Salt Reabsorption

Question 1

1. name 2 benefits of using frog skin as an epithelial model
2. name 3 epithelia that frog skin acts as a relevant model for

Answer 1

1. large and easily dissected
2. renal collecting duct, distal colon and airway surface epithelium

Question 2

1. in ussing chamber experiments, what solutions were initially used?
2. what was used to measure the net movement of sodium?
3. what type of ion flux was seen?
4. what was this sodium flux equal to? what does this mean?
5. what happened when an electride was placed in the apical membrane and the EC [Na] was changed?
6. What do these experiments suggest?

Answer 2

1. symetrical solutions. any ion movement is due to active transport
2. ²⁴Na
3. active sodium flux - influx
4. equal to I_SC, indicating that all of the current measured is caused by the movement of sodium
5. Vm changed in respect to [Na]
6. there is a sodium channel on the apical membrane which promotes active sodium absorption (indirect evidence)

Question 3

1. in what samples were the effects of amiloride measured? (2)
2. what were the effects of amioride on Vte? what does this suggest?
3. what does sodium absorption tend to do to the membranr potential?
4. what was the effect of amiloride on the membrane potential?
5. overall what do these studies suggest?

Answer 3

1. human colonic biopsy samples (Vte) and human nasal biopsy samples (apical Vm)
2. brought it closer to zero, suggesting that a sodium channel had been inhibited
3. depolarise
4. hyperpolarise
5. suggests that a Na channel is present in the membrane (indirect evidence)

Question 4

1. what does conventional cloning involve?
2. why couldn’t conventional cloning be used to clone ENaC?
3. name and describe the type of cloning that was used to clone ENaC.

Answer 4

1. an amiloride affinity column that a cell extract is passed down
2. because ENaC is expressed in low abundance
3. Functional Cloning

mRNA from rats fed a low salt diet (aldosterone upregulates ENaC expression in response to low salt) is isolated and cut into pools. These poos are injected independently into xenopus oocytes, and 2 electrode voltage clamp technique is used to see which pool confered amiloride sensitive sodium currents

when the appropriate pool was found, it was further restructed until the ENaC encoding gene was found

the protein was made from this gene and sequenced.

Question 5

1. what did the alpha clone contain? what did the polyA clone contain?
2. what would be expected if 2ng of the alpha clone and the poly A clone are expressed into the cells?
3. why was this expected result not true?

Answer 5

1. purely ENaC; ENaC and other genes
2. that currents would be bigger with the alpha clone, as there would be more ENaC expressed
3. ENaC consists of alpha, beta and gamma subunits. the alpha subunit can form functional channels but with suboptimal activity. The other genes in the polyA clone encoded the beta and gamma subunits

Question 6

1. How is ENaC regulated by hormones?
2. How is ENaC regulated by proteolytic cleavage. Give 3 examples
3. How does Nedd4-2 regulate ENaC?
4. what is Usp-45? What is it upregulated by?
5. what is feedback inhibition?

Answer 6

1. aldosterone increases the apical targeting of ENaC, by acting through kinase cascades (SGK1 and GILZ)
2. proteolytic cleavage enhances ENaC activity
   - amiloride sensitive Na influx is partially blocked by a general protease inhibitor
   - patch clamping shows proteolytic cleavage increases the Po of ENaC
   - a decrease in molecular weight of ENaC is seen in rats fed a low salt diet.
3. ubiquitinates ENaC which is required for its endocytosis and degradation
4. a deubiquitinating enzyme upregulated by aldosterone - reverses the effect of Nedd4-2 to keep ENaC in the membrane
5. the inhibition of ENaC in response to high IC [Na]

Question 7

1. what type of protein is SGK1?
2. what is it upregulated by?
3. how does it act to upregulate ENaC activity?
4. what is the phenotype of KOs?

Answer 7

1. S/T kinase
2. aldosterone
3. phosphorylates Nedd4-2 therefore preventing it from interracting with ENaC
4. unable to conserve Na as apical ENaC expression is diminished (lack of reabsorption)

Question 8

1. what is GILZ stimulated by?
2. what activity promotes the interraction of ENaC with Nedd4-2?
3. How does Gilz act to upregulate ENaC?

