Lecture 6 Atypical CF

Question 1

What percentage of CF patients don’t possess 2 CFTR mutations

Answer 1

1-2% in Northern Europ

Question 2

What is the significance of some patients with CF not having a mutation in both CF alleles yet still having symptoms

Answer 2

CF is recessive and carriers should be asymptomatic. 1 or no CFTR mutation should cause symptoms in these patients

Question 3

Atypical CF usually has less severe symptoms T or F

Answer 3

T

Question 4

What would be the predicted hypothesis for how mutations in ENaC could cause CF-like symptoms

Answer 4

A gain of function in ENaC would cause more Na+ to be absorbed in the airways. This would cause a decrease in the height of the ASL and subsequent CF symptoms

Question 5

Mutations in which ENaC subunits have been identified in atypical CF patients

Answer 5

Α β and γ subunits as well as other genes

Question 6

What are the two ENaC α subunit common genetic backgrounds

Answer 6

T663 is what is normally seen in the ENaC α subunit (wild type) however A663 is a common background seen in the population. A663 doesn’t impact the overall function of ENaC

Question 7

Give some examples of ENaC α subunit gain of function mutations

Answer 7

V114I (in A663 background) L180L-R181W (in A663 background) and W493R (in both backgrounds)

Question 8

Which ENaC α subunit mutations seems to give the most severe gain of function effects

Answer 8

W493R

Question 9

Which ENaC α subunit mutations have been found to be loss of function

Answer 9

F61L and A334T (in the A663 background)

Question 10

What can be said about the total levels of the ENaC α subunit protein in the loss of function and gain of function mutations

Answer 10

Overall protein levels remain the same in both the gain of function and loss of function mutations. Hence there is no change in the total number of channels

Question 11

How could a loss of function in ENaC account for atypical CF symptoms

Answer 11

If there is a loss of ENaC function then decrease Na+ reabsorption could lead to an increase in the height of the PCL. This prevents the cilia from beating as effectively and may result in defects in the ability to clear mucus.

Question 12

What was the effect of amiloride on the nasal potential of patients with classical CF compared to healthy patients why was this the case

Answer 12

There was a much bigger positive shift in nasal potential with amiloride compared to healthy patients. This means that the function of the ENaC channel must be greater in the patients. This is because the CFTR mutant protein can’t supress the function of ENaC thus resulting in a greater ENaC function in CF patients

Question 13

What was the effect on the nasal potential of switching to a Cl- free environment in patients with classical CF compared to healthy patients why was this the case

Answer 13

Low Cl- caused no change in the nasal potential of the classical CF patients. This is because they have no functional CFTR protein hence there will be no effect on extracellular Cl- concentrations on the membrane potential

Question 14

What was the effect of amiloride on the nasal potential of patients with atypical CF compared to healthy patients and those with classical CF why was this the case

Answer 14

There was a bigger positive shift in nasal potential with amiloride compared to healthy patients this was a similar degree as the patients with classical CF. This is most likely through a more direct gain of function mechanism in the ENaC channel itself rather than the effects of mutant CFTR as these patients still retained one wild type copy of the gene

Question 15

What was the effect on the nasal potential of switching to a Cl- free environment in patients with atypical CF compared to healthy patients and those with classical CF why was this the case

Answer 15

Low Cl- a change in the nasal potential reminiscent of the healthy patients. This is because they have retained 50% of functional CFTR protein sufficient for normal functioning of the channel

Question 16

Describe the subunit structure and composition of ENaC

Answer 16

ENaC is a channel consisting of 4 subunits made up of 2 transmembrane domains and an extracellular loop

Question 17

Where is the W493R mutation in the ENaC α subunit found

Answer 17

In the extracellular loop of the subunit

Question 18

Using the data below discuss the impact of the W493R mutation in the ENaC α subunits on the currents generated in both the genetic backgrounds

