Salt Reabsorption and epithelial Na+ channel

Question 1

Why is frog skin a good model tight epithelium?

Answer 1

Frogs absorb Na+ from their external environment
This model also has a transepithelial resistance of more than 2000ohms/cm2
Also large, cheap and easy to use

Question 2

Ussing chamber method with frog skin - how does it work?

Why is a -ve potential seen?

Answer 2

Current is injected into chamber –> causes change in transepithelial potential –> resistance can then be calculated
A negative potential is seen –> this is due to a loss of Na+ from apical to basolateral membrane

Question 3

How is Na+24 used in the method?

Answer 3

Radioactive isotope of Na –> added to one side of the chamber and concentration of isotope is measured in other side
This is done over a known time period > can work out the amount of Na+ transport per unit time

Question 4

There was net absoprtion from the pond to frog – what does this show?

Answer 4

Membrane potential produced from membrane is due to the permeability to Na+!

Question 5

In a Human Colon Biopsy, there was a less negative transepithelial potential generated than in the frog skin - what does this show?

Answer 5

• Less reabsorption of Na+ is seen

- More leaky than frog skin

Question 6

For the patch clamp technique, what is recorded and how is this shown on the graph?

Answer 6

Records single channel conductance of Na+ channels
A deflection on a graph is the opening of a channel
More Na+ in cell = more deflections as more ions available so more current can be generated

Question 7

How was expression cloning of Enac carried out?

Answer 7

• Took RNA from cells of SALT DEPLETED RATS and chopped into pools
• injected chopped up RNA into oocytes
• identified which sections produced amiloride-blockable currents
• repeat until smaller and smaller sequence isolated –> gene coding sequence isolated

Question 8

What did results from expression cloning of Enac show?

Answer 8

The pools of RNA with highest percentage of coding sequence produced the largest currents
Enac found to be made up of 3 sub units –> 3 genes code for 3 sub units that together produce the largest currents

Question 9

GOF mutations of ENac – what is this caused by and how is it characterised?

Answer 9

Mutations in B/y sub units
LIDDLE’S SYNDROME -> too much enac in membrane!
Hypertension

Question 10

LOF mutations of ENac – what is this caused by and how is it characterised?

Answer 10

Pseudohypoaldosteronism

LOF mutations –> loss of function of Enac so loss of Na+ in the urine

Question 11

Sub unit activation –> how does this structure impact Enac function?

Answer 11

a, B and y together enhances function. Sub units separately can form functional channel but need all 3 for full activity

Question 12

What other Na+ transport proteins are found in epithelia?

Answer 12

• Na+/H+ exchanger
• Na+/ HCo3- cotransporter
• Thiazide sensitive Nacl transporter
• Na+/K+/2cl- co transporter (a loop diuretic sensitive protein)