Lecture 7- Atypical CF Flashcards
Describe ENaC mutations observed in patients
Learning Objective
Discuss the data that supports a change in ENaC function.
Learning Objective
Discuss the data that highlights the altered mechanisms in these mutations
Learning Objective
Using a mouse model describe the likely impact
of mutations on the lungs.
Learning Objective
Atypical CF
- Doesn’t fit the normal profile
- CF symptoms
CFTR mutation determines Severity- Atypical profile
- Mild symptoms
- 1 or no CFTR mutations
- carriers typically no symptoms
Describe the cell model for Atypical CF
APICAL -PCL/ASL - ENAC - CFTR Paracellular water movement BASOLATERAL - Na/K pump - NKCC2 -K channel
Hypothesis
is that other genes are mutated in these patients.
ENac Hypothesis mutations in the airways
- These are gain of function mutations
- More Na absorbed
- Depletion ASL( PCL)
- CF like symptoms
ENac Gene screen
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 many genes are there for ENac ?
3
What are the 3 ENac Genes ?
- SCNN1A
- SCNN1B
- SCNN1G
Impact of Mutations on ENac function -
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
Test in patients-
- 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
Transepithelial potential in normal individual
-15 mv
Testing ENac mutations in patients -
classic
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
Testing ENac mutations in patients - W493R/F508
- 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
Testing ENac mutations in patients -
W493R/E528E
- MILD CFTR ploymophism
-normal CFTR function - only one copy
ENac mutation – big shift in potential with amiloride
GOF Characteristics in ENac mutations
- Excess sodium absorption from liquid layer
- water follows the NA
- drop int the liquid layer height
- mimics what we see in CF
Summary half way through lecture
- Some GoF mutations in ENaC in patients
- These could explain the CF like symptoms seen in these patients.
- Some LoF mutations in ENaC in patients.
- How do these impact leading to CF like symptoms?
W493R mutation – mechanism?
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
follow up study - W493R mutation
overexpression
- bigger function of ENac in the mutant
- amiloride sensitive current hugely enhanced in the mutant - evidence for GOF
Mechanisms of W493R mutation
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 **
GOF has nothing to do with
A lack of endocytosis- Feedback inhibition still taking place
Cleaved Channels - Uncleaved
Near silent
low Po
Generate Smaller ENac currents
Cleaved channels - Cleaved
Very active
High Po
Generate larger ENaC currents
Response to Chymotrypsin
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
Single current recordings
- 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
Sodium Self inhibition
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»_space; gives enhanced magnitude of Na currents
Alpha W493R summary-
- W493R increases ENaC currents
- No change in Na feedback
- High currents not due to increased cleavage
- Loss of Na self inhibition
- High currents, greater water absorption, CF like symptoms.
BetaV348M mutation mechanism of action
- Change in Open probability
- GOF
ENac
Very small single channel conductance so difficult to measure
BV348M - Po Study
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
MTSET
- Maintains ENaC channels in open state
MTSET results-
Amiloride sensitive Short Circuit current - ratio WT Po- 0.24 Mutant Po- 0.33 mutant channel causes an increase in Po
Western Blot Analysis- BV348M
No change in channel number
BV348M Summary
- V348M increases ENaC currents
- No change channel number.
- Increase in channel Po
- High currents, greater water absorption, CF like symptoms.
Mouse Model for Atypical CF- Over expression SCNN1B
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
Significance of the Beta subunit
- Beta subunit expression is the rate limiting step in ENac function
- if you want to increase ENac function you overexpress the Beta subunit
Mouse model - Bacterial clearance
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
Beta subunit summary
- subunit over expression increases Na currents (not shown lecture)
- Depletion ASL
- Increase mucous plugs
- Increase inflammation (not shown lecture)
- Poor bacteria clearance
- CF like symptoms!
