Salt Reabsorption 1 Flashcards
Why is frog skin a good model for epithelia?
It is cheap, easy to get, large tissue and is robust
It has similar molecular components to the collecting duct, distal colon, salivary gland, sweat duct and airway surface epithelia
How was sodium transport in frog skin validated using the Ussing chamber technique?
They used Na24 which is radioactive
This was put in chamber one on the apical side
Over time it was found in chamber 2 and the concentration was measured
They then did this the other way round to measure overall sodium transport
What data did Ussing ans Zerhman collect in 1951?
They worked out how many moles had been transported: 1 micro amp is equal to 10.4pmol/s
They worked out sodium influx and outflux in this way
This told Ussing that the net transport of sodium was from apical to baso i.e. in to the frog
Additionally, flux was not = 0
This shows that there is an active component in this pathway which is Na+/K+ ATPase
What was the hypothesis about Na+ reabsorption and ENa?
If the apical membrane has an Na+ channel then the membrane potential for Na+ should move with ENa
i.e. concentrations of Na+ are changed outside the celll
How did Ussing chamber work on human colon biopsies show the apical membrane has a sodium channel?
Adding amiloride causes Vte to shift more positively
This shows that there is an amiloride sensitive current as less Na+ is being transported so there is less loss of positive ions
What was the experiment in human nasal biopsies which suggests the presence of an apical Na+ channel?
Na+ entry in to a cell depolarises it
When amiloride was added hyperolarisation occured as there is a shift away from ENa
This shows there is an Na+ selective channel in the apical membrane
What was the first DIRECT evidence of an apical sodium channel?
Direct evidence came from the development of the patch clamp technique
This allows you to directly look at currents moving through the channels
The size of currents shown from single channel recordings increased when sodium concentration was increased on the outside of the cell
Why did expression cloning of ENaC not work?
Its protein abundance was too low for the existing method to work - this was done using an amiloride affinity column
Through what method was ENaC cloned?
Functional expression
How did the functional expression method work?
As we knew the channel properties of ENaC the mRNA of cells were divided in to pools
Pools were then injected in to the Xenopus oocytes and were then looked at which one produced an amiloride sensitive current
Pools were then divided again
Why were the rats where the mRNA came from on a low salt diet?
So they produced aldosterone = more ENaC expression
Why did the pure clone of ENaC mRNA give a smaller current than poly mRNA?
The alpha subunit can make a functional pore by itself but for correct function it requires the expression of the beta and gamma subunit also
What happened when all three subunits were expressed together?
When alpha and beta or alpha and gamma were expressed together current was 200 amps
When all three were expressed current was 2000 amps
What are the effects of gain of function ENaC mutations?
Liddle’s syndrome
Hypertension
Increase in ENaC
Due to Nedd4 not being able to retrieve the channel from the membrane
What are the effects of loss of function ENaC mutations?
PHA1
Hypotension
ENaC is lost = volume depletion
