L10 - Aquaporins

Question 1

Where is AQP1 found?

Answer 1

Kidney

RBCs

Question 2

Where is AQP2/3/4 found?

Answer 2

Collecting duct

Question 3

Where is AQP5 found?

Answer 3

Sweat

Question 4

What is diabetes insidious caused by?

Answer 4

Issues in AQP2

Question 5

What is the diffusional water permeability?

Answer 5

Permeability to water when there is no osmotic gradient

Question 6

What happens if a cell is in isotonic solution?

Answer 6

Influx = efflux

No change in volume

Question 7

What is the osmotic water permeability?

Answer 7

Permeability to water when an osmotic gradient is applied

Question 8

What happens if a cell is in hypertonic solution?

Answer 8

Water leaves to balance out osmotic pressure

Cell will shrink

Question 9

What happens if a cell is in hypotonic solution?

Answer 9

Water enters cell to balance out osmotic pressure

Cell will swell –> may burst

Question 10

What does it mean if the Pf/Pd ratio is greater than 1?

Answer 10

A water pore is present in the cell membrane
Could be 
- Aquaporin 
- Urea transporter – UTV 
- Na/glucose transporter

Question 11

What type of cells have they measured Pd and Pf in?

Answer 11

Frog Ovarian Egg
Frog Body Cavity Egg
Xenopus Body Cavity Egg
Zebra Fish Ovarian Egg
Zebra Fish Shed Egg
Amoeba
Trout Egg – unusual properties and composition 
- These are all large single cells

Question 12

What is a Cartesian diver balance?

Answer 12

Small glass funnel with cells in – diver
At the top they put an air bubble – depending on the density of the air bubble the diver will come to a level in the isotonic solution
Then either apply pressure or suction

Question 13

What happens when you apply pressure to a Cartesian diver balance?

Answer 13

Increases pressure in air above solution

Air bubble gets smaller so diver sinks

Question 14

What happens when you apply suction to a Cartesian diver balance?

Answer 14

Decreases pressure in air above solution

Air bubble gets bigger so diver rises

Question 15

How do you keep the diver in a Cartesian diver balance at the correct height?

Answer 15

1. Cells equilibrated in H2O
2. Cells placed into solution containing D2O (heavy water)
3. D2O exchanges with H2O in cells
4. Cells become heavier – diver starts to sink
5. Apply suction to keep the diver at a constant height
6. Measure the pressure needed to keep the diver at a constant height with time

Question 16

What does a Cartesian diver balance allow you to calculate?

Answer 16

Measure the pressure needed to keep the diver at a constant height with time
Pressure/suction proportional to increase in weight of diver
Can then work out how much heavy water has entered the cell

Question 17

How do you calculate Pd from Cartesian diver balance data?

Answer 17

The change in equilibrium pressure was correlated to the change in weight
- Curves off as diffusion comes to a steady state
From this correlation the change in the cell weight could be measured –> now known how much D2O has moved into the cell and the Pd can be calculated

Question 18

What were the results of the Cartesian diver balance with all different cell types?

Answer 18

Rapid exchange of cell water in all cases

1/2 time of the exchange < 4.5 minutes for all the cell types except trout egg cells

Question 19

What were the results of the Cartesian diver balance with trout egg cells?

Answer 19

After 5 hours there was no evidence of D2O moving into these cells
Implies the trout egg cell membrane has no diffusional H2O permeability
- Water does not cross the membrane very easily
- Likely to have a high proportion of cholesterol

Question 20

How do you measure Pf?

Answer 20

Measure the change in cell volume over time when exposed to a hypertonic or hypotonic solution
Measure cell diameter of cell – from this work out volume
- Easy to apply to spherical egg cells
Then use equation
∆V = Pf · SA · t · ∆C

Question 21

What does the equation… mean?

∆V = Pf · SA · t · ∆C

Answer 21

SA – surface area
T – time
C – different in starting concentration between inside and outside cells

Question 22

What were the results of the Pf experiment with all different cell types?

Answer 22

All the cells types swell and have a Pf/Pd ratio >1
- Except trout egg cells  
These cells (except trout egg cells) have some kind of water pore in their membrane

Question 23

Why are the results with trout egg cells different than all other cells?

Answer 23

The cells have no water permeability
Trout is a fresh water fish
- Eggs exposed to high osmotic gradient
- If it had water permeability – egg would take in too much water – swell and burst

Question 24

What is the Pf/Pd ratio for RBC’s?

Answer 24

2.5
RBC’s have water pores 
- Have highest water permeability of any cell – along with proximal tubule cell
  -- Allows Na/Cl reabsorption 
Now know it is AQP1

Question 25

What is the diameter of the water pore in a RBC?

