Lecture 08 Atypical Cystic Fibrosis Flashcards
Compare atypical and classical cystic fibrosis
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
What could account for the differences seen in mutation severity?
modifier genes
If not CFTR, what was hypothesised to cause atypical CF?
What sort of mutation?
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
What subunit if the ENaC GoF mutation in?
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)
How was the impact of mutations on ENaC function studied?
What GoF ENaC mutations were identified?
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)
What is the impact of different mutations in terms of chloride secretion and amiloride on ion channel function?
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
How did the researchers confirm that the alpha-W493R GoF mutation was affecting ENaC?
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
What are the common variants of sodium channels?
What is the impact of this?
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
How does Sodium Feedback Inhibition work?
- ENaC is in the membrane and opens to allow sodium influx into the cell, increasing the intracellular [Na+]
- 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+]
What is the impact of the alpha-W493R mutation on Sodium Feedback Inhibition?
What experimental evidence is there for this?
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
How does cleavage affect ENaC?
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
Give an example of a protease that cleaves ENaC
Chymotrypsin
Does the alpha-W493R mutation lead to a higher proportion of cleaved ENaC?
What is the experimental evidence for this?
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
What is Sodium Self Inhibition?
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
What is the impact of sodium self inhibition on ENaC?
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
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?
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
Does the beta-V348M mutation affect the number of channels at the membrane?
How was this confirmed experimentally?
no change in number of channels at the membrane
Western Blot Analysis
Why does over-expressing the beta-subunit lead to CF-like symptoms?
How was this shown experimentally?
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
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?
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
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?
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
