Molecular physiology of salt reabsorption Flashcards
Which epithelia is froh skin a suitable model for
Renal collecting duct, distal colon, salivary gland, sweat duct, airway surface epithelia
What is the ussing chamber technique
The technique is used to measure the short-circuit current as an indicator of net ion transport taking place across an epithelium. There are two chambers containing two experimental solutions, separated by the epithelia under investigation. There are two electrodes in each solution, connected to an amplifier which allows us to measure the trans-epithelial voltage (Vte). There are two more elctrodes which inject current which allows us to measure the trans-epithelial resistance and from Ohm’s law, derive the short circuit current (SCC).
What was the first experiment done on frog skin to investigate Na transport across the epithelium
They added 24Na (radioactive) into one of the chambers. This enabled them to detect radioactive counts in the other chamber and allowed them to measure Na transport (the reverse experiment can be done in order to determine the amount of Na leakage)
What did the experiment on frog skin epithelia show about the movement of Na ions
By measuring the amount of Na being transported from the pond side to the body side, as well as Na movement back to the pond side, they could calculate net Na movement. This showed that the net movement of Na was reabsorption into the body. It was shown that the total current generated was due to Na because the current of Na influx - Na outflux was almost identical to the total current generated.
What is a requirement of the ussing chamber method and how is this achieved in frog skin epithelia
Requires there to be an active component in the reabsorption of the investigated ion. In this case it is the Na/Katpase creating a driving force for the net reabsorption of Na acros the apical membrane by ENaC.
What electrophysiology experiments were performed on frog skin epithelia and what did they show>
Used to investigate if the apical membane of the frog skin epithelia was permeable to Na. If it is and electrogenic, then opening of these channels will drive the Vte towards the nernst potential of Na. If the extracellular concentration of Na is then changed then the Nernst potential will also change, changing the Vte recorded as a result. They found that as the extracellular conc of Na was increased, so did the Vte, this showed there were Na channels in the apical membrane.
What effect does amiloride have on cells from human colon biopsies
The colon absorbs Na (it has an apical Na channel and a basolateral NaKATPase). Amiloride blocks ENaC in the epithelium and the Vte shifts towards 0 because we can no longer absorb the positive Na ions. If the amiloride was placed on the basolateral side then no change was seen because ENaC is only present in the apical membrane.
What studies were performed on nasal epithelia and what did it show
Nasal epithlia are a good representitive epithelia for the upper airway. Amiloride applied to the apical membrane of the epithelia caused a hyperpolarisation shift in the Vte. This shows that amiloride is causing a shift away from the nernst of Na, and that human epithelial cells have ENaC on the apical membrane and this channel is helping Na to be reabsorbed.
What did patch clamp analysis of ENaC reveal
This directly measures ENaC single channel function. They did an outisde - out patch in renal collecting duct cells and say many steps up and down, indicating that ENaC channels are opening and closing.
Why was the convention al cloning method at the time unsuccessful in cloning ENaC
After isolating ENaC by using an amiloride affinity column, huge amounts of protein are needed to perform the sequencing, however in native cells the amount of ENaC is very small. Not enough to be able to perform the sequencing.
What approach successfully cloned ENaC
Native tissues were taken and performed a functional expression approach. They took salt depleted rats, meaning aldosterone expression is high and subsequently so is ENaC mRNA. They then took polyARNA from kidney cells and chopped it into bits and split it into 10 pools. Each pool was injected into oocytes and the amount of amiloride sensitive current was measured. The one pool that exhibits ASC contains ENaC so this pool is split further until a small section of mRNA is left. This was discovered to be the alpha subinit of ENaC
How were the beta and gamma subunits of ENaC discovered.
Less pure samples produced bigger amiloride sensitive currents even though there was less alpha ENaC present. The same process was performed to investigate these which were the beta and gamma subunits which come together to form a fully functional ENaC channel.
What diseases are associated with ENaC
LOF = PHA - hypotension due to loss of Na and water reabsorption
Liddles syndrome GOF - hypertension due to too much ENaC and water reabsorption.
