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A BMS346 Epithelial Physiology > CF and small molecules I > Flashcards

CF and small molecules I Flashcards

1
Q

What is the CF gene product?

A

A Cl channel in the apical membrane of a variety of different epithelial cells

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2
Q

What is the structure of the CFTR channel?

A

12 transmembrane spanning domains:

  • 2 groups of 6
  • Regulatory domain
  • 2 nucleotide binding domains
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3
Q

Where is the regulatory domain in the CFTR channel found?

Why is this domain important?

A

Between each group of 6

It is the site at which the CFTR is PHOSPHORYLATED by PKA

Plays an important role in the GATING of the channel

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4
Q

What are the nucleotide binding domains in CFTR and where are they found?

A

NBD1 and NBD2

Plays an important role int he gating of the channel

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5
Q

Describe the mutations that cause CF

A

> 1200 mutations that have the potential of causing CF

With NBD1 and NBD2 being HOTSPOTS for mutations

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6
Q

What is the most COMMON mutation that leads to CF?

What is the allelic frequency?

A

Δ (Delta) F508 mutation in NBD1

70-90% of individuals with CF have this mutation

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7
Q

What are the different classes of CF mutations?

A

6 different classes

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8
Q

How are different CF mutations grouped into different classes?

A

Depends on the impact of the mutation on the protein and mRNA (impact on the CFTR channel)

One mutation may belong to more than one class

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9
Q

Describe class I of CF mutations

A

NULL mutation:

  • mRNA produced is broken down
  • No CFTR protein is made
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10
Q

Describe class II of CF mutations

A

TRAFFICKING mutation:
- CFTR protein is made but not trafficked to the apical membrane

  • Eg. protein may be missfolded and targeted for degradation before reaching the membrane
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11
Q

Describe class III of CF mutations

A

REGULATION mutation:
- Protein is made and gets to the membrane but is not regulated appropriately

(Normally activated by PKA mediated phosphorylation)

BUT, protein doesn’t respond/phosphorylation doesn’t occur

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12
Q

Describe class IV of CF mutations

A

CONDUCTION mutation:
- Gating mutation - channel doesn’t open effectively as should do

  • Regulation may be normal but the protein doesn’t respond normally
  • Po of the channel is lower
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13
Q

Describe class V of CF mutations

A

PARTIAL REDUCTION mutation:

- mRNA is made but the amount is reduced compared to normal –> amount of protein produces is lower than control

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14
Q

What mutation class does the F508 mutation belong to?Why?

A

Class II:
- Missfolded protein due to mutation –> targeted for degradation instead of trafficking to the membrane

Class VI:
- One in the membrane is removed quicker than normal

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15
Q

What mutation class does the F508 mutation belong to?Why?

A

Class II:
- Missfolded protein due to mutation –> targeted for degradation instead of trafficking to the membrane

Class VI:
- One in the membrane is removed quicker than normal

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16
Q

What determines the severity of CF in patients?

A

The type of mutation

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17
Q

What is used as a measurement of CF severity?

A

Sweat Cl concentration

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18
Q

What is the normal sweat Cl concentration?

A

Around 20mm/L

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19
Q

What is the CF diagnostic sweat Cl concentration? (That allows the diagnosis of CF)

A

60mm/L

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20
Q

What are the severe CF mutations?

What do these mutations correlate with?

A

Class I-III mutations

Correlate with:

  • High sweat Cl concentration
  • Low CFTR protein function
  • Pancreas not functioning
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21
Q

What is the SC concentration of severe mutations?

A

SC concentration - 100mm/L

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22
Q

In what mutations do patients have pancreatic SUFFICIENCY?

A

Classes IV and V

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23
Q

Are classes IV and V above or below the clinical threshold?

A

Above (SC conc is around 70mm/L)

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24
Q

Why are very severe mutations in CF (I-III) associated with poor pancreatic sufficiency?

A

In severe mutations there is no secretion of the digestive enzymes from the pancreas

Due to the role of CFTR in BICARBONATE secretion

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25
Q

What is the function of CFTR in classes I-III?

A

0%

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26
Q

What is the function of CFTR in classes IV and V?

A

10%

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27
Q

What is CBVAD?

A

Condition with 2 CF mutaitons

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28
Q

Describe the CBVAD condition

A

Rarer and less severe

Patients BELOW the diagnostic threshold

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29
Q

Describe the sweat test conclusion for CBVAD

Why?

What might be done next?

A

Inconclusive

Patients are BELOW the diagnostic threshold but SC concentrations are still very high

Alternative tests may be done next:

  • Genetic testing
  • Nasal potential difference testing
  • Biopsy of the gut (see if the response to acetyl choline is lost)
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30
Q

Why is an inconclusive swear test not good?

A

My not have access to treatments for CF

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31
Q

What % of CFTR normal function in carries?

A

50% normal function of the protein because 50% normal protein

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32
Q

What % of normal function is needed for the normal function of the CFTR channel?

A

About 15%

33
Q

What happens in the cell if CFTR is non-functional/has a reduced function in the UPPER AIRWAY?

