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BMS346 - Epithelial Physiology > L10-13 Aquaporins > Flashcards

L10-13 Aquaporins Flashcards

1
Q

What is the diffusional water permeability

A

The permeability at which there is no osmotic gradient

Pd

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2
Q

Describe the movement of water at the diffusional water permeability

A

There is no net movement of water
Influx = Efflux
No change in cell volume as a result

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3
Q

Describe what is meant by osmotic water permeability

A

Where water will move to balance out and resolve the osmotic gradient
Pf

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4
Q

What is seen when a cell is placed in a hypertonic solution

A

High solute

So water moves out of the cell

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5
Q

What is seen when a cell is placed in a hypotonic solution

A

Low solute

Water moves in and cell can lyse

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6
Q

Pd

A

Diffusional water permeability

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7
Q

Pf

A

Osmotic water permeability

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8
Q

What is known if Pf/Pd is greater than one

A

Water pore present in the membrane

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9
Q

Who performed experiements looking at Pd and Pd in various cell types

A

Prescott and Zeuthen

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10
Q

What system allows the measurement of Pd

A

Using a carteisan diver

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11
Q

Describe the experimental set up of the cartesian diver

A

Small glass funnel with cells on and air bubble on top

DEPENDING ON THE DENSITY OF THE AIR BUBLE THE DIVER WILL COME TO A SPECIFIC LEVEL

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12
Q

Describe what happens when suction is applied to the diver

A

Suction - negative pressure
Air bubble will expand
Diver will move up

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13
Q

Describe what happens when pressure is applied to the diver

A

Pressure - positive pressure
Air bubble will expand
Diver moves up

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14
Q

Describe how Pf can be determined from the cartesian diver system

A

Equilibriate the system with D2O (heavy water)
Place cells in solution so D2O is exchanged with H2O
Diver will start to sink as getting heavier
Suction applied
MEASURE THE AMMOUNT OF SUCTION THAT IS NEEDED TO KEEP THE DIVER AT A CONSTANT LEVEL
From this you can calculate the ammount of D2O that has entered the cells and can then calculate the Pf

IN A SERIES OF EXPERIMENTS THE CHANGE IN PRESSURE WAS CORRELATED TO THE CHANGE IN WEIGHT
Know how much D2O has moved into the cell and the Pd could be calculated

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15
Q

In the vast majority of cel types what was seen in the diver expt

A

Rapid exchange of cell water in vast majority of cases

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16
Q

In most of the cell types where water exchange happened what was the t1/2 of exchange

A

4.5 mins

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17
Q

What cell type was there no movement of water

A

Trout egg cell

After 5 hours there was no evidence of D2O moving into the cell
Implies that the membran has no diffusionaly H2O permeability

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18
Q

What would we expect to be present in high quanitities in the trout egg cell membrane

A

High levels of cholesterol - reduction in permeability

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19
Q

Describe a much simpler method for measuing Pf

A

Measure the change in cell volume when exposed to a hyper/hypo tonic solution

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20
Q

Formaula for the change in cell volume approach

A

DeltaV = Pf . SA. t. deltaC

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21
Q

Why is the change in cell volume easy to use on egg cells

A

Since cells are spherical

Can simply measure the change in cell diameter

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22
Q

What is seen in all of the cells (except the trout egg cells) when they are placed in a hypotonic solution

A

All swell - this suggests that they all have a water pore in the membrane

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23
Q

Why may the trout egg cell not have a water pore in the membrane

A

Since trout will lay in fresh water - so will be exposed to a large osmotic gradient

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24
Q

Describe how the Pf of the zebrafish egg canges

A

High in mother - since will be in an isotonic environment

Drops when laid since laid into freshwater and will be exposed to a high osmotic gradient

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25
Q

What is the Pf/Pd of a red blood cell - what does this tell us?

A

2.5
So must have a water pore in the membrane
We now know that this is AQP-1

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26
Q

Approx how big is the AQP1 pore

How many copies per cell

A

3.5A

100’000 copies per cell

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27
Q

AQP1 was first characterised as

A

CHIP28

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28
Q

Functional characteristics of AQP1

A

Water channel
Mercury sens
Tetrameric - four functional units

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29
Q

How was the AQP1 protein initially discovered

A

By accident
When running Rhesus proteins on a gel - found a 28kD that co precipitated with a 32kD Rhesus polypeptide
Isolation and production of an antibody

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30
Q

What did the AB produced to recognise the 28kD NOT DETECT

A

The 32KD protein

Concluded that these two proteins are not related

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31
Q

Why are two bands of CHIP28 often seen

A

Glycosylation

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32
Q

Describe why bands were seen at various mutliples of 28kD

A

Because there is evidence for oligomerisation of AQP1 in the membrane

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33
Q

Describe IHC of the a-CHIP28

A

Stains the proximal tubule and the thin descending limb

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34
Q

Describe how CHIP28 was cloned

A

N-termianl portion was sequenced
With this information a combination of PCR and library screening was used to find the sequence for CHIP28

