Lecture 7- Atypical CF

Question 1

Describe ENaC mutations observed in patients

Answer 1

Learning Objective

Question 2

Discuss the data that supports a change in ENaC function.

Answer 2

Learning Objective

Question 3

Discuss the data that highlights the altered mechanisms in these mutations

Answer 3

Learning Objective

Question 4

Using a mouse model describe the likely impact

of mutations on the lungs.

Answer 4

Learning Objective

Question 5

Atypical CF

Answer 5

• Doesn’t fit the normal profile

- CF symptoms

Question 6

CFTR mutation determines Severity- Atypical profile

Answer 6

• Mild symptoms
• 1 or no CFTR mutations
• carriers typically no symptoms

Question 7

Describe the cell model for Atypical CF

Answer 7

APICAL 
-PCL/ASL 
- ENAC 
- CFTR
Paracellular water movement 
BASOLATERAL 
- Na/K pump
- NKCC2 
-K channel

Question 8

Hypothesis

Answer 8

is that other genes are mutated in these patients.

Question 9

ENac Hypothesis mutations in the airways

Answer 9

• These are gain of function mutations
• More Na absorbed
• Depletion ASL( PCL)
• CF like symptoms

Question 10

ENac Gene screen

Answer 10

Alpha, beta, gamma
31 patients who were atypical - one or no CFTR mutations
- screened for mutations in the alpha,beta or gamma
- 11 mutations in the alpha subunit
-7 mutations in the Beta subunit
- 8 mutations in the gamma
- predicted that they are GOF

Question 11

How many genes are there for ENac ?

Answer 11

3

Question 12

What are the 3 ENac Genes ?

Answer 12

 - SCNN1A
 - SCNN1B
 - SCNN1G

Question 13

Impact of Mutations on ENac function -

Answer 13

measure Na currents generated in over-expression mutants
- graph shows amiloride sensitive Na current of a percentage of WT

• 3,4,6,7 GOF but not in 8,9,10 showed nothing
• 2 an 5 not GOF show reduced Na currents- unexpected
• no change in number of channels in membrane - suggests the mutation affects open probability

Question 14

Test in patients-

Answer 14

• nasal electrodes - classical CF recording, atypical and normal
• squirt in low chloride solution indicated how much chloride secretion is going on
• then added amiloride- size of shift tells you function of ENac

Question 15

Transepithelial potential in normal individual

Answer 15

-15 mv

Question 16

Testing ENac mutations in patients -

classic

Answer 16

Classic CF - starting potential hyperpolarised – no change when chloride added no open cl channel on apical surface CFTR channels not working – expect to see a bigger response to amiloride which is what we see - shift of -19 mv

Question 17

Testing ENac mutations in patients - W493R/F508

Answer 17

• ENac Mutation
• Not normal but not classic
• Cftr cl channels functional normally no problems with CFTR
• F508 carrier
• Add amiloride 20mv shift in potential similar to classic cystic fibrosis

Question 18

Testing ENac mutations in patients -

W493R/E528E

Answer 18

• MILD CFTR ploymophism
  -normal CFTR function - only one copy
  ENac mutation – big shift in potential with amiloride

Question 19

GOF Characteristics in ENac mutations

Answer 19

• Excess sodium absorption from liquid layer
• water follows the NA
• drop int the liquid layer height
• mimics what we see in CF

Question 20

Summary half way through lecture

Answer 20

1. Some GoF mutations in ENaC in patients
2. These could explain the CF like symptoms seen in these patients.
3. Some LoF mutations in ENaC in patients.
4. How do these impact leading to CF like symptoms?

Question 21

W493R mutation – mechanism?

