Lecture 02 Salt Absorption I Flashcards
What does frog skin model? What is it a relevant model of?
tight epithelium - outside as apical and inside as basolateral membrane
renal collecting duct, distal colon, salivary gland, sweat duct, airway surface epithelium
What are the advantages of using frog skin?
large surface area, cheap, easy to get hold of, robust
What experiment did Ussing and Zerahn carry out in 1951?
used ussing chamber with chamber 1 representing the outside and apical side of the frog skin
chamber 2 representing the inside and the basolateral side of the frog skin
both chambers full of Krebs (bicarb) solution so there was no net movement of ions
injected radioactive sodium-24 into chamber 1 and measured the radioactivity in both chambers over time
What did Ussing and Zerahn’s 1951 experiment allow them to calculate?
how many moles of sodium-24 had been transported per unit time
this allowed them to work backwards to calculate the SCC
What was required to calculate the SCC for sodium?
an active step
i.e. sodium-potassium ATPase providing active transportation of sodium on the basolateral membrane
What experiment did Ussing and Zerahn carry out in 1958?
electrophysiology experiment looking at the properties of the apical membrane
used an intracellular microelectrode and injected different sodium concentrations into the extracellular chamber to cause changes in the Nernst potential
What did Ussing and Zerahn predict for their experiment in 1958?
the presence of a sodium channel in the apical membrane and a sodium-potassium pump in the basolateral membrane to allow net uptake of sodium across the cell
if there was a sodium channel present in the apical membrane the membrane potential would move with the Nernst potential for sodium
What was the result of the 1958 experiment by Ussing and Zerahn?
by calculating the Nernst potential it was shown that the apical membrane was permeable to sodium and therefore a sodium channel must be present in the apical membrane allowing net uptake of sodium across the cell
What did the experiment by Mall et al in 1999 show?
used human colon cells from biopsy which showed a transepithelial potential of -1mV
known action of amiloride to block ENaC
when amiloride added shift in transepithelial potential to 0 and therefore a sodium channel must be present in the apical membrane
What are the disadvantages of frog skin as a model?
frog skin has a very negative transepithelial potential in humans it is -1
more leak-back in humans and less tight epithelia
frog skin almost only absorbs sodium in human colon there are many different ion uptake that will effect the potential
What did the experiment by Mall et al in 1998 show?
used human nasal cells as a model for the upper airway epithelium
added amiloride and observed hyper polarisation moving the membrane potential away from the Nernst potential for sodium
This means there are sodium-selective channels in the apical membrane of the nasal epithelium
What evidence showed direct evidence of the presence of the sodium channel in the apical membrane?
patch clamp technique by Neher and Sackmann, 1981
using single channel analysis on renal collecting duct cells
What did the experiment by Canessa et al in 1995 show?
expression cloning using a screen by functional expression
took all mRNA from cells chopped into up and divided into 10 pools
took each pool and injected into Xenopus oocytes to see which pool would present the sodium-selctive amiloride-inhabitable currents for functional expression
repeated this until couldn’t go smaller
How was ENaC stimulated in rat colon cells? (Canessa 1995 experiment)
rats fed on a low sodium diet
salt depletion caused aldosterone release
aldosterone induced production of ENaC mRNA
What is the structure of ENaC? How does this affect the function of the channel?
3 subunits needed for normal function alpha beta and gamma
alpha subunit forms the channel but needs beta and gamma subunits for full activity
when all subunits present together the currents measured are 10 times larger that two subunits alone