A
  • Less water secretion BETWEEN the cells
  • Height of the ASL DROPS
  • Cilia bend over
  • Mucocillary clearance is not normal/is slowed
  • THICK mucus with bacteria and viruses

–> Resulting in airway problems and increased risk of infection

34
Q

How do CFTR and ENaC interact in the UPPER AIRWAY?

A

When CFTR is ACTIVE ENaC is INACTIVE and vice versa

35
Q

What sets the height of the ASL? (Upper A)

A

The BALANCE between Na absorption (ENaC function) and Cl secretion (CFTR function) across the apical membrane of the cell

36
Q

What happens to ENaC when CFTR is upregulated? (Upper A)

A

Inactivated by the active CFTR

37
Q

What happens to ENaC when CFTR is non-functional? (Upper A)

A
  • Inhibition of ENaC by CFTR is relieved
  • ENaC has a gain of function
  • ENHANCED Na absorption
  • Exacerbates the impact on the ASL
  • Height of the layer drops further
38
Q

How is Na and Cl handling different in the alveolar compared to upper airway cells?

Why?

A

1) It is REVERSED:
- Cl moves INTO the cell through CFTR

Due to a KCl CO-transport protein on the basolateral membrane that transports K and Cl OUT of the cell (takes Cl out dependant on the K gradient)

  • -> Intracellular Cl is LOW
  • -> Driving force for Cl to move INTO the cell
  • -> Net ABSORPTION of Cl from the ASL

2) CFTR ACTIVATES ENaC

39
Q

What is the height of the ASL in the alveolar optimum for?

A

Gas exchange

40
Q

What is CF associated with in the alveoli?

What does this compromise?

A

Alveolar odema (increases fluid)

Compromised gas exchange (ASL is increased in heigh, no longer optimum)

41
Q

Why is salty sweat a characteristic of CF?

A

Due to the role of CFTR channels in absorbing Cl ions from the human swear gland

42
Q

Describe NORMAL Cl handling in the sweat gland

A
  • Cl is secreted into the lumen of the sweat gland INDEPENDENTLY of CFTR
  • Distal sweat gland absorbs Cl and Na from the sweat being produced
  • Whatever is left in the lumen is excreted as sweat
43
Q

Why is Cl secretion into the lumen of the sweat gland in CF patients normal?

What is different in Cl handling in the sweat gland in CF patients?

A

Because CL –> lumen of the sweat gland is CFTR INDEPENDENT

Instead, reduced reabsorption of Na and Cl from the lumen of the distal sweat gland
–> High levels of NaCl lost in the sweat

44
Q

Where is the CFTR channel present in the distal sweat gland?

A

In the apical AND the basolateral membrane

45
Q

How is Cl reabsorbed in the distal sweat gland?

A

Lower concentration in the cell compared to the apical membrane

Lower concentration at the basolateral membrane compared to inside the cell

46
Q

What is the problem with the current CF treatments?

A

They all treat the SYMPTOMS and not the genetic cause (eg. mycolytics, bronchodilators, pancreatic enzymes)

47
Q

What are the new ideas for treatment for CF?

A

Small molecules

48
Q

What are some of the clinically approved drugs (small molecules) to treat CF?

A
  • Ivacaftor
  • Ivacaftor/Lumacaftor (Orkambi)
  • Ivacafor/Tezacaftor
  • Ivacaftor/Tezacaftor/Elexacaftor
49
Q

What type of molecule is Ivacaftor?

A

A POTENTIATOR - increases the Po of the channels

50
Q

What mutation type is Ivacaftor useful in?

Example?

A

Useful in mutations with gating defects

G551D mutation

51
Q

How common is the G551D mutation?

A

Relatively common

15% of patients have this mutation

52
Q

What are type of molecules are Lumacaftor, Tezacaftor and Elexacaftor?

A

CORRECTORS - traffics the mutant protein to the membrane

53
Q

What small molecule treatments are used to treat F508 HOMOzygote mutations?

A

1) Ivacafor and Lumcafor (Orkambi)
2) Ivacaftor and Tezacaftor
3) Ivacaftor, Tezacaftor and Elexcaftor

54
Q

What small molecule treatments are used to treat F508 HETEROzygote mutations?

A

1) Ivacaftor and Tezacaftor

2) Ivacaftor, Tezacaftor and Elexcaftor

55
Q

What was the original name for Ivacaftor?

A

VX-770

56
Q

What happens in unstimulated SCC in WT CFTR expressing rat thyroid cells?

A

SCC - 7microAmpscm2

57
Q

What happens to the WT CFTR expressing rat thyroid cells when stimulated with forskolin?

Why?

A

SCC increase dramatically (to 50microAmpscm2

Why?
- Forskolin activates PKA

  • PKA phosphorylates CFTR –> up regulates CFTR function and therefore Cl secretion
58
Q

What happens in unstimulated SCC in G551D mutant CFTR expressing rat thyroid cells?

Why?

A

SCC is lower than the WT (2.5microAmpscm2)

Why?
- Gating defects (open probability of the CFTR channel is reduced)

59
Q

What happens to the SCC in G551D mutant CFTR expressing rat thyroid cells when stimulated with Forks?

