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Epithelial Physiology > L10 - Aquaporins > Flashcards

L10 - Aquaporins Flashcards

1
Q

Where is AQP1 found?

A

Kidney

RBCs

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2
Q

Where is AQP2/3/4 found?

A

Collecting duct

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3
Q

Where is AQP5 found?

A

Sweat

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4
Q

What is diabetes insidious caused by?

A

Issues in AQP2

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5
Q

What is the diffusional water permeability?

A

Permeability to water when there is no osmotic gradient

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6
Q

What happens if a cell is in isotonic solution?

A

Influx = efflux

No change in volume

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7
Q

What is the osmotic water permeability?

A

Permeability to water when an osmotic gradient is applied

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8
Q

What happens if a cell is in hypertonic solution?

A

Water leaves to balance out osmotic pressure

Cell will shrink

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9
Q

What happens if a cell is in hypotonic solution?

A

Water enters cell to balance out osmotic pressure

Cell will swell –> may burst

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10
Q

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

A 
A water pore is present in the cell membrane
Could be 
- Aquaporin 
- Urea transporter – UTV 
- Na/glucose transporter
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11
Q

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

A 
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
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12
Q

What is a Cartesian diver balance?

A

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

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13
Q

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

A

Increases pressure in air above solution

Air bubble gets smaller so diver sinks

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14
Q

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

A

Decreases pressure in air above solution

Air bubble gets bigger so diver rises

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15
Q

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

A
  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
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16
Q

What does a Cartesian diver balance allow you to calculate?

A

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

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17
Q

How do you calculate Pd from Cartesian diver balance data?

A

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

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18
Q

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

A

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

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19
Q

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

A

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

20
Q

How do you measure Pf?

A

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

21
Q

What does the equation… mean?

∆V = Pf · SA · t · ∆C

A

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

22
Q

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

A 
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
23
Q

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

A

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

24
Q

What is the Pf/Pd ratio for RBC’s?

A 
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
25
Q

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

A

2.5A

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

26
Q

What is the protein name for AQP1?

A

CHIP28

27
Q

What are the functional characteristics of CHIP28?

A

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

28
Q

What did initial studies trying to identify AQP1 look at?

A

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

29
Q

What did the antibody to the 28Kd protein find?

A

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
30
Q

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

A

Higher molecular weight bands appear in multiple of 28

31
Q

What area of the body did the antibody stain?

A

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

32
Q

How did they clone AQP1?

A

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

33
Q

What did sequence analysis of AQP1 predict?

A

Protein with a molecular weight of 28kd

34
Q

What did structural analysis of AQP1 predict?

A

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

35
Q

Which tandem repeat did all the clones of CHIP28 have?

A

NPA

36
Q

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

A 
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
37
Q

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

A

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

38
Q

The water permeability of RBCs is very sensitive to?

A

HgCl2 or the organic mercurial pCMBS

39
Q

What is the experimental evidence that AQP1 is mercurial sensitive?

A

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

40
Q

How do mercurial agents exert their action?

A

By binding to cysteine residues

41
Q

Does AQP1 contain cysteine residues?

A

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

42
Q

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

A

Individually mutate each cysteine residues to serine

Test effect of HgCl2 on water permeability in these mutants

43
Q

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

A

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
44
Q

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

A

Hourglass model

45
Q

What is the hourglass model of AQP1?

A

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

46
Q

How does mercurial sensitivity fit in with the hourglass model?

A

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

Epithelial Physiology (10 decks)

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