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Epithelial Physiology > L7 - Atypical CF > Flashcards

L7 - Atypical CF Flashcards

1
Q

What is classical CF?

A

Severe symptoms
2 mutations in CFTR
- Normally delta F508 and one other

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2
Q

What is atypical CF?

A

Mild symptoms
1 or no CFTR mutations
1-2% northern European - 2 mutations not observed

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3
Q

What other mutated genes could be present in atypical CF?

A

Might be other mutated genes - modifier genes

  • Might code for proteins that regulate CFTR
  • Might play a role in how the airway cell works normally
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4
Q

What are the two reasons the height of the ASL is lower in CF patients?

A

No Cl secretion
GOF mutations in ENaC
- More Na+ absorbed –> reduced ASL –> CF like symptoms

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5
Q

Over many ENaC gene screens in CF patients overall what was discovered about the mutations?

A

A range of mutations observed

  • Some of these had a greater incidence than in controls
  • Some polymorphisms were observed in controls
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6
Q

What factor did they monitor when looking at the over expression of a range of different ENaC mutations?

A

Amiloride sensitive Na current as a % of the control

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7
Q

What are the two different common variants patients can have?

A

A663 or T663

  • Two different amino acids that people can have – different backgrounds
  • Do not impact ENaC function
  • Looked at if having a different background impacted ENaC if you have another mutation
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8
Q

Results of looking at the over expression of a range of different ENaC mutations?

A

In some mutations - size of current the same – mutation unlikely to be underlying symptoms in patients
In some mutations – size of current increased –> function of ENaC enhanced
In 2 mutations – size of current decreases –> function of ENaC decreased

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9
Q

If the size of the amiloride sensitive Na current increases when overexpressing an ENaC mutation what does this show?

A

Gain of function

What you would expect from symptoms

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10
Q

If the size of the amiloride sensitive Na current decreases when overexpressing an ENaC mutation what does this show?

A

Loss of function –> less Na absorption –> height of ASL increases
CF like symptoms as height of ASL too high (rather than too low like normal)

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11
Q

Normal individual nasal transepithelial potential difference, Cl current, amiloride current

A

PD = -15.2
Cl = 16.3
Amilroide - 6.5

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12
Q

Classic CF patient nasal transepithelial potential difference, Cl current, amiloride current

A 
PD = 31.1
Cl = 0.08 (decreased)
Amiloride = 19.4 (increased)
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13
Q

One CF mutation and one ENaC mutation patient nasal transepithelial potential difference, Cl current, amiloride current

A 
PD = -25
Cl = -15 
Amiloride = 2- (increased)

alphaW493R – GOF ENaC mutation
F508del – CFTR mutation

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14
Q

What does nasal transepithelial potential difference, Cl current, amiloride current show?

A

Amiloride – change in potential indicates function of ENaC

Isotprotenolol – stimulates Cl handling – indicates CFTR function

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15
Q

What is the alphaW493R mutation and where is it found?

A

Found on extracellular loop of subunit

Previous study showed the mutation increased ENaC currents

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16
Q

What did the results with the alphaW493R mutation and amiloride show?

A

Currents greater in absence of amiloride

Currents greater in W493R mutant (mutation in ENaC alpha subunit)

17
Q

What two things could cause greater currents in the alphaW493R mutant?

A

Number of channels – not this

Open probability of channels

18
Q

What does Na feedback inhibition lead to?

A

Works to promote endocytosis of ENaC from membrane
ENaC opens –> rise in intracellular Na –> promotes endocytosis of ENaC
from membrane

19
Q

How did they test if Na feedback inhibition was inhibited in alphaW493R mutant?

A

Tested by activating the Na channels with low extracellular Na
If extracellular Na is low –> little Na entering cell –> low intracellular Na –> little ENaC endocytosis triggered –> then go to high extracellular Na

Certain % of ENaC channels will be removed from membrane
Compare low Na currents with high Na currents
- Get idea of how much endocytosis occur
Do this with wild-type and mutant and see if % are similar
- Should be less endocytosis in mutant

20
Q

What were the results of Na feedback inhibition experiments on the alphaW493R mutant?

