L4 CF and Small Molecules (Part 1) Flashcards
How many TMD CFTR
12 - 2 groups of six
What are the important regions of the CFTR protein?
R domain - where phosphorylation occurs
NBD1/2 where nucelotides are able to bind
Describe the pattern of mutations if CFTR
No obvious pattern
NBDs are common places for mutations to be observed
What is a common mutation
F508
How many mutations have the potential to cause CF
over 1200
Describe a type 1 mutation
Null production
mRNA is unstable and no CFTR is made
Describe a type 2 mutation
Trafficking
CFTR is made but not trafficked effectively to the membrane
Example of a type 2 mutation
F508 example
Misfolding leads to degradation
Describe a type 3 mutation
Regulation
Protein is made and gets to the membrane but is not regulated properly e.g. protein doesnt respond to phosphorylation or is unable to be phosphorylated
Describe a type 4 mutation
Gating
Channels don’t open effectively - Po is lower
Describe a type 5 mutation
Partial reduction in mRNA
Some mRNA is present so some is made but this is reduced - reduction in the ammount present in the apical membrane
Describe a type 6 mutation
High turnover of CFTR
Ammount of time CFTR spends in the apical membrrane is reduced
Example of a type 6 mutation
F508 - remoced equikcly from the membrane
What is the normal level for sweat chloride
20mmol
What is the diagnostic threshod for sweat chloride
60mmol
What classes of mutations are very serious leading to a sweat chloride in excess of 100mmol
Classes 1-3
Correlated with a massive loss of function in CFTR
What do class 4-5 mutations tend to be associated with?
Pancreatic insufficiency - failure to secrete digestive enzymes
for the less severe mutations that dont cause diagnostic threshold to be exceeded what tests could be used
Nasal potential difference
Biopsy from the gut - test the response of this to ACh
Do you need 100% function for normal function
No
Carriers asymptomatic with 50% of the protein produced
Where is the f508 mutation found
NBD1
What is the minimum ammount of functional CFTR required for normal function
15%
Describe the channels seen in the upper airway cell
APICAL
ENaC - Na into the cell
CFTR - Cl secretion
BASAL
Na/K ATPase
NKCC1 (Na 2Cl and K all IN)
K+ channels
What is the effect of activation of CFTR on ENaC
CFTR inhibits mucous
Effect on Non functional CFTR
ASL drops - PCL drops
Muco-ciliary clearance is slowed
Thick mucous with bacteria and viruses
CFTR and ENaC _____
INTERACT
CFTR is a regulator of ENaC
CFTR is a regulator of ENaC what does that mean for the case of CF
In CF reduced inhibition of ENaC (seems to be gain of function)
Reduced Cl secretion AND enhanced Na absorption
Alveolar cell model
APICAL
ENaC - Na reabsorption
CF - CL REABSORPTION
BASAL
KCl cotransporter
Na/K ATPase
K channel
Why is Cl reabsorbed in the Alveolar cells
Because KCl cotransporter on the basolateral membrane - sets up a low IC Cl driving Cl reabsrption across the apical membrane
In alveolar cells how does CFTR interact with ENaC
CFTR ACTIVATES ENaC
Why is the height of the ASL important in the alveolar cells
Has to be optimum for gas exchange to occur
What is the effect of CF on the alveolar cells of the lungs
Oedema - fluid in the lungs
Cell model: Distal Sweat Gland
APICAL
ENaC (Na IN)
CFTR (Cl IN)
BASAL
Na/K ATPase
CFTR (ALSO ON BASAL SIDE)
K Channel (OUT)
Describe the formation of Sweat in the distal sweat glands
CFTR independent secretion of CL into the lumen of the sweat gland
Absorption of Na and Cl from the lumen
Water follows
Describe why CF patients have salty sweat
Failure to get Cl absorption from the sweat
ENaC not activated
What is the interaction between CFTR and ENaC in the distal sweat gland
CFTR ACTIVATES ENAC
What are the driving forces seen in the distal sweat gland
One for Cl to move through cFTR on the apical membrane
ANOTHER one for Cl to move across the basolateral membrane through CFTR
Why is antibiotic resistance common in CF sufferers
Suffer from frequent infections
What do all of the CF treatments have in common
All of them treat the symptoms
Ivacaftor is a
Potentiator
Lumacaftor is a
Corrector
Elexacator is a
Corrector
Tezacaftor is a
Corrector
Orkambi contains
Ivacaftor and lumacaftor
Ivacaftor alone used to treat
G551D
Orkambi used to treat
F508 homozygotes
What is a potentiator used for
To increase the Po
Used to treat gating mutations
What is a corrector used for
Traffic the mutant protein to the membrane and increase the N number
What was the intial step in vertex pharmaceeticals to find CF drigs
100’000 drugs were screened
FIRST CLINICAL TRIAL FROM LECTURE
Vx-770/Ivacaftor for a G551D mutant
What is forskolin added for
Stimulate CFTR
Stimulation of PKA - phosphorylation of CFTR
What is the effect on SCC in G551D of FSK
Unstimulated WT has a basical level which is heavily increased by presence of FSK
In G551D initial level much lower, less of an increase in presence of FSK
What is the effect of VX-770 (lumacaftor) on the ISC of G551D cells
Increase above WT unstimulated in the presence of FSK
Describe the trace seen when adding FSK, VX-770 then CFTR inhibitor to the G551D cells
What was performed after this?
Add FSk, small increase, add VX-770 = masive increase, removed when CFTR inhibitor is applied
Performed in a different order - VX-770, FSK and CFTRinhibotor
What is the function of single channel recordings
Investigate Po
Descibe what seen in the single channel recordings for patients with G551D
In mutant not as many channel opening - less frequent
What was seen in the single channel recordings when the cells were exposed to ivacaftor
Many more deflections seen - increase in Po
Recovery?
When looking at short circuit currents from CFTRwhy add amiloride
ENaC would contaminate the recordings
Describe what was seen in the short circuit recordings when ivacaftor added
Increase in current which increases as the concentration of ivacaftor is increased
Describe the experimental conditions when looking at the effect of ivacaftor of the ASL
Describe the results seen
ASL starts very high and then drops to optimum level
VIP added - stimulates PK-A –> CFTR
G551D alone with VIP - ASL falls way too low
With ivacaftor present height is shifted closer to the height of the wildtype
Describe the experimental conditions used to look at the effect of ivacaftor on the cililary beat frequency
What were the results seen
DMSO used as a control - VIP added
Without any VX-770 CBF was very low
Vx770 increased the CBF but with VIP and VX770 the CBF was brought closest to the wild type levels
CBF
Ciliary beat frequncy
Ivacaftor clinical trials were
Randomised, double blind clinical trials
Ivacaftor clinical trials stats
Sufferers with at least the 1 G551D mutation
Lasted 48 weeks
What was the doseing protocol for the ivacaftor trials
150mg every 12 hours
How was change in FEV1 value expressed in the ivacaftor clinical trial data
AS CHANGE OF % PREDICTED
Positive value - closer to predicted and this is good
Describe the effect of ivacaftor on lung function
Within two weeks 10% imporvement
Describe the effect of ivacaftor on the number of pulmonary exacerbation events
Decrease in the proportion of the number of subjects who had an event of pulmonary exacerbation
Describe the effect of ivacaftor on sweat NaCl
When taking ivacaftor sweat Cl drops BELOW THE CLINICAL TRESHOLD
Describe the effect of ivacaftor on nasal potential difference
Increase in the concentration of ivacafor lead to an improvement of change in the baseline nasal epitheliun
What is the effect of using isoproterenol
It is a beta agonist
Which activates CFTR through cAMP
