Salt secretion and cftr

Question 1

Cystic fibrosis - what is it? Incidence? Symptoms?

Answer 1

Most common lethal genetic disease in caucasians - airway disease!!
- Exocrine pancreatic insufficiency, increased sweat Cl- concentration, male infertility (Cl- channel needed in development –> absence of vas deferens!)

Question 2

Model for basis of secretion - why??

Where are leaky epithelia found?

Answer 2

Shark rectal gland! Large, robust and lasts for a long time
Model for leaky epithelia –> secretion of Nacl rich fluid such as what is seen in the choroid plexus, upper airway and proximal tubule

Question 3

What are Ouabain and Barium and what are their impacts in normal cells?

Answer 3

OUABAIN - Na+/K+/ATPase pump blocker –> when blocked secretion is at 0 as this sets up the driving force for the secretion of Cl-!!
BARIUM - K+ channel (basolateral membrane) blocker –> inhibits Cl- secretion !! Causes membrane to depolarise and membrane potential shifts +vely; ions not recycled again

Question 4

Relevance of K+ and membrane potential in Cl- secretion?

Answer 4

• Low intracell Na+ concentration

- -ve membrane potential - both critical for secretion of Cl-!!!

Question 5

What is FUROSEMIDE and what is it used for?

Answer 5

Na+/K+/2Cl- transport blocker –> causes secretion to shift to 0 –> no Cl- being brought in from basolateral side!!

Question 6

Role of Na+/K+/ATPase in Cl- secretion?

Answer 6

Sets up the driving force for the influx of Na+ through NKCC1. Blocking this therefore causes a reduction in the function of NKCC1

Question 7

Role of NKCC1 in Cl- secretion?

Answer 7

Brings Cl- into cell depending on the functioning of ATPase pump (sets up the driving force for influx of Na+ via NKCC1)

Question 8

What does it mean if Cl- is ‘above electrochemical equilibrium?’

Answer 8

An active component is involved and causing accumulation of Cl- – this is seen when the ic [Cl-] > calculated value!!
Did this to work out what the pathway of Cl- would be on the apical membrane

Question 9

How can you use the nerst potential to work out IC [Cl-]??

Answer 9

Take the membrane potential and ec [Cl-], rearrange the nerst equation to find out the ic [Cl-] IF CL- IS PASSIVELY DISTRIBUTED (no active componant involved in Cl- being brought into the cell). If they are the same, no active componant involved. If higher, active componant involved

Question 10

Outline completed model for Cl- secretion?

Answer 10

• NKCC1 –> brings in Cl- under the influence of the driving force set up by the pump in the basolateral membrane
• PUMP - sets up the driving force for the activity of NKCC1 by maintaining a low ic [Na+]
• K+ channel - recycles K+ back out of the cell via the basolateral membrane, which causes hyperpolarisation of the membrane and stimulates secretion of Cl- via CFTR

Question 11

What are the 6 classes of CFTR mutations

Answer 11

1 - null production
2 - trafficking (dF508!!)
3 - regulatory mutation
4 - conduction - gating mutation - channel doesnt open and close properly
5 - partial reduction - less mRNA is produced
6 - high turnover CFTR - reduced time at membrane so less protein there at any given time point

Question 12

Properties of the CFTR channel?

Answer 12

12 tmds, 1 sub unit, 1 regul. domain, 2 nucleotide binding domains (VERY IMPORTANT –> commonly mutated!!)

Question 13

Diagnostic cut off point and mutation severity –> what are they are what classes of mutations are normally associated with what?

Answer 13

> 60mmol/L [Cl-] in the sweat = diagnostic cut off!
Class 1-3 of mutations –> pancreatic insufficiency , individuals most ill
Class 4 and 5 –> less severe

Question 14

How is the isolated rat colonic crypt a good model for Cl- secretion?

Answer 14

Determines water content of rat faesces –> lower 2/3 of colon secretes Cl- –> H2o follows. Too much secretion = diarrhoea.

Question 15

Role of K+ and Na+ in the colon?

Answer 15

K+ channels = hyperpolarisation of the membrane, increasing the driving force for the secretion of Cl-
Na+ ions undergo paracellular secretion

Question 16

Ach receptors and Prostoglandins on epithelial cells - what are their roles?

Answer 16

Increase in Ca2+ inside cell stimulates Ach receptors, which activates K+ channels to stimulate Cl- secretion!
Prostoglandin receptor (PGE2) are activated by cAMP --> stimulates pKA --> Cl- secretion

Question 17

Effects of stimulation with ; Forskolin, Carbachol, Indomethacin, IBMX?

Answer 17

Forskolin –> increase in cAMP so increase in secretion?
Carbachol –> Achr activator by increasing i.c Ca2+
IBMX –> increase cAMP by inhibiting Phosphodiesterase enzyme
Indomethacin –> inhib PG production and decrease cAMP levels

Question 18

Impact of phosphodiesterases?

Answer 18

Cause break down of prostoglandins –> less stimulation of PGE2 –> break down cAMP, decreasing Cl- secretion!!

Question 19

Impact of adenylate cyclase?

Answer 19

Increase cAMP levels to increase Cl- secretion!

Question 20

CFTR not full story- other Cl- channels?

Answer 20

CLCs - voltage gated Cl- channels

Caccs - Ca2+ activated Cl- channels

Question 21

Pathology of CF newborns colonic mucosa

Answer 21

Lack of Cl- channel is seen –> results in blockages caused by thick mucus –> no Cl - secretion so water doesn’t follow and thick mucus. Causes blockages in digestive system and death

Question 22

UPPER AIRWAY - MODEL role of CFTR?

Answer 22

CFTR —| Enac –> loss of function in CFTR therefore thick mucous produced!

Question 23

ALVEOLAR MODEL - role of CFTR?

Answer 23

Enac and CFTR active together!!
No NKCC2 in basolateral membrane, instead K+/Cl- contrasporter moves Na+/Cl- out which creates driving force for ABSORPTION of Cl- via CFTR!!
When not working can cause alveolar oedema (fluid in alveoli)

Question 24

DISTAL SWEAT GLANDS MODEL - role of CFTR

Answer 24

CFTR stimulates Enac allowing Na+ to be reabsorbed
Faulty CFTR means salty sweat!!
In the sweat glands, fluid moves down sweat duct and ions are secreted, then they are reabsorbed at more distal point (via CFTR —> Enac)