what is frog skin a good model for?
Frog skin is a good model for tight epithelium
- good model for RCD, DC, salivary gland, sweat duct, airway surface epithelium
how is radioactive sodium used in ussing chamber?
Radioactive Na used in ussing chambers, put in chamber 1 and look at amount of RA sodium in chamber 2 to see how much has transported through frog skin
- also tested to see is Na could be transported the other way
is frog skin sodium absorbing or secreting epithelium?
Frog skin is sodium absorbing epithelium (apical to basal)- transport of sodium is directed into the frog - is a small leak out of frog
what is needed for the net transport of sodium?
Active transport is needed for the net movement of sodium
- active process is sodium potassium atpase on basolateral membrane
can you meausre recycling with an ussing chamber?
No, can only measure net transport with an ussing chamber - cannot measure the recycling or leaking of ions
what is Isc?
Short circuit current - measure of the net transport taking place across epithelium (representative of Vte)
what is cell driven towards if sodium channels are open?
Driven towards nernst for sodium. - is change EC sodium - nernst changes so membrane potential also changes is apical selective sodium channels
what happens when amiloride is added to human colonic biopsy?
potential shifts to 0, less sodium absorption into the cell
What does Na entry do to the cell?
depolarises it
what happens to the cell is amiloride is added to nasal biopsy cells?
- cell hyperpolarises as Na+ absorption/entry via ENaC is blocked (potassium channels predominate resting potential)
- when amiloride washed out resting potential returns to normal
what can patch clamp analysis of ENaC show?
- can directly measure the function of a channel
- shows was a Na+ selective channel
why did conventional cloning of ENaC not work?
There was not enough ENaC protien in native cells
How did caressa et al clone ENaC?
- expression cloning
- tissue source colon - feed rats low salt diet - induces ENaC via increased aldosterone - this maximises amount of ENaC in colon
- extract polA RNA - make into mRNA
- cDNA cloned in expression vector
- cDNA divided into 10 pools - inject into xenopus oocyte and use patch clamp to see if amiloride sensitive (is were sodium selcetive channels in pools)
- Took positive pool and split into pools again
- amount of mRNA got smaller until found peice of mRNA which coded for ENaC (a subunit)
how may subunits does ENaC have?
3 subunits, a,b,y
- a is the channel but b and y are both needed for full activation/function of the channel
what can GOF mutations in ENaC cause?
Liddles syndrome
Give some of the features/symptoms of liddles symtoms
- hypertension
- increase in ENaC
- ## Nedd4 (channel retriever) cannot endocytose ENaC from membrane
what can be used to treat liddles syndrome?
amiloride - diuretic
What can LOF in ENaC cause?
PHA - pseudohypoaldosteronism
Give some symptoms of pseudohypoaldosteronism
- hypotension
- salt wasting
- increased urine flow rate
- ENaC inactive? isnt working properly
- failure in response to aldosterone