Answer 8

1. mineralcorticoid receptor activiaton by aldosterone binding
2. direct phosphorylation of ENaC by MAP-K
3. prevents MAP-K activation (hence preventig the phosphorylation of ENaC, therefore preventing its interraction with Nedd4-2) by inhibiting the phosphorylation of MAP-KKK by ras

Question 9

1. what is the height of the periciliary layer (PCL) important for?
2. what happens when the PCL is too low?
3. what happens when the PCL is too high?
4. what is PCL height determined by?
5. describe the 2 mechanisms that control the height of the PCL?

Answer 9

1. mucous clearance
2. the cilia are bent therefore can’t beat properly
3. the volume is too large for the cilia to move the mucous
4. sodium absorption and chloride secretion
5. passive - movement of water into the mucous layer; active - ion transport

Question 10

1. following the addition of water, what was the change in the volume of the PCL?
2. what were the effects of amiloride and bumetanide on Vte at 0 hours?
3. what were the effects of amiloride and bumetanide on Vte at 48 hours?

Answer 10

1. 30μL to 7μL
2. amiloride had a large inhibitory effect; bumetanide had little/no effect
3. amiloride had a smaller effect than at 0hrs; bumetanide had a large effect.

Question 11

1. what was the difference in the Vte following the treatment with amiloride when cells were pretreated with the respiratory syncytial virus?
2. what was the effect on amiloride sensitiveI_SC when cells were pretreated with RSV?
3. what does this imply about RSV?
4. name 4 inhibitors that were used to investigate the effects of the RSV?
5. what were the effects of these inhibitors?
6. what do the results of this imply?

Answer 11

1. shift in Vte was smaller following pre-treatment with RSV
2. amiloride sensitive SSC were also smaller following treatment with RSV
3. it inhibits ENaC
4. BIM - PKC inhibitor

NA - inhibits the binding to glycoproteins

PPMP - inhibits binding to glycolipids

5. BIM and PPMP restored amiloride sensitive SSCs following treatment with RSV. NA had no effect
6. PKC and glycolipids are important for the ability of RSV to inhibit ENaC

Question 12

1. Name and describe the 3 components of the influenza virus.
2. what were the effects of expressing M2-GFP in oocytes and human airway cell lines? What was the control experiment?
3. what did single channel patch clamping show?
4. what did western blotting show?
5. what were the effects of the lysosomal/proteosomal inhibitors?
6. what were the effects of M2 on the liddle’s mutant ENaC?
7. what was used to detect ROS? what was the effects of M2-RFP expression on ROS? What was the control experiment?
8. what were the effects of antioxidants and PKC inhibitors?
9. what were the conclusions of this experiment?

Answer 12

1. haemagluttin - binds to sialic acid and activates PKCM1 matrix proteinM2 matrix protein - forms acid activated amantadine inhibited H⁺ channels which inserts into the host apical membrane
2. significantly inhibited ENaC currents; expression of GFP alone had no effect
3. decreased number of activ ENaC channels following M2 expression
4. decreased surface expression of ENaC following M2 expression
5. prevention of ENaC inhibition bt M2
6. no effect
7. DCF-DA was used to reveal ROS. It produces green fluorescence in the absence of ROS. It showed that overexpression of M2-RFP caused an increase in ROS that colocalised with M2. RFP alone had no effect on ROS
8. prevented ENaC inhibition by M2
9. M2 downregulates ENaC by enhancing its ubiquitination and degradationM2 increases levels of ROS which is responsible for ENaC downregulation

Question 13

1. name 4 symptoms of pseudohypoaldosteronism
2. what form do dominant mutations cause?
3. what form do recessive mutations cause?
4. in what protein are recessive mutations found?
5. name 5 characteristics of the recessive form.

Answer 13

1. salt wasting, hypotension, hypokalaemia, high renin and aldosterone
2. renal form. mutations in mineralcorticoid receptor
3. systemic form.
4. mutations in ENaC
5. frequent lower resp tract infectionssignificantly increased wet weight of nasal fluidconcentration of nasal fluid significantly higherVte less negative (loss of sodium contribution)percentage inhibition of Vte by amiloride is significantly reduced.