L7 Atypical CF Flashcards

1
Q

What are the two types of cystic fibrosis

A

Classical and atypical

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2
Q

Stats regarding atpyical CF

A

Mild symptoms observed with only 1 or NO CFTR mutations

In Northern Europe - small incidence of two mutations and no symptoms

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3
Q

Upper airway cell model

APICAl

A

ENaC - Na absorption

CFTR - Cl secretion

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4
Q

Upper airway cell model

A

NKCC1
Na/K ATPase
K Channel - recycle K to allow function of NKCC1

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5
Q

What is the effect of increased Na absorption

A

Bring down the height of ASL - just in the CFTR mutants

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6
Q

Normal subunit composition of ENaC

A

abg(amma)

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7
Q

Where can mutations occur in ENaC

A

In all three o the mutants

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8
Q

How many ENaC mutations have been screened

A

45 different mutations

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9
Q

What is a common ENaC variant

A

A663 - no effect on function

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10
Q

What is an effect of the A663 mutation

A

This mutation predisposes to other mutations

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11
Q

How were the EnaC mutants screened

A

OE express a number of common ENaC mutations and record the amiloride sensitive Na current

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12
Q

What were the results of the ENaC mutant screening

A

Some - size of the current is exactly the same so unlikely this mutation is having any affect on function

Some there an increase in the function - could cause symptoms

Two there is a loss of function
- height of the ASL would increase

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13
Q

What is the activity of ENaC in classical CF - why is this

A

ENaC enhanced

No longer inhibition of ENaC

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14
Q

Where does the aW493R occur

A

In the effector loop of one of the subunits

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15
Q

Describe the trace seen for aW493R compared to the wildtype

A

When amiloride removed greater current seen in the mutant compared to the wildtype

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16
Q

In the aW493R mutant is there a difference on the T/A backgrounds

A

No effect on these results

17
Q

What is Na feedback inhibition

A

As Na moves into the cells and [Na] increases

This promotes the endocytosis of ENaC from the membrane

18
Q

What is the question asked regarding Na feedback inhibition

A

Is there a change in this in mutants

Does the mutations prevent the endocytosis of ENaC

19
Q

Describe how the question of the mutations affecting Na feedback inhibition was adressed

A

Use ECF of low Na - Na still able to move in but endocytosis prevented

20
Q

Does aW493R affect Na feedback inhibition

A

Comparing low and high Na
Decreased currents with high Na outside
This is also seen in the mutant
Percentage drop is similar low to high

21
Q

Describe how else ENaC channel activity may be activated

A

Cleavage by proteases to increase the Po

22
Q

Two classes of cleavage

A

Uncleaved

Cleaved

23
Q

Describe the uncleaved channels

A

Near silent
Low Po
Generate smaller currents

24
Q

Describe the cleaved channels

A

Very active
High Po
Generate larger currents

25
Q

How is cleavage investigated?

A

Looking at the response to chemotrypsin

26
Q

Describe the response of the wildtype channels to chemotrypsin

A

Near silent channels now have a much increased Po

From one open to 5

27
Q

Describe the response of the mutant channels to chemotrypsin

A

Already have a high Po which is unchaged

28
Q

Conclusions drawn form the chemotrypsin expts

A

No activation of the near silent channels in the mutant. Reduced response to chymotrypsin not due to increase endogenous cleavage before chymotrypsin is added

Channels are resistant to cleavage but this doesnt have an effect

29
Q

What is Na self inhibition

A

That the Na moving through the pore actuall blocks the pore reducing the size of current flow

30
Q

When stepping from 1mM Na to 110 mM Na what is the effect seen in WT and the mutants

A

WT peaks and then quickly declines due to Na self inhibition
Mutant - peak current is much biggger and dont get the peak followed by inhibition
Higher Po because Na going through the pore doesnt block more Na moving throguh

31
Q

What is a ENaC mutation found in the beta subunit

A

BV348M

32
Q

What is the effect of BV348M on current

A

Steps up from 4uA to 5.5uA

33
Q

Describe how the Po of the BV348M-ENaC channels was measured

A

Measure the amiloride sensitive whole cell currents - add MSET this stabilises the open state of ENaC by binding to cystenine the open state of ENaC
We assume this gives a Po of 1
Compare current before and after MTSET addition to work out the apparent Po

34
Q

In order to measure Po using MTSET what must first be done

A

Have to make a background cysteine mutant - to allow MTSET to bind

35
Q

What is the effect of BV348M on Po

A

WT Po of .24
BV348M Po of .33

So Po of this mutant channel (BV348M) has increased

36
Q

Descrbe what was seen in single channel recordings of WT and BV348M mutants

A

BV348M Increased Po - enhanced function

37
Q

What was seen in a western blot of WT and BV348M cells

A

No signigicant different

No change in N number

38
Q

With atypical mutations more likely to affect ___ than ____

A

Po than N number

39
Q

Describe the effects of overexpressing SCNN1B (B subunit)

A

Decreased height of the PCL
Mucous clearance is decreased
Mouse mutants die sooner - due to problems with mucociliary clearance - plugging the airways