Lecture 8 - Molecular Basis of Cystic Fibrosis

Question 1

On which chromosome is the CFTR gene?

Answer 1

Cr 7

Question 2

Length of CFTR gene

Answer 2

70kB

Question 3

Number of exons in CFTR gene

Answer 3

27

Question 4

What is the CFTR protein?

Answer 4

A large, integral, glycosylated, membrane-spanning protein

Question 5

Weight of CFTR protein

Answer 5

170kD

Question 6

Is CFTR responsible for active transport?

Answer 6

No.

Despite using ATP, CFTR moves Cl- with concentration gradient

Question 7

Which family does CFTR belong to?

Answer 7

ATP-Binding Cassette (ABC) superfamily of membrane transporters

Question 8

How is CFTR channel regulated?

Answer 8

By cAMP-dependent phosphorylation

Question 9

Which cells express CFTR?

Answer 9

Epithelial cells, normally on apical surface

Question 10

What are the domains of the CFTR protein?
1)
2)
3)
4)
5)

Answer 10

1) Membrane-spanning domain 1
2) MSD 2
3) Nucleotide-binding domain 1
4) NBD 2
5) Regulatory domain

Question 11

Function of membrane-spanning domains

Answer 11

Form pore through which Cl- move

Question 12

Function of nucleotide-binding domains

Answer 12

Bind and hydrolyse ATD

Question 13

Function of regulatory domain

Answer 13

Several sites that can be phosphorylated by cAMP-dependent kinases

EG: Protein kinase A

Question 14

Examples of different direction of Cl- flow through CFTR

Answer 14

Lungs: Cl- flows out of cell

Sweat duct epithelial cells: Cl- flows into cells

Question 15

How does CFTR interact with other proteins?

Answer 15

Largely through C-terminal, which is anchored to cytoskeleton

Question 16

Example of protein with which CFTR interacts

Answer 16

ENaC sodium channel

Question 17

Domain on C-terminal of CFTR which interacts with other proteins

Answer 17

TRL - Threonine - Arginine - Leucine

Question 18

Normal function of CFTR in cell in lung
1)
2)

Answer 18

1) Cl- moves out of cell through CFTR

2) Na+, H2O move into cell down concentration gradient

Question 19

Defective function of CFTR in cell in lung
1)
2)
3)

Answer 19

1) Cl- ions can’t escape cell through CFTR
2) Buildup of Cl- in cell results in a greater concentration gradient.
3) ENaC is not inhibited by CFTR. Unregulated uptake of Na+ into cells, leading to water osmotically being absorbed into cells. Dehydration of ASL

Question 20

Normal function of CFTR in a sweat duct
1)
2)

Answer 20

1) Cl-, Na+ and H2O enter cell down concentration gradient

2) Sweat is secreted, but ions are largely reabsorbed by cells

Question 21

Defective function of CFTR in a sweat duct
1)
2)

Answer 21

1) Cl- can’t enter cell, so Na+ ions don’t either

2) Buildup of Cl- and Na+ ions in sweat, leading to abnormally salty sweat

Question 22

Number of known mutations in CFTR

Answer 22

Over 1900

Question 23

Where are most mutations found in CFTR?

Answer 23

Exons 4, 8, 14, 20

Question 24

Most frequent mutation in CFTR

Answer 24

Missense

Question 25

Class I CFTR defect

Answer 25

No protein production

Question 26

Class II CFTR defect

Answer 26

Defective processing
(maturation, premature degradation)

Protein can’t leave ER, Golgi

Question 27

Class III CFTR defect

Answer 27

Defective regulation
(defective ATP binding, hydrolysis)

Channels don’t open

Question 28

Class IV CFTR defect

Answer 28

Defective or reduced opening of ion channel, ion conductance

Channels can open, but not much

Question 29

Class V CFTR defect

Answer 29

Reduced protein production

promoter or splicing abnormality

Question 30

Class VI CFTR defect

Answer 30

Accelerated turnover from cell surface

Quite a rare type of defect

Question 31

Common mutations leading to a class I defect

Answer 31

Nonsense, missense, frameshift mutations

Question 32

Do mutations in CFTR gene have to be pathogenic?

Answer 32

No.

Some are non-pathogenic, some have reduced penetrance

Question 33

What could lead to a class VI defect?

Answer 33

Nonsense mutation placing a stop codon near the C-terminal

Protein instability at cell surface

Question 34

What could lead to a class III defect?

Answer 34

Mutation in nucleotide-binding domain 2.
ATP can’t be hydrolysed

Or mutation in R domain

Question 35

What could lead to a class II defect?

Answer 35

Mutation in nuclear binding domain 1.
Defective cell processing

This is where F508del occurs.

Question 36

What could lead to a class IV defect?

Answer 36

Mutation in membrane-spanning domain 1.

Cl- have more trouble moving through pore

Question 37

What could lead to a class I defect?

Answer 37

Mutation in membrane-spanning domain 1.

Normal levels of mRNA, absent protein

Question 38

What could lead to a class V defect?

Answer 38

Mutation in membrane-spanning domain 1.

Reduced number of transcripts

Question 39

Most common mutation

Answer 39

F508del

Question 40

Which class of defect results from F508del?

Answer 40

Class II

Protein is misfolded, retained in ER, degraded

Question 41

Way to correct class I defects

Answer 41

Aminoglycosides

Allow ‘read through’ of mRNA

Question 42

Way to correct class II defects

Answer 42

‘Correctors’ to improve protein processing

Question 43

Way to correct class III defects

Answer 43

‘Potentiators’ to activate protein

Question 44

Way to correct class IV defects

Answer 44

Flavinoid compounds to improve channel conductance

channel more likely to be open

Question 45

Way to correct class V defects

Answer 45

Improve number of correctly-spliced mRNA molecules

Question 46

How does F508del occur?
1)
2)
3)

Answer 46

1) C from end of isoleucine and TT from phenylalanine are deleted.
2) Last amino acid of phenylalanine codon (T) combined with first two amino acids of isoleucine codon (AT) leads to isoleucine codon.
3) 3-base, out of frame deletion, but only phenylalanine has been removed.

Question 47

Frequency of F508del mutation

Answer 47

~50% CF sufferers homozygous for F508del

F508del accounts for ~75% of CFTR mutations in northern Europe

Question 48

How was CFTR first discovered to be the protein involved in CF?

Answer 48

Positional cloning

Question 49

Least common place in CFTR gene for a mutation to occur.

Answer 49

Promoter