AQUAPORINS

Question 1

Whats the diffusional water permeability?

Answer 1

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

Question 2

What is the osmotic water permeability?

Answer 2

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

Question 3

What is the pf/pd ratio indicative of?

Answer 3

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

Question 4

How do you measure Pd?

Answer 4

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

Question 5

What was the Pd conclusions?

Answer 5

There was rapid exchange of water in all cases- with a half time of under 4 minutes
except the trout eggs cells

Question 6

What were the Pd results in the trout egg cells?

Answer 6

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

Question 7

How can we measure Pf?

Answer 7

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

Question 8

What were the results of the Pf experiments?

Answer 8

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

Question 9

Why is there a difference in the zebrafish ovarian egg and the zebrafish laid egg?

Answer 9

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

Question 10

What are the water permeability characteristics of red blood cells?

Answer 10

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

Question 11

What did they discover when they were studying the rhesus proteins?

Answer 11

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

Question 12

What else did they discover about the mystery protein?

Answer 12

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

Question 13

How did they clone AQP1?

Answer 13

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)

Question 14

What did the analysis of CHIP28 predict about its function?

Answer 14

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

Question 15

What does MIP26 do?

Answer 15

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

Question 16

What was the circumstantial evidence that CHIP28 was a water pore?

Answer 16

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

Question 17

What was the experimental evidence that CHIP28 was a water channel?

Answer 17

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

Question 18

What is AQP1 water permeability sensitive to?

Answer 18

Mercury

Hgcl2/ pCMBS

Question 19

Whats the experimental evidence for AQP1 mercury sensitivity?

Answer 19

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

Question 20

What agent reverses the Hg induced inhibition?

Answer 20

Beta- mercaptoethanol - same change in cell volume as oocytes just expressing AQP1

Question 21

How does mercury effect the AQP, which AQP is mercury insensitive?

Answer 21

Binds to the cysteine residues

AQP4

Question 22

What experiment did they carry out to find out how the cysteine residues were interacting with mercury?

Answer 22

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

Question 23

What did the hourglass model say about the structure of AQP1?

Answer 23

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

Question 24

How did they find the shape of AQP1- what did they think was happening?

Answer 24

Cyro-electron microscopy work
AQP1 is a tetramer
one central pore and looks like each monomer has its own pore

Question 25

What experiment established whether AQP1’s functional unit was the monomer or tetramer?

Answer 25

Created tandem dimers of WT AQP1 and C189S mutant
express AQP1-AQP1, AQP1-CI189S, mutant-mutant
Add mercury:
WT- W.P goes down to background levels
wt-mutant: 50% drop in WP
mutant-mutant: W.P doesnt change
conclusion: each AQP1 monomer is a functional H20 channel

Question 26

How are protons prevented from moving through the AQP1 channel?

Answer 26

As H20 enters the channel-
the overlapping NPA motifs in the channel decouple hydrogen bonding in the h20 molecules
h20 forms H bonds with the NPA motifs
protons can’t move through the channel with h20

Question 27

Where does mercury bind in AQPs?

Answer 27

Made a crystal structure of the pore with mercury bound
mercury binds to the middle of each subunit pore
-mercury binding is a common pattern in aquaporins

Question 28

Characteristics of AQP3?

Answer 28

Part of the aqua glycerine family
-transports glycerol and urea as well as h20
expressed in the skin
found on the basolateral membrane
mercury sensitive- probably has cysteine in a key place

Question 29

How did they show AQP3 permeability to urea and glycerol?

Answer 29

Took oocytes expressing AQP3
dropped them in a solution containing radio-labelled glycerol and urea
take the oocytes out- measure how much radiolabelled glycerol and urea is in the oocytes
control cells= low permeability
AQP3 oocytes= high permeability

Question 30

Where is AQP6 found and what are its main characteristics?

Answer 30

Unorthodox
found in the kidney- not in the cell membrane
co-localises with the proton pump in intracellular vesicles
mercury stimulated rather than inhibited
saw chloride conductance through the channel
when they expressed AQP6 in oocytes- didnt seeing any increases in h20 permeability
stimulated at acidic pHs

Question 31

What is the function of AQP6?

Answer 31

AQP6 is acting to balance charge
as the proton pump brings in H+- AQP6 brings in Cl-
balances the charge

Question 32

What are the 3 classes of aquaporin

Answer 32

1. Aquaporins= primary links to h20 transport
2. Aqua- glyceroporins= permeable to h20 and small molecules like urea and glycerol
3. Unorthodox= NPA motif may be slightly different in these, AQP8 falls inbetween unorthodox and normal
   - also linked to transporting the gas ammonia

Question 33

What is the disease linked to AQP2?

Answer 33

Diabetes insipidus
mutations in AQP2- reduces channel permeability to water
unable to concentrate urine
doesnt respond to vasopressin

Question 34

What occurs in classic reabsorption?

Answer 34

Movement of Na, cl and solute across the apical membrane which exits via the basolateral membrane
lumen (kidney) becomes slightly hypotonic
gives you an osmotic gradient across the PT- drive water movement through AQP1

Question 35

Evidence for near isoosmotic fluid absorption?

Answer 35

Isolate a PT segment
mount it on pipettes
droplet of mineral oil surrounds the outside of the PT
apical and basolateral side perfused independently of each other
start perfusing down the tubule- small droplet of water forms in the oil- absorbed water

Question 36

Whats another technique for looking at the absorption in the kidney?

Answer 36

Micropuncture technique- done in vivo
exposed kidney of an animal- pipettes into the capillaries and the tubule itself
perfused the lumen of the tubule and capillaries with a solution containing 154mM NaCl

Question 37

What is the osmolality of the plasma at the end of the PT in AQP1 KO mice?

Answer 37

Much more hypertonic than usual
as solute is reabsorped into the plasma
h20 cant move across to nullify the gradient
so generated gradient is much larger

Question 38

What channel mediates water movement in the acinar cells of the salivary gland?

Answer 38

AQP5

Question 39

What did they see in terms of salivary secretion and composition in AQP5 KO mice?

Answer 39

Measures salivary secretion- stimulated secretion with pilocarpine
the amount of secretion was reduced in the KO
osmolality of the saliva in the KO- much higher than WT
WT- was nearly isotonic