Answer 18

The αW493R mutation causes increased ENaC currents. These bigger currents can be seen in both ENaC backgrounds

Question 19

What is meant by Na+ feedback inhibition in the ENaC channel

Answer 19

If ENaC channels are opened in the membrane and Na+ goes into the cell it leads to a rise in intracellular Na+. This increase in intracellular Na+ triggers the endocytosis of ENaC from the membrane

Question 20

What would you expect to be the effects of a gain of function mutation on Na+ feedback inhibition

Answer 20

In order to be a gain of function mutation you’d expect a mutation to lead to decreased feedback inhibition and thus less endocytosis of the channel. This would lead to more ENaC channels in the membrane and thus greater Na+ currents

Question 21

How can Na+ feedback inhibition be prevented experimentally

Answer 21

Feedback inhibition can be prevented by low extracellular Na+. With less Na+ outside the cells there is a smaller influx of Na+ into the cells as the driving force for Na+ entry is less

Question 22

What can be said about the surface expression of the α ENaC channel harbouring the W493R mutation

Answer 22

It is the same as wild type hence the same number of channels are present in the membrane

Question 23

How can the degree of endocytosis as a result of Na+ feedback inhibition be measured experimentally

Answer 23

By measuring the ratio of the amiloride-sensitive current in response to high or low extracellular Na+ concentrations

Question 24

Below is some data collected investigating if the W493R mutation in ENaC changed the degree of Na+ feedback inhibition. Discuss what this data shows

Answer 24

The graph shows that the W493R ENaC channel generated bigger currents in both the high and low Na+ extracellular solutions. However the ratio of Iamil in the two solutions was the same as wild type. Hence there was actually the same degree of endocytosis of wild type and mutant channels

Question 25

What can be said about the effects of the W493R mutation in the ENaC α subunit in regards to Na+ feedback inhibition

Answer 25

There was no effect Na+ feedback inhibition is still happing in the mutant to the same degree as wild type

Question 26

What two forms of ENaC are there

Answer 26

Cleaved and uncleaved

Question 27

How do the two forms of ENaC differ in their properties

Answer 27

Uncleaved ENaC is a near silent channel with a low P0. These channels subsequently generate smaller ENaC currents. In contrast cleaved ENaC is very active with a high P0 and generates larger ENaC currents

Question 28

What is the effect of chymotrypsin on ENaC

Answer 28

Chymotrypsin cleaves ENaC increasing the P0 and currents generated

Question 29

Below is the data showing the effects of chymotrypsin on the currents generated by ENaC. Discuss what this data shows

Answer 29

The traces show that for wild type the addition of chymotrypsin increases currents generated. This is assumingly because ENaC is going from its uncleaved to cleaved form which increases the P0. In contrast for the W493R ENaC the addition of chymotrypsin doesn’t increase the current. This implies that the channels are already cleaved (although this is not the case) and that the response to chymotrypsin lost in the mutant.

Question 30

Below is the data collected from the single channel recordings of the wild type ENaC channel before and after chymotrypsin. Discuss the effects of chymotrypsin on these recordings

Answer 30

The addition of chymotrypsin to the wild type ENaC increases the number of channels flickering from open/closed from ~2 to 5. This corresponds to the cleavage of these channels and the increase in open probability. Following chymotrypsin treatment there is an increase in ENaC channels flickering from open and closed represented by the rapid upward/downward deflections. This feature is classic of cleaved ENaC

Question 31

Using the data below compare the effects of adding chymotrypsin to the wild type and W493R channel

Answer 31

Before adding chymotrypsin the channels are open more/for longer (red boxes) than wild type indicating that the P0 must already be higher. The addition of chymotrypsin to W493R ENaC subsequently doesn’t increase the number of channels opening as is seen in the wild type indicating there is no activation of the near silent channels. Hence if promoting the cleavage of ENaC doesn’t increase the flickering of the mutant channels it suggests that the reduced response to chymotrypsin is not due to increased prior cleavage of the channel due to the mutation

