History of the water pore

Question 1

Pd - Diffusional water permeability - what does it refer to? Why can we measure this?

Answer 1

Permeability to water in the absence of an osmotic gradient - can measure this because there is continuous exchange of water across a gradient

Question 2

What conditions can be seen in an isotonic solution?

Answer 2

Influx of water = efflux.
No change in volume of cells is observed
Rate of movement of water = rate of diffusion of water

Question 3

What conditions can be seen in hypotonic solution?

Answer 3

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

Question 4

Pf - osmotic water permability - what does this refer to?

Answer 4

Permeability to water when an osmotic gradient is applied

Question 5

What does it mean when the ratio of Pf:Pd is more than 1?

Answer 5

A water pore is present –> provides a pathway for water to move along an osmotic gradient

Question 6

CARTESIAN DIVER BALANCE - impact of placing cell in D20 solution?

Answer 6

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

Question 7

CARTESIAN DIVER BALANCE - impact of applying suction to the solution?

Answer 7

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)

Question 8

How can you calculate the Pd of a cell using the diver balance method?

Answer 8

Calculate how much D20 has moved into the cell over time

Question 9

Cell types used to measure Pd though diver balance method?

Answer 9

• Frog egg
• Amoeba
• Zebrafish ovarian egg
• Xenopus body cavity egg

Question 10

What was found regarding the different cell types for the diver method? Why was this cell different?

Answer 10

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)

Question 11

Equation for measuring change in cell volume?

Answer 11

change in Vol = Pf x Surface area x time x change in concentration gradient

Question 12

Red blood cell - how big is its Pd and Pf? Why is this the case?

Answer 12

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!!!

Question 13

Why are eggs used to measure Pf ?

Answer 13

Round in shape so easy to measure diameter therefore change in cell volume when placed in different concentrat

Question 14

Why did zebrafish eggs not swell in a hypotonic solution?

Answer 14

Fresh water animal so low permeability to H20 as the animal exists in a low osmolarity solution –> otherwise eggs would swell up and burst!

Question 15

AQP1 - strucure, location, why is it hard to clone?

Answer 15

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

Question 16

What suggested that AQP2 was a water pore after cloning?

Answer 16

-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

Question 17

Experimental evidence for AQP2 - what happened when the protein was overexpressed in xenopus oocytes and then the eggs exposed to hypotonic shock?

Answer 17

Eggs swelled and burst!
Rapid change in volume of cells
THE PERMEABILITY TO WATER HAD INCREASED

Question 18

AQP1 experimental evidence - background for procedure?

Answer 18

The water permeability of red blood cells is very sensitivity to HgCl2 or the organic mercurial pCMBS

Question 19

AQP1 experimental evidence - what was shown when oocytes were pre-incubated with HgCl2?

Answer 19

Pre-incubation in HgCl2 slows the volume change in oocytes expressing AQP1

Pre-incubation in HgCl2 has no effect upon control oocytes

Question 20

AQP1 - what is meant by the hourglass model?

Answer 20

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.

Question 21

Testing basis of mercurial action on AQP1 – how was this carried out? What was the basis for this?

Answer 21

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.