Answer 21

Found on EC loop of subunit
Previous study - increased ENaC currents
This study confirmed those findings
Bigger function of Enac in mutant 
Amiloride sensitive current is massively enhanced in mutant – GOF

Question 22

follow up study - W493R mutation

Answer 22

overexpression

• bigger function of ENac in the mutant
• amiloride sensitive current hugely enhanced in the mutant - evidence for GOF

Question 23

Mechanisms of W493R mutation

Answer 23

Na Feedback inhibition?!
ENac opens - Na floods into the cell - transient rise in IC Na before Na/K atpase upregulated to get rid of Na
- IC Na increase promotes Endocytosis
- ENac channels endocytosed from the membrane
- If this process affected in the mutant expect to see high Na current
- WT low to high Na currents drop
- Mutant higher but same pattern - drop of current with the high Na shows feedback inhibition is taking place - ***% drop the same just bigger starting point **

Question 24

GOF has nothing to do with

Answer 24

A lack of endocytosis- Feedback inhibition still taking place

Question 25

Cleaved Channels - Uncleaved

Answer 25

Near silent low Po Generate Smaller ENac currents

Question 26

Cleaved channels - Cleaved

Answer 26

Very active High Po Generate larger ENaC currents

Question 27

Response to Chymotrypsin

Answer 27

Wt- increase in current shows increase in function of ENac - ENac cleaved so Po goes up Mutant- Response to Chymotrypsin is lost - BUT turns out Gating of channels has the effect and the higher Po has nothing to do with cleavage

Question 28

Single current recordings

Answer 28

- mutant sustained opening events - different to Wt irrespective of Cleavage - chymotrypsin doesnt have an effect due to the gating difference - number of channels the same though

Question 29

Sodium Self inhibition

Answer 29

Property of ENac - amount of Na going into the cell dictates the Po - when channel opens and Na goes in the high Na at IC reduces channels Po - mutant - influx no decay away - loss of Na self inhibition >> gives enhanced magnitude of Na currents

Question 30

Alpha W493R summary-

Answer 30

1. W493R increases ENaC currents 2. No change in Na feedback 3. High currents not due to increased cleavage 4. Loss of Na self inhibition 5. High currents, greater water absorption, CF like symptoms.

Question 31

BetaV348M mutation mechanism of action

Answer 31

- Change in Open probability | - GOF

Question 32

ENac

Answer 32

Very small single channel conductance so difficult to measure

Question 33

BV348M - Po Study

Answer 33

MTSET used – sulfhydryl agent, binds to cysteine, stabilises open state of channel Therefore stabilises Po at ~1 – measure current before and after MTSET gives an estimate of Po e.g. 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

Question 34

MTSET

Answer 34

- Maintains ENaC channels in open state

Question 35

MTSET results-

Answer 35

``` Amiloride sensitive Short Circuit current - ratio WT Po- 0.24 Mutant Po- 0.33 mutant channel causes an increase in Po ```

Question 36

Western Blot Analysis- BV348M

Answer 36

No change in channel number

Question 37

BV348M Summary

Answer 37

1. V348M increases ENaC currents 2. No change channel number. 3. Increase in channel Po 4. High currents, greater water absorption, CF like symptoms.

Question 38

Mouse Model for Atypical CF- Over expression SCNN1B

Answer 38

PCL down and cilia bent over - PCL heigh reduced in bronchus and Trachea - Mucous clearance also lower in mutant - lots of mucous plugs and plaques also - significant postnatal mortality

Question 39

Significance of the Beta subunit

Answer 39

- Beta subunit expression is the rate limiting step in ENac function - if you want to increase ENac function you overexpress the Beta subunit

Question 40

Mouse model - Bacterial clearance

Answer 40

Given intratracheal injection bacteria clearance of bacteria monitored after 3 days - wt bacteria almost completely gone - mutant - little or no clearance of bacteria from the lungs

Question 41

Beta subunit summary

Answer 41

1.  subunit over expression increases Na currents (not shown lecture) 2. Depletion ASL 3. Increase mucous plugs 4. Increase inflammation (not shown lecture) 5. Poor bacteria clearance 6. CF like symptoms!