Question 32

What is meant by Na+ self-inhibition

Answer 32

This is the phenomena whereby Na+ ions moving into the cell induce the closing of the pore of the channel

Question 33

Draw a current-time curve of the wild type ENaC channel showing the effects of Na+ self-inhibition

Answer 33

See diagram below

Question 34

Use a diagram to illustrate the effects of a loss of Na+ self-inhibition of the ENaC channel

Answer 34

See traces below

Question 35

What are the effects of the W493R mutation on ENaC function

Answer 35

The W493R mutation causes increased ENaC currents. This isn’t due to a change in Na+ feedback nor increased cleavage. What is seen is a loss of Na+ self-inhibition that causes the high currents a greater absorption of water and subsequent CF like symptoms

Question 36

What specific feature of these two current traces shows that the V348M mutation in the ENaC β subunit is a gain of function mutation

Answer 36

There is an increased response to removing amiloride. Hence alleviation of inhibition of ENaC generates bigger currents which in turn means that the activity of ENaC is greater.

Question 37

What is the effect of MTSET on ENaC

Answer 37

MTSET is a sulfhydryl agent that binds to cysteine residues and stabilises ENaC in its open state effectively setting P0 at ~1

Question 38

How can the P0 of the wild type and βV348M ENaC channel be investigated using MTSET

Answer 38

With the rationale that after the addition of MSET the P0 of the channel is 1 the ratio of currents generated before and after MSET is proportional to the ratio of P0 before and after MSET. Ib/Ia = P0b/P0a. Using this equation and the knowledge that Ia = 1 you can measure the currents generated before and after MTSET and rearrange that to calculate Ib

Question 39

How did the two open probabilities of the wild type and βV348M ENaC channels compare from the MTSET experiments

Answer 39

The open probability of the βV348M ENaC channel increased compared to wild type (Wild type P0 ~ 0.24 βV348M P0 ~ 0.33)

Question 40

What did single channel recording reveal about the open probability of the βV348M ENaC channel compared to wild type

Answer 40

Single channel recordings showed that the βV348M ENaC channels were open more often than wild type. Hence the mutant channels were open for more of the time

Question 41

What are the effects of the V348M mutation in the ENaC β subunit

Answer 41

Increased ENaC currents due to an increase in channel P0

Question 42

The V493M mutation in the ENaC α subunit increases the open probability of the channel T or F

Answer 42

T – but by a different mechanism than the V348M β subunit mutation

Question 43

Describe the mouse model of atypical CF and how it models the disease

Answer 43

The mouse model of atypical CF is an overexpression model. The mice are overexpressing the β subunit of the ENaC channel rather than expressing a mutant ENaC α subunit. The β subunit of ENaC acts as rate limiting factor and as such overexpression of this subunit alleviates its rate limiting effects resulting in an increased ENaC activity

Question 44

What is the limitation of the mouse model of atypical CF

Answer 44

It is an overexpression model rather than a mutant model. Overexpression isn’t seen in patients and so may not be representative of what is seen in vivo

Question 45

Give an overview of the symptoms seen in the mouse models of atypical CF

Answer 45

Mice overexpressing βENaC have an enhanced function of ENaC and a decreased height of PCL in bronchus and trachea. This means that the cilia bent over and mucus clearance is also compromised. SNN1B overexpression mice have lots of mucus plagues and plugs and other blockages in the airways. This corresponds to a significant post-natal mortality with just under a 50% survival rate after 28days

Question 46

Which bacterial infection are CF patients extremely vulnerable to

Answer 46

Pseudomonas aeruginosa

Question 47

Mouse models of atypical CF were given intra-tracheal injections of bacteria and the clearance of this bacteria monitored after 3 days. Use the data below to describe the effects of this overexpression model

Answer 47

Wild type mice had cleared the bacteria whereas Β subunit overexpressing mice had significantly high levels of the bacteria still