L7 Atypical CF

Question 1

What are the two types of cystic fibrosis

Answer 1

Classical and atypical

Question 2

Stats regarding atpyical CF

Answer 2

Mild symptoms observed with only 1 or NO CFTR mutations

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

Question 3

Upper airway cell model

APICAl

Answer 3

ENaC - Na absorption

CFTR - Cl secretion

Question 4

Upper airway cell model

Answer 4

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

Question 5

What is the effect of increased Na absorption

Answer 5

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

Question 6

Normal subunit composition of ENaC

Answer 6

abg(amma)

Question 7

Where can mutations occur in ENaC

Answer 7

In all three o the mutants

Question 8

How many ENaC mutations have been screened

Answer 8

45 different mutations

Question 9

What is a common ENaC variant

Answer 9

A663 - no effect on function

Question 10

What is an effect of the A663 mutation

Answer 10

This mutation predisposes to other mutations

Question 11

How were the EnaC mutants screened

Answer 11

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

Question 12

What were the results of the ENaC mutant screening

Answer 12

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

Question 13

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

Answer 13

ENaC enhanced

No longer inhibition of ENaC

Question 14

Where does the aW493R occur

Answer 14

In the effector loop of one of the subunits

Question 15

Describe the trace seen for aW493R compared to the wildtype

Answer 15

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

Question 16

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

Answer 16

No effect on these results

Question 17

What is Na feedback inhibition

Answer 17

As Na moves into the cells and [Na] increases

This promotes the endocytosis of ENaC from the membrane

Question 18

What is the question asked regarding Na feedback inhibition

Answer 18

Is there a change in this in mutants

Does the mutations prevent the endocytosis of ENaC

Question 19

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

Answer 19

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

Question 20

Does aW493R affect Na feedback inhibition

Answer 20

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

Question 21

Describe how else ENaC channel activity may be activated

Answer 21

Cleavage by proteases to increase the Po

Question 22

Two classes of cleavage

Answer 22

Uncleaved

Cleaved

Question 23

Describe the uncleaved channels

Answer 23

Near silent
Low Po
Generate smaller currents

Question 24

Describe the cleaved channels

Answer 24

Very active
High Po
Generate larger currents

Question 25

How is cleavage investigated?

Answer 25

Looking at the response to chemotrypsin

Question 26

Describe the response of the wildtype channels to chemotrypsin

Answer 26

Near silent channels now have a much increased Po | From one open to 5

Question 27

Describe the response of the mutant channels to chemotrypsin

Answer 27

Already have a high Po which is unchaged

Question 28

Conclusions drawn form the chemotrypsin expts

Answer 28

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

Question 29

What is Na self inhibition

Answer 29

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

Question 30

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

Answer 30

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

Question 31

What is a ENaC mutation found in the beta subunit

Answer 31

BV348M

Question 32

What is the effect of BV348M on current

Answer 32

Steps up from 4uA to 5.5uA

Question 33

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

Answer 33

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

Question 34

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

Answer 34

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

Question 35

What is the effect of BV348M on Po

Answer 35

WT Po of .24 BV348M Po of .33 So Po of this mutant channel (BV348M) has increased

Question 36

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

Answer 36

BV348M Increased Po - enhanced function

Question 37

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

Answer 37

No signigicant different | No change in N number

Question 38

With atypical mutations more likely to affect ___ than ____

Answer 38

Po than N number

Question 39

Describe the effects of overexpressing SCNN1B (B subunit)

Answer 39

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