduction in the liquid layer

Question 4

What subunit if the ENaC GoF mutation in?

Answer 4

one ENaC (SCNN1) screen in 31 atypical CF patients identified mutations in all three subunits:
11 mutations in alpha
7 mutations in beta
8 mutations in gamme
(since further mutations have been identified)

Question 5

How was the impact of mutations on ENaC function studied?

What GoF ENaC mutations were identified?

Answer 5

mutations taken and express in cells
looked at the function by measuring the sodium current using amiloride

V114I (SCNN1A-A663); L180L-R181W (SCNN1A-A663); W493R (SCNN1A-A663); W493R (SCNN1A-T663)

Question 6

What is the impact of different mutations in terms of chloride secretion and amiloride on ion channel function?

Answer 6

normal/wt - no mutation:
resting membrane potential ~-15mV
response to Cl- ~-16mV = normal function of CFTR
response to amiloride ~+6.5mV = normal function of ENaC

classic CF:
resting membrane potential much lower ~-31mV
no change in potential in response to chloride - no CFTR function (expected)
big response to amiloride ~+19mV

atypical CF (W493R/delta-F508 and W493R/E528E):
resting membrane potential lower than normal but not as severe as classic CF ~-19-25mV
normal CFTR response to chloride ~-15-20mV
big shift in response to ENaC ~+18-20mV
expect this is GoF ENaC mutation

Question 7

How did the researchers confirm that the alpha-W493R GoF mutation was affecting ENaC?

Answer 7

measuring current in the channel in the absence and presence of ENaC
only channel activity when amiloride is absent
much later current measured in the mutant

Question 8

What are the common variants of sodium channels?

What is the impact of this?

Answer 8

T663 and A663 variants - show different backgrounds but have no impact on the function of the channel
confirmed by measured the sodium currents in wt and mutant forms of both variants
comparison showed no effect between the two variants

Question 9

How does Sodium Feedback Inhibition work?

Answer 9

1. ENaC is in the membrane and opens to allow sodium influx into the cell, increasing the intracellular [Na+]
2. transient increase of sodium causes activation of ENaC retrieval from the membrane leading to ENaC endocytosis

feedback inhibition is prevented by low extracellular sodium - i.e. negative feedback loop acting to maintain IC [Na+]

Question 10

What is the impact of the alpha-W493R mutation on Sodium Feedback Inhibition?

What experimental evidence is there for this?

Answer 10

no impact on sodium feedback inhibition in terms of decreasing the current, therefore mutation does not affect this mechanism

take wt or mutant channels expressed at the surface with low EC [Na+] to keep them there. Add high EC [Na+] solution and should see the sodium current decrease as the number of channels decreases (endocytosis)
despite much higher starting current in the mutant the % decrease in sodium current = sodium feedback inhibition is the same

Question 11

How does cleavage affect ENaC?

Answer 11

cleaving ENaC with proteases causes it to have a higher open probability (Po) - i.e. higher current
uncleaved ENaC is near silent - low Po and small currents
cleaved ENaC is very active - high Po and high currents

Question 12

Give an example of a protease that cleaves ENaC

Answer 12

Chymotrypsin

Question 13

Does the alpha-W493R mutation lead to a higher proportion of cleaved ENaC?

What is the experimental evidence for this?

Answer 13

no, the increased currents are not due to increased cleavage

response to chymotrypsin:
wt - when chymotrypsin is added there is an increased function of ENaC (increased current)
mutant - absence of amiloride already very high current, no additional response to chymotrypsin - i.e. response is lost

patch clamp technique
wt - ‘flickery’ behaviour - channels open and close very quickly. many silent channels. increased opening response to chymotrypsin but still ‘flickery’
mutant - sustained opening when add chymotrypsin no effect in increase/decrease Po or number of channels

Question 14

What is Sodium Self Inhibition?

Answer 14

a property of ENaC channels meaning sodium moving into the cell regulates the Po of the channel
when sodium moves into the cell, the IC [Na+] reduces the channel Po

Question 15

What is the impact of sodium self inhibition on ENaC?

Answer 15

wt - subtle change observed - tail
mutant - large sodium current and no ‘tail’ indicating that sodium self inhibition has been lost

lose of sodium self inhibition whilst subtle is enough to give the enhanced ENaC function seen in patients causing increased open probability

Question 16

What is the impact of the beta-V348M mutation?

How was the open probability calculated?

How was this technique applied to the beta-V348M mutant?

Why couldn’t the patch clamp technique be used?

Answer 16

gain-of-function (GoF) mutation

used MTSET which stabilises ENaC in the open state giving a Po of 1
measure the amiloride-sensitive current to give the current of ENaC channels in the absence of MTSET
add MTSET and remeasure the amiloride-sensitive current
take a ratio of the two currents to give the apparent Po of ENaC

added a mutation to wt and mutant beta-V348M: beta-S520C to add a cysteine allowing MTSET to work (controls carried out to check no effect of additional mutant)
showed wt Po to be 0.24 or 24%
showed mutant Po to be 0.33 or 33%
increased activity of ENaC in mutant form - increase Po

very difficult to measure single channel conductance of ENaC. If there is lots of background interference don’t get a clear result

Question 17

Does the beta-V348M mutation affect the number of channels at the membrane?

How was this confirmed experimentally?

Answer 17

no change in number of channels at the membrane

Western Blot Analysis

Question 18

Why does over-expressing the beta-subunit lead to CF-like symptoms?

How was this shown experimentally?

Answer 18

the amount of beta-subunit of ENaC present is the rate-limiting step for ENaC activity
the beta-subunit upregulates ENaC function - increasing Na+ absorption and therefore water absorption which decreases the depth of the liquid layer

over-expression of beta-subunit in mice (SCNN1B)
take histological samples of lung tissue shows decrease in the PCL layer and cilia bent over in the mutant
decreased mucous clearance in mutant - CF-like

Question 19

What is the impact of the beta-subunit over-expression in mice in terms of mucous clearance?

What experimental evidence is there to support this?

Answer 19

increased associated mucous plaques and plugs - tubular structure of lungs full with mucous - seen in histological samples

this is associated with a significant decrease in postnatal mortality - study looked at % survival comparing wt and beta-over-expressing mutant
mutation led to over half the mice dying within 28 days of birth
can be said that over-expressing beta-subunit has a major impact on the animal’s ability to sustain life

Question 20

What is the impact of beta-subunit over-expression in mice in terms of bacterial infections?

What experimental evidence is there to support this?

Why is this seen?

Answer 20

beta subunit over-expression causes decrease ability in clearance of bacteria from the lungs

mice injected with either influenza or aeruginosa bacteria and amount of bacteria still present in the lungs was measured after 3 days comparing wt and mutant
wt - cleared almost all bacteria from the lungs
beta over-expressing mutant - very high levels of bacteria present in the lungs

decreased PCL leads to decrease cilia movement and so limited mucous movement and clearance which leads to decreased bacteria clearance