Molecular physiology of salt reabsorption Flashcards
Why is frog skin a good model for salt reabsorption?
The mechanism they use to absorb salt from their external environment is essentially the same as the one we use to absorb salt from our:
upper airway cells, collecting duct cells and principal cels
What other cells mimic frog skin?
Renal collecting duct cells distal colon salivary glands sweat ducts airway surface epithelium
What experiment did they do to validate the results from the Ussing chamber?
Radioactive tracer experiment- Na24
Na24 in chamber 1- then take a sample from chamber 2
see how much radioactivity has been transported across the epithelium
then do the reverse way- and work out how much Na has been transported per unit of time in each direction= NET movement of sodium
How did they know there was a bit of sodium leak back?
There was sodium outflux current calculated thru the radioactive tracer experiment (basolateral to apical membrane)
What did the Ussing chamber experiment + Radioactive tracer experi lead to the conclusion of?
The net transport across the frog skin epithelium was mainly sodium
sodium transport is taking place across the apical membrane and is being reabsorbed via the basolateral membrane
What electrophysiological experiment did they carry out in 1958, what did it conclude?
Impaled an electrode in the apical membrane and then changed the concentration of Na outside the cell
conclusions:
theres net absorption of sodium into the frog
the membrane is permeable to sodium
na channels are present in the membrane
How did amiloride effect cells from a human colon biopsy”
Vte moved to zero
Why do humans have a smaller Vte than frogs?
Human epithelium isn’t as tight
and there are more ions transported across the epithelium, mainly sodium in frogs
What did work on a human nasal biopsy conclude?
Sodium entry into the cell leads to depolarisation
adding amiloride leads to hyperpolarisation of the cell
What did patch-clamp analysis of ENac conclude?
Patch-clamp- way of looking at individual sodium channels- other studies looked at the epithelium as a whole
took single channel recordings from renal collecting duct cells
higher concs of Na added= the size of the deflections increased
*direct evidence for epithelial sodium channels
Why was it initially so hard to clone Enac?
Barely any Enac in native cells- so hard to clone
conventional cloning= isolate the protein and sequence it- e.g by using amiloride affinity column-
there wasnt enough Enac so it didnt work
What approach did they use instead to clone Enac?
Functional expression screen took the mrna from enac producing cells chop up the mrna into small pools chop the mrna up smaller every time- untill they got the smallest amount possible injected into oocytes
What did they see when they measured the current of cells with the pure enac clone and a mixed clone?
Saw bigger currents from the mixed clone- which was confusing
Why was there bigger currents from the mixed clone?
There are 3 genes that code for Enac- each codes for a subunit
The pure clone= alpha, can make a functional Enac channel
but the alpha subunit needs the other subunits (beta and gamma) for full activity
the mixed clone= had more/all of the subunits
What are the mutations in Enac?
GOF= liddles syndrome
hypertension
LOF= PHA
hypotension
