AQUAPORINS Flashcards
Whats the diffusional water permeability?
Pd= The permeability to water when there is no osmotic gradient-cells in a isotonic solution- continous exchange of water accross the membrane influx= efflux
What is the osmotic water permeability?
Pf= the permeability to water when an osmotic gradient is applied
cells in a hypertonic solution- cells will shrink as water moves out
cells in a hypotonic solution- cells will swell as water moves in
What is the pf/pd ratio indicative of?
If the ratio is greater than 1- a water pore is present in the membrane- doesn’t have to be an aquaporin
e.g sodium-glucose cotranspoter- has water transport properties
How do you measure Pd?
The catesian diver balance
1- diver in a solution with D20- measure the pressure needed to keep the diver at a steady height- correlate this with the weight of the diver
2- separate set of experiments- used divers of a known weight- correlated the equilibration pressure to the change in weight
3- from this correlation- can measure the change in cell weight- can then work out how much D20 has exchanged into the cells- and thus what the water permeability of these cells is
What was the Pd conclusions?
There was rapid exchange of water in all cases- with a half time of under 4 minutes
except the trout eggs cells
What were the Pd results in the trout egg cells?
After 5 hours- no evidence for D20 moving into the cells- implies that these cells have no water permeability
must be something different about the lipid composition of these cells
How can we measure Pf?
Measure the change in cell volume over time when cells are exposed to a hypertonic or hypotonic solution
use equation: change in volume= Pf.surface area.time.change in concentration gradient
What were the results of the Pf experiments?
All the cells swelled in the hypotonic solution- except the trout egg cells
the osmotic pf/pd ratio was greater than 1 - all these cell types have some sort of water pore
Why is there a difference in the zebrafish ovarian egg and the zebrafish laid egg?
ovarian egg= pf of 29.3
shed egg= 0.45
osmotic water permeability plummets
zebrafish are fresh water fish- lay egg in fresh water- if there was high water permeability- eggs would swell and burst
What are the water permeability characteristics of red blood cells?
Pf/pd ratio of 2.5
very high permeability
RBCs have water pores
They tried to identify the water pore in RBCs- ended up being AQP1
What did they discover when they were studying the rhesus proteins?
A 28kd protein kept co-precipitating with the 32kd rhesus protein
this protein didnt show up in traditional staining for RBC proteins
isolated the protein and made an antibody- the antibody recognises the 28kd protein (and a higher mw protein) but not the 32kd one
*the 28kd and 32kd proteins arent related
What else did they discover about the mystery protein?
The higher MW protein is a glycosylated version of the protein
there was evidence that the protein exists as oligomers in the membrane- lots of bands present
the antibody also stained the PT and the descending thin limb in the kidney- lots of water permeability here
How did they clone AQP1?
Took the 28kd protein- had the N terminal residues sequenced
with this sequence- combination of PCR and library screening was used to identify the message of CHIP28 (AQP1)
What did the analysis of CHIP28 predict about its function?
Sequence analysis predicted a weight of 28kd
predicted 6 TMD, an intracellular end and a C terminal
found that a few proteins- including MIP26- had a similar homology to this protein- but nobody knew what these other proteins did
What does MIP26 do?
MIP26 makes 60% of the protein in the lens of the eye
all these proteins (including CHIP28) have the tandem repeat of the amino acid sequence- NPA
*turns out this tandem repeat is critical for the function and formation of the water pore
What was the circumstantial evidence that CHIP28 was a water pore?
Actual number of copies of CHIP28 in a RBC and the estimation of the number of copies though biophysical calculations is the same
The predicted size of CHIP28 (28.5kd) is similar to the size of the 30kd functional unit of the PT water channels
CHIP28 transcripts correspond to the RNA fraction from the kidneys that produces the greatest water channel activity
CHIP28 is resistance to enzymatic degradation- as was the RBC water channel they knew of
What was the experimental evidence that CHIP28 was a water channel?
CHIP28 was expressed in xenopus oocytes- the oocytes were then expressed to hypotonic shock- eventually the cells burst
- in control cells- change in volume is slow
-CHIP28 oocytes- volume change is rapid and the oocytes explode quickly
*CHIP28 confers high water permeability on RBCs and the proximal tubule
Renamed AQP1
What is AQP1 water permeability sensitive to?
Mercury
Hgcl2/ pCMBS
Whats the experimental evidence for AQP1 mercury sensitivity?
When cells exposed to hypotonic shock-
control oocytes + HgCl2 preincubation= no change in cell volume
oocytes expressing AQP1 = fast change in cell volume oocytes expressing AQP1 + HgCl2 preincubation= slower change in cell volume
What agent reverses the Hg induced inhibition?
Beta- mercaptoethanol - same change in cell volume as oocytes just expressing AQP1
How does mercury effect the AQP, which AQP is mercury insensitive?
Binds to the cysteine residues
AQP4
What experiment did they carry out to find out how the cysteine residues were interacting with mercury?
Made 4 mutants- each one had a mutated cysteine residue
all mutants had a WT like water permeability- function isnt affected by the mutation
3 out of the 4 mutants- adding mercury- see a drop in WP
C189S mutant- unaffected by mercury- this cysteine is the mercury-sensitive cysteine in WT AQP1
What did the hourglass model say about the structure of AQP1?
6 TM domains
loops B and E loop into the membrane but dont cross it
in the 3D structure of the channel- loop B and E overlap and forms the pore
NPA motif in the middle of the loops
as the protein folds in the membrane- 2 NPA motifs come together to form the pore
cysteine 189 is very close to the opening of the pore
the size of the residues- particularly cysteine and alanine is very important
How did they find the shape of AQP1- what did they think was happening?
Cyro-electron microscopy work
AQP1 is a tetramer
one central pore and looks like each monomer has its own pore
