Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

BMS 346- Epithelial physiology in health and disease > Lecture 7- Atypical CF > Flashcards

Lecture 7- Atypical CF Flashcards

1
Q

Describe ENaC mutations observed in patients

A

Learning Objective

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

Discuss the data that supports a change in ENaC function.

A

Learning Objective

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

Discuss the data that highlights the altered mechanisms in these mutations

A

Learning Objective

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Using a mouse model describe the likely impact

of mutations on the lungs.

A

Learning Objective

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Atypical CF

A
  • Doesn’t fit the normal profile

- CF symptoms

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

CFTR mutation determines Severity- Atypical profile

A
  • Mild symptoms
  • 1 or no CFTR mutations
  • carriers typically no symptoms
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Describe the cell model for Atypical CF

A 
APICAL 
-PCL/ASL 
- ENAC 
- CFTR
Paracellular water movement 
BASOLATERAL 
- Na/K pump
- NKCC2 
-K channel
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Hypothesis

A

is that other genes are mutated in these patients.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

ENac Hypothesis mutations in the airways

A
  • These are gain of function mutations
  • More Na absorbed
  • Depletion ASL( PCL)
  • CF like symptoms
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

ENac Gene screen

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

How many genes are there for ENac ?

A

3

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What are the 3 ENac Genes ?

A

 - SCNN1A
 - SCNN1B
 - SCNN1G

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Impact of Mutations on ENac function -

A

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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Test in patients-

A
  • 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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Transepithelial potential in normal individual

A

-15 mv

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Testing ENac mutations in patients -

classic

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

Testing ENac mutations in patients - W493R/F508

A
  • 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
18
Q

Testing ENac mutations in patients -

W493R/E528E

A
  • MILD CFTR ploymophism
    -normal CFTR function - only one copy
    ENac mutation – big shift in potential with amiloride
19
Q

GOF Characteristics in ENac mutations

A
  • Excess sodium absorption from liquid layer
  • water follows the NA
  • drop int the liquid layer height
  • mimics what we see in CF
20
Q

Summary half way through lecture

A
  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?
21
Q

W493R mutation – mechanism?

A 
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
22
Q

follow up study - W493R mutation

A

overexpression

  • bigger function of ENac in the mutant
  • amiloride sensitive current hugely enhanced in the mutant - evidence for GOF
23
Q

Mechanisms of W493R mutation

A

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 **

24
Q

GOF has nothing to do with

A

A lack of endocytosis- Feedback inhibition still taking place

25
Q

Cleaved Channels - Uncleaved

A

Near silent
low Po
Generate Smaller ENac currents

26
Q

Cleaved channels - Cleaved

A

Very active
High Po
Generate larger ENaC currents

27
Q

Response to Chymotrypsin

A

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

28
Q

Single current recordings

A
  • 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
29
Q

Sodium Self inhibition

A

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&raquo_space; gives enhanced magnitude of Na currents
30
Q

Alpha W493R summary-

A
  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.
31
Q

BetaV348M mutation mechanism of action

A
  • Change in Open probability

- GOF

32
Q

ENac

A

Very small single channel conductance so difficult to measure

33
Q

BV348M - Po Study

A

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

34
Q

MTSET

A
  • Maintains ENaC channels in open state
35
Q

MTSET results-

A 
Amiloride sensitive Short Circuit current 
- ratio 
WT Po- 0.24 
Mutant Po- 0.33
mutant channel causes an increase in Po
36
Q

Western Blot Analysis- BV348M

A

No change in channel number

37
Q

BV348M Summary

A
  1. V348M increases ENaC currents
  2. No change channel number.
  3. Increase in channel Po
  4. High currents, greater water absorption, CF like symptoms.
38
Q

Mouse Model for Atypical CF- Over expression SCNN1B

A

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
39
Q

Significance of the Beta subunit

A
  • Beta subunit expression is the rate limiting step in ENac function
  • if you want to increase ENac function you overexpress the Beta subunit
40
Q

Mouse model - Bacterial clearance

A

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
41
Q

Beta subunit summary

A
  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!

BMS 346- Epithelial physiology in health and disease (13 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions