L10-13 Aquaporins Flashcards
What is the diffusional water permeability
The permeability at which there is no osmotic gradient
Pd
Describe the movement of water at the diffusional water permeability
There is no net movement of water
Influx = Efflux
No change in cell volume as a result
Describe what is meant by osmotic water permeability
Where water will move to balance out and resolve the osmotic gradient
Pf
What is seen when a cell is placed in a hypertonic solution
High solute
So water moves out of the cell
What is seen when a cell is placed in a hypotonic solution
Low solute
Water moves in and cell can lyse
Pd
Diffusional water permeability
Pf
Osmotic water permeability
What is known if Pf/Pd is greater than one
Water pore present in the membrane
Who performed experiements looking at Pd and Pd in various cell types
Prescott and Zeuthen
What system allows the measurement of Pd
Using a carteisan diver
Describe the experimental set up of the cartesian diver
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
Describe what happens when suction is applied to the diver
Suction - negative pressure
Air bubble will expand
Diver will move up
Describe what happens when pressure is applied to the diver
Pressure - positive pressure
Air bubble will expand
Diver moves up
Describe how Pf can be determined from the cartesian diver system
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
In the vast majority of cel types what was seen in the diver expt
Rapid exchange of cell water in vast majority of cases
In most of the cell types where water exchange happened what was the t1/2 of exchange
4.5 mins
What cell type was there no movement of water
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
What would we expect to be present in high quanitities in the trout egg cell membrane
High levels of cholesterol - reduction in permeability
Describe a much simpler method for measuing Pf
Measure the change in cell volume when exposed to a hyper/hypo tonic solution
Formaula for the change in cell volume approach
DeltaV = Pf . SA. t. deltaC
Why is the change in cell volume easy to use on egg cells
Since cells are spherical
Can simply measure the change in cell diameter
What is seen in all of the cells (except the trout egg cells) when they are placed in a hypotonic solution
All swell - this suggests that they all have a water pore in the membrane
Why may the trout egg cell not have a water pore in the membrane
Since trout will lay in fresh water - so will be exposed to a large osmotic gradient
Describe how the Pf of the zebrafish egg canges
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
What is the Pf/Pd of a red blood cell - what does this tell us?
2.5
So must have a water pore in the membrane
We now know that this is AQP-1
Approx how big is the AQP1 pore
How many copies per cell
3.5A
100’000 copies per cell
AQP1 was first characterised as
CHIP28
Functional characteristics of AQP1
Water channel
Mercury sens
Tetrameric - four functional units
How was the AQP1 protein initially discovered
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
What did the AB produced to recognise the 28kD NOT DETECT
The 32KD protein
Concluded that these two proteins are not related
Why are two bands of CHIP28 often seen
Glycosylation
Describe why bands were seen at various mutliples of 28kD
Because there is evidence for oligomerisation of AQP1 in the membrane
Describe IHC of the a-CHIP28
Stains the proximal tubule and the thin descending limb
Describe how CHIP28 was cloned
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
Describe the results of the structural analysis of CHIP28
6 TMDs
N and C found IC
42% homology to MIP26 and high homology to several proteins with no known function
What was found in common between CHIP28 and several other proteins
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
Describe the four pieces of circumstantial evidence that CHIP28 acts as a water channel
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
Describe the experimental evidence tha CHIP28 acts as a water channel
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
Conclusions of the OE of CHIP28 in Ex Oocytes
CHIP28 is able to confer high water permeability of the RBC and proximal tubule
What is the background to the discovery that mercury can act on the AQPs
water permeability of the red blood cells is very sensnitive to HgCl2 or the organic mercurial pCMBS
Experimental evidence that mercury has an affect on the water channel AQP1
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
Icing on the cake expt for the mercurial sensitivity studies
Reducing agent b-mercaptoethanol reverses the inhibition since it is able to reverse the binding of Hg to cystenine residues
How does the mercurial agent act
By binding cysteine residues
Where are the cys residues located in AQP1
87 102 152 189
Describe how it was determined where Hg is binding to AQP1
Make mutants at each of the positions …. Where swap C for S
Test for sensitivity
Describe the reuslts of the CXXS mutant studies
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
Describe the hourglass model of AQP1
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
What is the effect of mutating alanine in the B loop NPA motif
Mutate to larger residue - blocks the movement of water indicating that there is a pore through the membrane
AQP1 is formed as a
Tetramer
When it wadiscovered that AQP1 was a tetramer what was the next step
To determine if the pore is through the middle of the tetramer of ife
Describe the experimental conditions required to to work out if the functional unit of AQP1 is a monomer or a tetramer
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
Describe the two things we would expect to see if the monomer/tetramer was the functional unit in C189S
If monomer - Expect a decrease of around 50% when Hg applied
If tetramer - expect and all-or-nothing decrease
Results of the tandem dimer studies
AQP1-C189s - decrease by 50%
C189S-C189S - no effect
WT-WT - full decrease
What was the conclusions of the tandem dimer studies
Each monomer is a functional H2O channel
Describe the selectivity of the AQP channels
Selective to water - not protons
Why would it be a suprise that protons werent able to be transported by AQP1
WHY IS THIS NOT THE CASE
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
What can also move through the pore
CO2
Describe an AQP1 bacterial homologue that is resitant to Hg
How was this used
What was found
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
How many Hg binding sites
2
Dont know the effect of the other one
What is AQP3 also permemable to
Glycerol and urea
How was it shown AQP3 is permeable to urea and glycerol
Take oocytes exp AQP3 in radio-glycerol solution
Can quantify radio-glycerol within the cell
What is the effect of Hg on urea and glycerol permemability?
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
What is AQP3 also known as
An aqua glycerol porin since is permeable to other solutes
Where is AQP6 seen to localise to
IC vesicles of the alpha intercalating cells
What type of condcutance does AQP6 show
Low water perm and a Cl conductance
Likely Cl moving
Through THE CENTRAL PORE OF THE TETRAMER
What is the effect of Hg on the conductance of AQP6
Hg increases the conductance
What else stimulates the H20 perm and Cl conductance
How was this seen
Acidic pH
Use a two electrode voltage clamp -
What in AQP6 is linked to the pH sens
Evidence
Specific histidine residue
If removed then pH sens lost
What is the AQP6 actually doing in vesicles
Acting as a charge balancer
As H+ moves in AQP6 activated and Cl moves in through AQP6 to balance the charge
How many members of the MIP family
Over 900 members
What are the 3 classed of MIPs
Aquaporins
Aquaglyceroporins
Unorthodox or superaquaporins
How many AQPs in humans
13
What are AQPs primarily selective for
Water - some gases
Where is AQP0 found
Lens
Location of 1-4
Kidney
Location of 3-7
Kidney
Location of 7
Role here
Proximal tubule
Responsible for the reabsoption of glycerol and urea
6 has a __ conduct
Cl
8 is a
Ammonia channel
11-12 tend to be
IC
11 linked to what disease
What was seen in KO studies
PCKD
KO leads to development of large vacuoles
AQP2 linked to what disease
Diabetes insipidus
Describe the insertion of AQP2 into the apical membrane
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
In absence of AVP What is the permeability of the alpha intercalated cell
V low
Two examples of reabsorptive epithelia
What predominantly mediates water transport in each one
Reabsorptive
Proximal tubule - AQP1
Small intestine - NO AQPS
An example of secretive epithelia - what AQP is present
AQP5
Water permeability of the proximal tubule
Very high
78% contributed by AQP1
Describe the effect of AQP1 KO on mice
Perfuse the lumen and basolateral side after mounting on a fusion pipette with an osmotic gradient
Reduction of water permeability
Describe the conditions that reabsoprtion occurs under
Near isotonic conditions gradieint still required but this is very small - 1/2 mosm
Describe what happens to the osmotic gradient in the proximal tubules
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
Describe what happens wher you replace the basolateral solition with mineral oil and perfuse normal saline through the lumen
Reabsorption of Na and other solutes
Absorbate is slightly hypertonic and this drives water reabsorption
Describe the micropuncture expts
What did they see
What were the conclusions
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
Overall conclusion from the micropuncture expt
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
What is seen in the AQP1 KO mouse - regarding the change in Osm
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
Describe the secretion of fluid from the saliveray acinar cells
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
Describe what is seen in the AQP5 KO
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