Answer 25

2.5A | At the time (using Cartesian diver and other experiments) they predicted 3.5A

Question 26

What is the protein name for AQP1?

Answer 26

CHIP28

Question 27

What are the functional characteristics of CHIP28?

Answer 27

CHIP28 is a water channel Mercurial Sensitivity of AQP1 – channel blocked by mercurial AQP1 - exist as tetramers

Question 28

What did initial studies trying to identify AQP1 look at?

Answer 28

Rhesus proteins Kept finding a 28kd protein that co-precipitated with a 32kd Rhesus polypeptide Isolated the 28kd protein and produced an antibody

Question 29

What did the antibody to the 28Kd protein find?

Answer 29

Antibody recognises a 28kd protein and a higher molecular weight band - Never found labelling of a 32kd protein - - 28kd and 32kd protein are not related - Higher molecular weight band - glycosylated form of 28kd

Question 30

What was the evidence that AQP1 exists as oligomers in the membrane?

Answer 30

Higher molecular weight bands appear in multiple of 28

Question 31

What area of the body did the antibody stain?

Answer 31

The proximal tubule and descending thin limb Protein present in two cell types with very high water permeability - Suggests protein is a water channel

Question 32

How did they clone AQP1?

Answer 32

of Aquaporin 1 N-terminal portion of 28kd protein was sequenced A combination of PCR and library screening was used to identify the DNA for CHIP28 - Can’t screen RBC library as no DNA

Question 33

What did sequence analysis of AQP1 predict?

Answer 33

Protein with a molecular weight of 28kd

Question 34

What did structural analysis of AQP1 predict?

Answer 34

6 transmembrane spans 42% homology to MIP 26 protein – major protein in lens fibre of eyes – didn’t know function High homology to several proteins with no known function All these related clones have the tandem repeat of the amino acid sequence NPA

Question 35

Which tandem repeat did all the clones of CHIP28 have?

Answer 35

NPA

Question 36

Overall what is the circumstantial evidence that CHIP28 is a water channel?

Answer 36

``` Actual copies of CHIP28 in RBC and biophysical calculations of channel number are in the same range 28.5 kd unit similar in size to 30kd functional unit of proximal tubule water channels CHIP28 transcript (2.9kb) corresponds to the RNA fraction from kidneys that produces greatest water channel activity CHIP28 is resistant to enzymatic digestion as is the RBC water channel ```

Question 37

Overall what is the experimental evidence that CHIP28 is a water channel?

Answer 37

CHIP-28 was expressed in xenopus oocytes and the oocytes were exposed to a hypotonic shock - In control oocytes - volume change is very slow - In oocytes expressing CHIP-28 - volume change is rapid and oocytes explode within minutes CHIP28 confers high water permeability on RBC’s and the proximal tubule

Question 38

The water permeability of RBCs is very sensitive to?

Answer 38

HgCl2 or the organic mercurial pCMBS

Question 39

What is the experimental evidence that AQP1 is mercurial sensitive?

Answer 39

Control oocytes - Pre-incubation in HgCl2 has no effect Oocytes expressing AQP1 - Pre-incubation in HgCl2 slows the volume change - The reducing agent ß-mercatoethanol reversed the Hg-induced inhibition

Question 40

How do mercurial agents exert their action?

Answer 40

By binding to cysteine residues

Question 41

Does AQP1 contain cysteine residues?

Answer 41

4 cysteine residues at amino acid positions 87,102,152 and 189

Question 42

How did they test which cysteine residue in AQP1 mercurial acted on?

Answer 42

Individually mutate each cysteine residues to serine | Test effect of HgCl2 on water permeability in these mutants

Question 43

Results of the test to see which cysteine residue in AQP1 mercurial acted on?

Answer 43

Mutants have the same water permeability as wild type AQP1 - HgCl2 reduced water permeability of C87S, C102S and C152S mutants - HgCl2 has no effect on C189S mutant - - Conclude that Hg binds to Cys 189

Question 44

What is the name of the model for the structure of AQP1?

Answer 44

Hourglass model

Question 45

What is the hourglass model of AQP1?

Answer 45

Amino and Carboxy ends of the protein are both intracellular Six transmembrane spanning domains Between TM 2-3 and TM 5-6 - Loops B and E which dip into the membrane - In the middle of each of these loops is the NPA motif As protein folds in the membrane the two NPA motifs come together --> produces a channel pore

Question 46

How does mercurial sensitivity fit in with the hourglass model?

Answer 46

Cysteine 189 confers mercurial sensitivity – in hourglass model this lies at the opening of the predicted pore