Why?

A

SCC increases from the unstimulated (due to the activation of the CFTR channel)

BUT is 10-fold less than the function in the WT when stimulated with Forks

60
Q

What happens to the SCC in G551D mutant CFTR expressing rat thyroid cells when treated with Ivaccaftor after being stimulated with Forks?

Why?

What is this compared to the WT? What is the result of this?

A

Increases the SCC to 20microAmpscm2

Ivacaftor is a POTENTIATOR:

  • Increases the Po of the mutant CFTR channels
  • -> Increase current

Although this is 40% of the normal function of the WT, only 15% of normal function of CFTR in needs to elevate the symptoms of CF in patients

61
Q

What happens to the SCC in G551D mutant CFTR expressing rat thyroid cells when treated with a CFTR inhibitor after being treated with Ivaccaftor and stimulated with Forks?

What does this show?

A

Reverses the increase in SCC

Shows the increase in SCC with Ivacaftor and Forks is mediate by the stimulation of CFTR

62
Q

What happens to the SCC in G551D mutant cells when add Ivacaftor BEFORE forks?

Why?

A

Little impact on the SCC

As no PKA mediated phosphorylation of the regulatory subunit of CFTR - not activated

63
Q

Does Ivacaftor work in cells taken from a G511D and F508 patient?

What is the evidence for this?

A

Yes,

  • Add Fsk to human bronchial epithelial cells –> increases the SCC slightly
  • Addition of increases concentrations of Ivacaftor –> get bigger and bigger SCC
64
Q

Why need to add amiloride to cells when looking at the current recordings in CFTR?

A

To prevent contamination of the recording by ENaC (amiloride blocks ENaC)

65
Q

What is the increase in the SCC in HBEs mediated by?

How can this be seen?

A

Activation of the mutant CFTR channel by Forks

Potentiation of the activated CFTR channel by Ivacaftor

As when CFTR inhibitor is added, the SCC decreases

66
Q

What is VIP and how is it used in this research?

A

Vasoactive intestinal peptide:

  • Simulates cAMP
  • cAMP activates PKA –> activates CFTR
67
Q

Following an experimental increase in the SCC, what happens to the ASL level in WT cells in the presence of VIP?

A

Decreases to an optimum

68
Q

Following an experimental increase in the SCC, what happens to the ASL level in mutant cells in the presence of VIP?

A

Height drops way below the normal WT level

69
Q

Following an experimental increase in the SCC, what happens to the ASL level in mutant cells in the presence of VIP and IVACAFTOR?

What does this show?

A

Level sits halfway between the mutant and the WT

Closer to the level of the WT and away from the height of the mutant (due to increases Cl secretion through CFTR)

70
Q

What is the affect of a CFTR mutation the ciliary beat frequency?

A

Large decrease compared to the WT

71
Q

What happens to the CBF when add VIP?

Ivacaftor?

VIP and Ivacaftor
Why?

A

Slight increase compared to the mutant

Even more of an increase compared to VIP

VIP + Ivacafor:

  • CBF is close to the WT level
  • Due to an increase in ASL
72
Q

Describe the clinical trials for Ivacafor

A

RANDOMISED, DOUBLE-BLIND, PLACEBO-CONTROLLED

Ivacaftor group:

  • 83 CF patients with G551D mutation
  • 48 weeks
  • 150mg every 12 hours

Placebo group:

  • 78 CF patients with G551D mutation
  • 48 weeks
  • Placebo
73
Q

How was the effect of Ivacaftor determined compared to the placebo?

A

1) Monitor FEV1 (lung function) as the % of predicted
- Express data as ‘change in % of predicted FEV1’
2) Occurrence of an ‘event’
3) Sweat Cl levels
4) Change in Vte with a beta receptor agonist that activates CFTR via cAMP

74
Q

What does it mean if the change in % of predicted FEV1 is 0?

A

No change

75
Q

What does it mean if the change in % of predicted FEV1 is a POSITIVE value?

A

Increase in lung function

76
Q

What does it mean if the change in % of predicted FEV1 is a NEGATIVE value?

A

Decrease in lung function

77
Q

What is the FEV1 % of the patients involved in the clinical trials without treatment?

SC levels?

A

~63%

~100mM

78
Q

What were the results of the Ivacaftor clinical trials?

A

With the placebo:
- Slow deterioration in lung function

  • 41% of patients have NO event
  • SC levels maintained at 100mM
  • No shift in Vte with cAMP is activated (CFTR channels aren’t working)

Treated patients:

  • Lung function improvement of 10% by week 2
  • Increase in lung function is MAINTAINED
  • 67% of patients have NO event
  • SC levels drop below clinical threshold but not completely to normal
  • Shift in the Vte when cAMP is activated (CFTR channels are functioning)
79
Q

What is an ‘event’?

A

Pulmonary exacerbation (infection, more coughs, poorer lung function measurement, loss of weight)

A BMS346 Epithelial Physiology (13 decks)

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