THIS LEAD TO THE DETECTION OF A SEQUENCE THAT WAS 28KD IN WEIGHT - AS TO BE EXPECTED

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35
Q

Describe the results of the structural analysis of CHIP28

A

6 TMDs
N and C found IC
42% homology to MIP26 and high homology to several proteins with no known function

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36
Q

What was found in common between CHIP28 and several other proteins

A

Tandem NPA repeat
Between spans 2/3 and 5/6 - this is seen as the calling code for aquaporins
Speculation that CHIP28 is involvd in water transport

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37
Q

Describe the four pieces of circumstantial evidence that CHIP28 acts as a water channel

A

Actual copies of CIP28 in RBC and calculations of channel numbers are in the region
28.5kD unit similar in size to the 30kd functional unit of the proximal tubule water channels
CHIP28 transcript corresponds to the RNA frction from kidneys that produces the greatest water channel activity
CHIP28 is resistanct to enzymatic digestion as it is the RBC water channel

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38
Q

Describe the experimental evidence tha CHIP28 acts as a water channel

A

In xenopus oocytes - CHIP28 exp - exposed to hypotonic shock
In control volume change is v slow - but in CHIP28 OE change is rapid - oocytes explode within a few mins

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39
Q

Conclusions of the OE of CHIP28 in Ex Oocytes

A

CHIP28 is able to confer high water permeability of the RBC and proximal tubule

40
Q

What is the background to the discovery that mercury can act on the AQPs

A

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

41
Q

Experimental evidence that mercury has an affect on the water channel AQP1

A

Pre-incubation with HgCl2 has no effect on control Oo

Pre inc with HgCl2 in oocytes OE AQP1 leads to a slowing of the volume change
Thus specific to AQP1

42
Q

Icing on the cake expt for the mercurial sensitivity studies

A

Reducing agent b-mercaptoethanol reverses the inhibition since it is able to reverse the binding of Hg to cystenine residues

43
Q

How does the mercurial agent act

A

By binding cysteine residues

44
Q

Where are the cys residues located in AQP1

A

87 102 152 189

45
Q

Describe how it was determined where Hg is binding to AQP1

A

Make mutants at each of the positions …. Where swap C for S
Test for sensitivity

46
Q

Describe the reuslts of the CXXS mutant studies

A

HgCl2 reduces water perm in the C87S, C102S and C152S to the same level in the WT - Hg is not binding here
HgCL2 has no effect on C189S — so Hg binding to the serine at position 189

47
Q

Describe the hourglass model of AQP1

A

N and C both foud IC
Six TMDs
B and E loops that dip into the middle - in these loops are the NPA motifs
As the protein folds the NPA motifs come together and this produces a pore

Cys 189 - confers Hg sensitivty and we know this since this lies in the opening of the pore

48
Q

What is the effect of mutating alanine in the B loop NPA motif

A

Mutate to larger residue - blocks the movement of water indicating that there is a pore through the membrane

49
Q

AQP1 is formed as a

A

Tetramer

50
Q

When it wadiscovered that AQP1 was a tetramer what was the next step

A

To determine if the pore is through the middle of the tetramer of ife

51
Q

Describe the experimental conditions required to to work out if the functional unit of AQP1 is a monomer or a tetramer

A

Construction of tandem dimers of WT AQP1 ans C189S mutants and express in xenopus oocytes
Forces the tetramer to have two WT units and two C189S SU

52
Q

Describe the two things we would expect to see if the monomer/tetramer was the functional unit in C189S

A

If monomer - Expect a decrease of around 50% when Hg applied
If tetramer - expect and all-or-nothing decrease

53
Q

Results of the tandem dimer studies

A

AQP1-C189s - decrease by 50%
C189S-C189S - no effect
WT-WT - full decrease

54
Q

What was the conclusions of the tandem dimer studies

A

Each monomer is a functional H2O channel

55
Q

Describe the selectivity of the AQP channels

A

Selective to water - not protons

56
Q

Why would it be a suprise that protons werent able to be transported by AQP1

WHY IS THIS NOT THE CASE

A

Since H-bonds would be expected to form with water and the H ions piggyback down on th water and through the channel

As the water moves down the H bonds are brikes and interact with Asp residues - this is similar to a SELECTIVITY FILTER

57
Q

What can also move through the pore

A

CO2

58
Q

Describe an AQP1 bacterial homologue that is resitant to Hg

How was this used

What was found

A

Homologue that lacks Cys 189 (also lacking in AQP4)
Mutate the C189 in
Becimes mercury sens
Can crystalise the strucutre with mercury bound

Found a mercuty binding site at the pore - when bound water unable to move through the pore

59
Q

How many Hg binding sites

A

2

Dont know the effect of the other one

60
Q

What is AQP3 also permemable to

A

Glycerol and urea

61
Q

How was it shown AQP3 is permeable to urea and glycerol

A

Take oocytes exp AQP3 in radio-glycerol solution

Can quantify radio-glycerol within the cell

62
Q

What is the effect of Hg on urea and glycerol permemability?

