History of the water pore Flashcards
Pd - Diffusional water permeability - what does it refer to? Why can we measure this?
Permeability to water in the absence of an osmotic gradient - can measure this because there is continuous exchange of water across a gradient
What conditions can be seen in an isotonic solution?
Influx of water = efflux.
No change in volume of cells is observed
Rate of movement of water = rate of diffusion of water
What conditions can be seen in hypotonic solution?
Water moves out of cells causing them to shrink. The opposite it seen in hypertonic conditions –> cells swell and burst as too much water moves in
Pf - osmotic water permability - what does this refer to?
Permeability to water when an osmotic gradient is applied
What does it mean when the ratio of Pf:Pd is more than 1?
A water pore is present –> provides a pathway for water to move along an osmotic gradient
CARTESIAN DIVER BALANCE - impact of placing cell in D20 solution?
D20 higher density so moves into cells and H20 moves out – causes the weight of the diver to go up and the diver to sink
CARTESIAN DIVER BALANCE - impact of applying suction to the solution?
Allows correllation of the pressure when D20 solution is used
Keeps the diver at an equilibrium pressure when D20 is applied (the longer spent in the D20)
How can you calculate the Pd of a cell using the diver balance method?
Calculate how much D20 has moved into the cell over time
Cell types used to measure Pd though diver balance method?
- Frog egg
- Amoeba
- Zebrafish ovarian egg
- Xenopus body cavity egg
What was found regarding the different cell types for the diver method? Why was this cell different?
All showed rapid exchange of water apart from trout eggs - lipid composition of trout egg membrane different (more cholesterol so tighter membrane and reduced fluidity observed)
Equation for measuring change in cell volume?
change in Vol = Pf x Surface area x time x change in concentration gradient
Red blood cell - how big is its Pd and Pf? Why is this the case?
Pf = 1.5x10 to power -14 cm3/s
Pd = 5.3x10 to power -3 cm3/s
There are loads of water pores- around 200,000 per cell!!!
Why are eggs used to measure Pf ?
Round in shape so easy to measure diameter therefore change in cell volume when placed in different concentrat
Why did zebrafish eggs not swell in a hypotonic solution?
Fresh water animal so low permeability to H20 as the animal exists in a low osmolarity solution –> otherwise eggs would swell up and burst!
AQP1 - strucure, location, why is it hard to clone?
4 sub units, 6 tmds
Found in proximal tubule and thick descending limb of the nephron
Hard to clone as it is difficult to isolate a protein linked to permability
Found to be a 28kd protein abundant at the cell membrane –> critical for h2o transport
What suggested that AQP2 was a water pore after cloning?
-N terminus found to contain NPA TANDEM REPEAT!!!!
This particular repeat sequence is critical for H20 transport
-resistant to enzymatic degradation (known RBC h20 channel also has this property)
-protein size similar to that of h20 channel found in the proximal tubule
Experimental evidence for AQP2 - what happened when the protein was overexpressed in xenopus oocytes and then the eggs exposed to hypotonic shock?
Eggs swelled and burst!
Rapid change in volume of cells
THE PERMEABILITY TO WATER HAD INCREASED
AQP1 experimental evidence - background for procedure?
The water permeability of red blood cells is very sensitivity to HgCl2 or the organic mercurial pCMBS
AQP1 experimental evidence - what was shown when oocytes were pre-incubated with HgCl2?
Pre-incubation in HgCl2 slows the volume change in oocytes expressing AQP1
Pre-incubation in HgCl2 has no effect upon control oocytes
AQP1 - what is meant by the hourglass model?
As the protein folds in the membrane the two NPA motifs come to lie together – produces a channel pore – the hourglass model.
The Cysteine residue at position 189 (C189) confers mercurial sensitivity – in the hourglass model this lies at the opening of the predicted pore.
Testing basis of mercurial action on AQP1 – how was this carried out? What was the basis for this?
Mercurial agents exert an action by binding to cysteine residues of AQP1
—> HgCl2 has no effect on C189S mutant - conclude that Hg binds to Cys 189.
