Lecture 8 - CF: Molecular Basis Flashcards Preview

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Flashcards in Lecture 8 - CF: Molecular Basis Deck (50):
1

In general, what does the mutation in CFTR lead to?

Abnormal cAMP-mediated regulation of chloride transport

2

Which gene is mutated?

CFTR gene
(CF transmembrane conductance regulator)

3

What is the CFTR protein?

Chloride channel protein

4

To which family does CFTR belong?

ABC superfamily of membrane transporters
(ATP-binding cassette)

5

Describe the energy requirements of the chloride channel

ATP required to open and close the channel
Ion movement through the channel is downstream (no ATP)

6

Describe and draw the structure of the CFTR protein

5 domains:
• 2 x MSD's (MSD 1, MSD 2) which form the channel
• Regulatory domain; R domain
• Nucleotide binding domains: NBD 1, NBD 2

Order:
• N terminus
• MSD 1
• NBD1
• MSD2
• NBD 2
• R domain
• C terminus

7

What is the important feature of NBDs?

ATP binding → channel opens

NB when ATP is hydrolysed, the channel closes again

8

What is the function of the R domain?

(Regulatory domain)
Phosphorylated by cAMP
Phosphorylation leads to fine tuning of the channel function

9

Describe the opening and closing of CFTR

ATP binds → opening of channel
ATP hydrolysis → closing of channel

10

What are some secondary roles of the CFTR channel?

Carboxy terminal region:
• regulation of ENaC (a sodium channel)
• conductance
• signal transduction
• cytoskeleton

11

Describe the role of CFTR in airways

How is this disrupted in CF?

Normally:
• Cl- ions moving out of the cell down a concentration gradient
(Na+ and water moving into cells)

In CF:
• Cl- ions maintained within the cell
• Increased Na+ and water movement into the cell
• Dehydrated mucous

12

Describe the role of CFTR in sweat ducts

How is this disrupted in CF?

Normally:
• Cl- ions moving into the cell down a concentration gradient

In CF:
• blockage of Cl- movement into the cells
• Na+ and water maintained outside the cell

13

What is the CF Mutation Database?

Collection of mutations that cause Cystic Fibrosis of varying and unknown significance

14

What are mutations of 'varying clinical consequence'?

Mutations that have decreased penetrance
Difficult for clinicians, because the severity of the mutation is often not known

15

Which type of mutation is most common in CF?

Missense (40%)

16

Which exons are most commonly mutated in CF?

Membrane spanning domains: 4, 8

Regulatory domain: 14

20

17

What are the classes of mutation?

6 classes
• Class I: no protein production
• Class II: Defective processing
• Class III: Defective regulation
• Class IV: Reduced ion conductance
• Class V: reduced protein production
• Class VI: accelerated turnover from the cell surface

18

What happens in Class I mutation?

Premature nonsense mutation
• STOP codon before the end of the protein
• truncated protein produced
• defective protein is unstable and tagged for degradation

There are also mutations whereby the mRNA is not produced properly

19

What happens in Class II mutation?

Gene is transcribed and translated into the ER
However, the protein never makes it out of the ER due to e.g. mutation in folding, targeting to cell surface

→ Degradation of the protein in the ER

20

What happens in Class III mutation?

CLOSED CHANNEL

• Protein makes it to the cell surface
• ATP doesn't bind properly to NBD
• Channel never opens

21

What happens in Class IV mutation?

REDUCED CONDUCTANCE
• Protein makes it to the cell surface
• Dysfunction of phosphorylation of the R domain
• Channel only opens somewhat, may be partially open / closed

22

What happens in Class V mutation?

Protein is produced, but not in the amounts required

23

What happens in Class VI mutation?

Protein is functional and makes it to the cell surface, but is removed too soon

24

What is the most common mutation in CF?

Describe it

F508del
• Class II mutation
• In NBD 1
• (Defective processing)
• Protein missfolds
• doesn't get out of ER and is targeted for degradation

25

What happens in S1255P?

• Class III mutation
• In NBD 2
• Defective regulation

NB don't need to memorise this

26

What happens in Q1412X?

• Class VI mutation
• Instability at the cell surface

NB don't need to memorise this

27

What happens in Splice mutation intron 4 donor site?

• Class I mutation
• In MSD 1
• Absent protein

NB don't need to memorise this

28

Why is important to know the mutations?

Therapies can be targeted to the particular mutation for better results

29

Which therapies are used for Class I mutation?

Aminoglycoside antibiotics:
• allow 'read through' of mRNA despite nonsense mutations

30

Which therapies are used for Class II mutation?

'Correctors'
• improve processing

31

Which therapies are used for Class III mutation?

'Potentiators'
active proteins, keep the channel open

32

Which therapies are used for Class IV mutation?

'Flavanoid'
Augment channel function

33

Which therapies are used for Class V mutation?

Increase levels of correctly spliced RNA

34

What is happening in F508del?

Phenylalanine n° 508 deletion
• three bases are deleted
• NB only two bases from the F508 codon deleted, and one for Isoleucine

35

How is CFTR mutation tested for?

Earlier:
1. RFLP analysis
S23
2. Multiplex PCR; SNP analysis
3. MALDI-TOF Mass Spectrometry

36

How many mutations are screened for in newborns?

12 mutations
More being added all the time

37

Describe the different frequencies of F508del in people of different ethnicities

Different ethnicities are affected to greater and lesser degrees by certain mutations.
For example Northern Europeans (esp. Danes) are particularly affected by the F508del mutation

38

How is Mass Spectrometry used for screening?

(MALDI-TOF MS)
• Large number of samples can be tested simultaneously
• Looking for a medium n° of SNPs

39

What is an RFLP?

Restriction fragment length polymorphism

• When DNA from different individuals is digested with restriction enzymes, one gets fragments with different lengths.

40

Describe RFLP analysis

• PCR + Restriction enzymes
• Run on an acrylamide gel
• Analysis of RFLPs

41

Describe Multiplex PCR

• Fluorscent tagging to see SNPs

42

Where is CFTR expressed?

Apical membrane of epithelium
In a wide range of tissues

43

What is the function of the MSD's?

Form the ion channel through the membrane

44

Draw a rough diagram of the CFTR gene

S 18
• 27 exons
• 2 x MSD coding regions
• 2 x NBD coding regions
• R domain coding region

45

What is the R117H mutation?

• Class IV mutation
• In MSD 1
• Defective conduction through the channel

46

Describe the difference between class III and IV mutation.
Which mutation is more severe?

Class III: ATP doesn't bind, hence channel doesn't open

Class IV: dysfunctional phosphorylation of R domain, channel has reduced conductance

Class III mutation is more severe

47

What is the importance of the external projections of the CFTR?

These are loops of the MBD that are located on the outside of the cell
These can interact with other, external molecules, altering the function of the CFTR channel

48

Which class of mutation is F508del?

Class II

49

How and when was the CFTR gene identified?

1989
Identified by positional cloning

50

How does the CFTR regulate ENaC?

The TRL (Threonine-Arg-Leu) sequence on the cytoplasmic C-terminus interacts with it

(Inhibition of ENaC)