CFTR and Salt Secretion

Question 1

Describe cystic fibrosis

Answer 1

Most common genetic mutation in Caucasians
Associated with symptoms e.g.
- Exocrine and pancreatic insufficiency
- Increased sweat Cl- = clinical diagnostic marker
- Male infertility (95% of males have this as their vas deferens does not develop)
- Airway disease
CF infants can be detected as they often show a failure to thrive

Question 2

List some examples of epithelia that secrete an NaCl rich fluid

Answer 2

Exocrine gland acini 
Sweat gland coli 
Small intestine 
Upper airway 
Choroid plexus 
Shark rectal gland

Question 3

Why is the shark rectal gland used as a model?

Answer 3

Labs used to have easy access to shark and the tissue is large and robust so lasts longer in an organ bath compared to mammalian tissues which are more sensitive

Question 4

Describe 2 pharmacological techniques which show that a low Na+ concentration and low mV is needed for Cl- secretion

Answer 4

1) Oubain blocks ATPase - required for low Na+, when blocked Cl- secretion was reduced
2) Barium blocks K+ channels - required for low -mV = reduction in Cl- secretion

Question 5

Which drug blocks NKCC1?

Answer 5

Furosemide

Question 6

What happens when NKCC1 is blocked?

Answer 6

Cl- secretion is reduced

Question 7

Describe how we know that Cl- is above its electrochemical gradient

Answer 7

mV was taken with known extracellular concentrations of Cl- on either the apical or basolateral side of the cell to work out intracellular Cl- concentration
This allows you to work out how much Cl- should be in a cell if there is no active component
e.g. basolateral = 17mM and apical = 28mM
So intracellular Cl- should be between these if there is no active component
However Intracellular Cl- is actually 70mM = there is an active component!
= Cl- is above its electrochemical gradient

Question 8

Why is it important that Cl- is above its electrochemical gradient inside the cell?

Answer 8

It means that when CFTR opens there is a driving force for Cl- to leave the cell

Question 9

Which protein is important for Cl- accumulation?

Answer 9

NKCC1

Question 10

How can CFTR be activated?

Answer 10

By cAMP

Question 11

What is the structure of CFTR?

Answer 11

It has 12 TMDs made of 1 subunit but other proteins ca interact with it
It is divided in to two domains where each one has a nuclear binding domain (NBD) which contains the amino acids where nucleotides bind to for regulation
There is also an R domain which is the site for regulation by phosphorylation by PKA for channel opening

Question 12

How many mutations have been found in CFTR which are linked to CF?

Answer 12

> 12,000

Question 13

What is the most common mutation found in CFTR and where is it located?

Answer 13

DeltaF508

Located in NBD1

Question 14

List the 6 classes of CFTR mutations and briefly describe them

Answer 14

1) Null production - mRNA is unstable so no protein is made
2) Trafficking - no protien is in the membrane due to misfolding
3) Regulatio - protein is made but not regulated e.g. by PKC so Po is low
4) Conduction - gating mutations where the channel does not open and close properly = decrease in Po
5) Partial reduction of mRNA - lower levels of mRNA is made
6) High turnover - reduced time in the cell membrane

Question 15

How much CFTR is needed for normal function?

Answer 15

Only 15 to 20%

Question 16

Why can you not have a drug that treats all CF patients?

Answer 16

Drugs need to be compounds which target specific mutations

Question 17

Why do some patients with the same mutation have a different severity of symptoms?

Answer 17

Severity if symptoms is linked to the patients compliance and their genetic background

Question 18

What is the diagnostic cut off for CF in terms of sweat chloride?

Answer 18

> 60mmol/L

Below this patients could have CF but with milder mutations = milder symptoms

Question 19

Which classes of mutations also give pancreatic insufficiency?

Answer 19

Classes 1 to 3

Their sweat Cl- can be as high as 100mmol/L

Question 20

What does Cl- secretion in the colon determine?

Answer 20

The water content of faeces

Question 21

Described the model for Cl- secretion in the colon

Answer 21

Basolateral membrane causes Cl- accumulation in the cell
Ca2+ activated K+ channels open = increased driving force for Cl- secretion as the K+ channels hyperpolarise the membrane
Na+ is secreted through a paracellular pathway
Ach receptor activation = increased Ca2+ = K+ channels activated
PGE2 receptor activation = increase in cAMP which stimulates CFTR

Question 22

List the 4 drugs used on human colonic mucosa studies

Answer 22

1) Carbachol (CCH) = Ach receptor activation = increased calcium
2) Indomethacin = Inhibition of prostaglandin production = decrease in cAMP
3) IBMX = inhibition of phosphodiesterases which breaks down cAMP = increase in cAMO
4) Forskolin = Activation of adenylate cyclase = increased cAMP
* When Forskolin and IBMX are used together there is an additive effect

Question 23

Describe the results from the experiment in Human colonic mucosa and the effects of these drugs on SSC

Answer 23

Stimulation of Cl- secretion gives a negative shift in Vte - this increased by CCH
In the presence of indomethacin and CCH there was no Cl- secretion as Cl- channel activity is low
Indomethacin and IBMX/Forskolin however caused a negative shift in Vte as Cl-channels are now activated e.g. the PGE2 receptor is being bypassed and we are still getting an increase in cAMP
Adding CCH to this then further increased Cl- secretion as calcium levels rise in the cell

Question 24

What were the results when this experiment was repeated with CF mutations?

Answer 24

In the mutations no Cl- secretion was seen

This is because the channels may not have been at the membrane or the Po was low i.e. at zero

Question 25

What is the problem with the colon in CF patients?

Answer 25

Because they do not secrete Cl- patients have blockages of the colon
e.g. 10% of CF newborns have meconium ileus which requires surgery to remove the blockage - an equivalent of this is seen in adults also

Question 26

How do CFTR mutations affect the upper airways?

Answer 26

CF patients have a thick mucous
In WT when CFTR is active ENaC is inhibited
So in the mutant ENaC is not inhibited = more Na+ reabsorption than there should be so height of the PCL is too low for cilia to beat

Question 27

Describe the effect of CFTR mutations in the alveolar model

Answer 27

NKCC1 is not expressed in the basolateral membrane - instead we have K+/Cl- co transport which moves K+ AND Cl- out of the cell
This means Cl- moves IN to the cell through CFTR
In this model ENaC and CFTR actually work together
CFTR mutations therefore can = alveolar oedema

Question 28

Describe the effect of CFTR mutations in the distal sweat gland model

Answer 28

One set of cells secretes NaCl which moved down to the distal sweat glands
The distal sweat glands are then meant to reabsorb this NaCl
However, if CFTR does not work then there is an increase of Cl- in sweat
ENaC and CFTR normally work together in this model