A

Mercury inhibits this increase in glycerol and urea permeability - not seen in AQP1
Suggests urea and glycerol going through the same pore since Hg blocks it

63
Q

What is AQP3 also known as

A

An aqua glycerol porin since is permeable to other solutes

64
Q

Where is AQP6 seen to localise to

A

IC vesicles of the alpha intercalating cells

65
Q

What type of condcutance does AQP6 show

A

Low water perm and a Cl conductance

66
Q

Likely Cl moving

A

Through THE CENTRAL PORE OF THE TETRAMER

67
Q

What is the effect of Hg on the conductance of AQP6

A

Hg increases the conductance

68
Q

What else stimulates the H20 perm and Cl conductance

How was this seen

A

Acidic pH

Use a two electrode voltage clamp -

69
Q

What in AQP6 is linked to the pH sens

Evidence

A

Specific histidine residue

If removed then pH sens lost

70
Q

What is the AQP6 actually doing in vesicles

A

Acting as a charge balancer

As H+ moves in AQP6 activated and Cl moves in through AQP6 to balance the charge

71
Q

How many members of the MIP family

A

Over 900 members

72
Q

What are the 3 classed of MIPs

A

Aquaporins
Aquaglyceroporins
Unorthodox or superaquaporins

73
Q

How many AQPs in humans

A

13

74
Q

What are AQPs primarily selective for

A

Water - some gases

75
Q

Where is AQP0 found

A

Lens

76
Q

Location of 1-4

A

Kidney

77
Q

Location of 3-7

A

Kidney

78
Q

Location of 7

Role here

A

Proximal tubule

Responsible for the reabsoption of glycerol and urea

79
Q

6 has a __ conduct

A

Cl

80
Q

8 is a

A

Ammonia channel

81
Q

11-12 tend to be

A

IC

82
Q

11 linked to what disease

What was seen in KO studies

A

PCKD

KO leads to development of large vacuoles

83
Q

AQP2 linked to what disease

A

Diabetes insipidus

84
Q

Describe the insertion of AQP2 into the apical membrane

A

VP binds to V2R on the basolateral membrane
Gs subunit
Activation of AC
Production of cAMP
Activation of PK-A
Phorpshory,ation of AQP2 in vesicles
Insertion of AQP2 into the apical membrane

85
Q

In absence of AVP What is the permeability of the alpha intercalated cell

A

V low

86
Q

Two examples of reabsorptive epithelia

What predominantly mediates water transport in each one

A

Reabsorptive
Proximal tubule - AQP1
Small intestine - NO AQPS

87
Q

An example of secretive epithelia - what AQP is present

A

AQP5

88
Q

Water permeability of the proximal tubule

A

Very high

78% contributed by AQP1

89
Q

Describe the effect of AQP1 KO on mice

A

Perfuse the lumen and basolateral side after mounting on a fusion pipette with an osmotic gradient
Reduction of water permeability

90
Q

Describe the conditions that reabsoprtion occurs under

A

Near isotonic conditions gradieint still required but this is very small - 1/2 mosm

91
Q

Describe what happens to the osmotic gradient in the proximal tubules

A

AQP1 in both apical and basolateral membranes
Reabsorption by a large number of cells
See the blood side is relatively hypertonic cf the lumen side
Gradient is dissipated alsmost immediateyly since water permemability is so high

92
Q

Describe what happens wher you replace the basolateral solition with mineral oil and perfuse normal saline through the lumen

A

Reabsorption of Na and other solutes

Absorbate is slightly hypertonic and this drives water reabsorption

93
Q

Describe the micropuncture expts

What did they see

What were the conclusions

A

Perfuse lumen and caps with a 154mM NaCl solution
PT begins reabsorbing
When collection made at the end the osmolality has gone down - is slighlty higer in the capillary is higher

OSM GDT OF AS A LITTLE AS TWO OSM IS DRIVING THIS

94
Q

Overall conclusion from the micropuncture expt

A

Transport by the proximal tubule generates a favourable osmotic graeitn for water reabsorption

Because of the extremtly high water permemability of the tubule this gradient is small and is probably due to the generation of a hypotonic lumen

95
Q

What is seen in the AQP1 KO mouse - regarding the change in Osm

A

In Wt smaller osmoitic gdt between lumen and the blood
In KO this larger gdt and the end of the PT is slightly hypotonic

This makes sense since we have rebaorption of solutes but water does not follow them

96
Q

Describe the secretion of fluid from the saliveray acinar cells

A

NKCC1 - brings Cl into the cells which is secreted across the apical membrane
Na follows through tight junctions
THIS MAKES THE LUMEN SLIGHTLY HYPERTONIC
Water moves across the apical membrane throguh AQP5
Near isosmotic fluid secretion

97
Q

Describe what is seen in the AQP5 KO

A

Loss prevents the dissipation of lumen hypertonicity

WT -
High rate of fluid sec, Na, K, Cl where we expect- water follows

KO -
Fluid secretion rate decrease
No pathway for the movement of water across the apical membrane
Hypertonic solutes in the lumen - production of a small volume of saliva with a high salt content

BMS346 - Epithelial Physiology (10 decks)

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