A

With high Na - more Na does enter but it quickly pulls ENaC out of membrane so currents soon drop
In mutant
- Na feedback inhibition is still occurring
- % drop is very similar between WT and mutant
- Starting surface expression unchanged
Therefore must be channel open probability affecting currents

21
Q

What are the two populations of ENaC channels?

A 
Uncleaved
- Near silent
- Low Po
- Generate smaller ENaC currents
Cleaved 
- Very active
- High Po
- Generate larger ENaC currents
22
Q

How did they test if the alpha W493R mutant increased the likelihood of cleavage?

A

Tested the response to chymotrypsin

23
Q

What were the results of the chymotrypsin experiment to investigate cleavage?

A

WT –> when chymotrypsin added increased cleaved channels –> increased open probability (1 –> 5)

Mutant

  • In absence of chymotrypsin already higher open probability
  • When chymotrypsin added no effect
24
Q

What did the results of the chymotrypsin experiment to investigate cleavage show?

A

Chymotrypsin cannot activate near silent channels - resistant to cleavage
Not the cleavage that is increasing the open probability
- Something else has already increased the open probability
Reduced response to chymotrypsin not due to increased endogenous cleavage before chymotrypsin added

25
Q

Overall what was the alpha W493R mutation doing to channel cleavage?

A

Mutation in ENaC was making the channels resistant to cleavage

26
Q

What is Na self-inhibition?

A

Where Na entering the cell blocks the pore of the channel –> reduces current flow

27
Q

What did the results of Na self inhibition with the alpha W493R mutation show?

A 
WT 
- 1mM to --> 110 mM 
- Decays to a new steady state - Na self-inhibition 
Mutant 
- 1mM to --> 110 mM 
- No decay/dip 
- Na self-inhibition is not occurring 
- This allows a bigger increase in currents --> higher open probability
28
Q

Overall, how was the alpha W493R mutation increasing open probability?

A

By blocking Na self inhibition

29
Q

What is the beta V348M mutation?

A

ENaC GOF mutation

30
Q

How did they look at the open probability of the beta V348M mutation?

A

Measured amilroide sensitive whole cell currents before adding MTSET

31
Q

What is MTSET?

A

Sulfhydryl agent

Binds to cysteine

32
Q

What does MTEST do to the ENaC channel?

A

Stabilises open state of channel –> ENaC will have an open probability of 1
Measure current before and after MTSET gives an estimate of Po

33
Q

Example calculation of current before and after MTSET

A

Amiloride sensitive current before MTSET = 12 µA
Amiloride sensitive current after MTSET = 20 µA
Ratio = 12/20 = 0.6
Apparent Po of channels = 0.6
Have to make an assumption that the number of channels isn’t changing

34
Q

Why did they have to modify ENaC to have a cysteine background when using MTSET to look at open probability?

A

Had to be used to ensure MTSET worked – it binds to cysteine

35
Q

How did they confirm that there was no change in beta ENaC channel number to prove it was changes in open probability affecting Na currents?

A

Western blot analysis

36
Q

What was the ASL like in a atypical CF mouse model (overexpression of beta ENaC subunit)?

A

Height of ASL reduced and cilia bent
- Mucous clearance reduced
- Leads to mucous plaques and plugs
28 days post birth - only 50% survival

37
Q

What did a slice through the lung of an atypical CF mouse model look like?

A

Mucous plugs

38
Q

When an atypical CF mouse model was given intra-tracheal injection of bacteria what happened?

A

Clearance of bacteria monitored after 3 days
Reduced clearance in atypical CF model
Increases chance of death

39
Q

Which of the following is correct when thinking about the causes of all forms of CF?

  • LOF in CFTR
  • LOF in ENaC
  • GOF in CFTR
  • GOF in ENaC
A
  • LOF in CFTR
  • LOF in ENaC
  • GOF in ENaC

Epithelial Physiology (10 decks)

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