Lecture 10 Structure and Function of AQP

Question 1

Describe the hourglass model of AQP

Answer 1

The structure of AQP consists of a protein with 6 transmembrane domains and intracellular amino and carboxy termini. Between transmembrane domains 2-3 and transmembrane domains 5-6 are the B-loop and E-loop which dip into the membrane. In the middle of these B-loop and E-loops are NPA motifs that dip back into the membrane and come together to make the pore of the AQP channel

Question 2

What is the relation between the mercurial sensitivity conferring residue and its location in the structure

Answer 2

C189 lies near the opening of the pore hence explaining why mercurial binding to this residue is associated with decreased permeability

Question 3

Other than cysteine residues near the pore what other key amino acids are present in AQP that seem to be involved in permeability

Answer 3

There is an additional alanine residue present in the intracellular side of the B loop which is also close to the pore. Changing this alanine for a larger residue prevents permeability to water and similarly switching this alanine for a cysteine makes the pore additionally mercury sensitive as Hg-binding to a residue at this position prevents permeability

Question 4

C189 is present in all AQPs T or F

Answer 4

F – mercurial sensitivity is different in each AQP

Question 5

What work showed that AQPs form as tetramers

Answer 5

Early studies using western blots showed bands that were multiples of the 28kDa protein. It was then confirmed to be the case using cryo-EM

Question 6

What two hypotheses were there for the way the AQP tetramer forms a pore

Answer 6

The first idea was that all 4 subunits associate to form 1 central pore. The other idea was that each subunit itself possessed a functional water pore

Question 7

How was it investigated whether the functional unit of an AQP was 1 monomer or the tetramer

Answer 7

Investigators constructed tandem dimers of wild-type AQP1 and C189S mutant channels that are insensitive to mercury and then expressed these dimers in Xenopus oocytes. The rationale was that upon injection of the tandem dimer containing 1 wild type and 1 mutant monomer into Xenopus oocytes you effectively make an APQ1 tetramer consisting of 2 wild type and 2 mutant monomers. Hence If each monomer forms its own pore the addition of mercury to oocytes expressing the tandem dimers would cause the water permeability to decrease by half. This is because only half of the monomers are mercury sensitive and the other half are C189S mutants. On the other hand if all four subunits form one central pore addition of mercury would cause a total inhibition of water permeability as mercury blockade of two of the subunits would be sufficient to inhibit the functioning of the whole tetramer

Question 8

Below are the results of the experiments investigating whether the functional unit of AQP1 was 1 individual monomer or indeed the tetramer itself. Describe what these results show and what this means

Answer 8

Hg reduced the H2O permeability of AQP1-AQP1 dimers to background levels. In contrast Hg had no effect on the H2O permeability of C189S-C189S dimers and reduced the H2O permeability of AQP1-C189S dimers by 50%. This hence shows that each of the AQP1 monomers is a functional H2O channel as there was no total inhibition of water permeability in the monomer expressing half mutant half wild type channels

Question 9

Although it was found that each AQP1 monomer did forms its own functional channel what can be said about this central pore that the monomers seem to associate around

Answer 9

The central pore was hypothesised to most likely be involved in NH3 CO2 CO(NH2)2 and Na+ transport

Question 10

What is the diameter of the AQP1 pore how does this relate to its function

Answer 10

The pore of AQP1 is between 1.8 and 2.2Å (1.8-2.2x10-10m) this is not much bigger than a water molecule

Question 11

How does AQP1 prevent H+ from entering the pore along the backbone of water molecules in order to remain H2O selective

Answer 11

As water molecules move through the pore as they come to the NPA motifs the hydrogen bonding between the molecules is broken. The water molecules then form bonds with the asparagine residues. This allows for a single file movement of water through the pore which also prevents H+ from hopping down the backbone

Question 12

What is the specific function of the NPA motif of the AQP1 pore

Answer 12

Water molecules form bonds with the asparagine residues in the NPA motifs. This is critical for the single-file movement of H2O through the channel

Question 13

Crystal structures of bacterial AQP1 attempting to find the site of Hg binding couldn’t find mercury binding to the channel why was this

Answer 13

Bacterial AQP1 is not mercury sensitive

Question 14

What is the significance of Thr183 in bacterial AQP1

Answer 14

This is the residue that is equivalent to C189 in humans. Switching this residues to a cysteine renders the AQP1 mercury sensitive

Question 15

Where-else has mercury been found to bind within AQP1

Answer 15

Hg has also been found to bind near to the central cleft this is thought to effect CO2 transport

Question 16

What is unusual about human AQP4

Answer 16

It isn’t mercury sensitive as it lacks C189 or any equivalent residues

Question 17

Where is AQP5 found

Answer 17

In the lungs and salivary glands

Question 18

Where are AQPs 2 3 and 4 primarily expressed

Answer 18

AQP2 is expressed on the apical membrane of the cells of the colleting duct whilst AQP3/4 is on the basolateral membrane of those cells

Question 19

What other class of proteins were the early AQPs found to be linked to

Answer 19

The glycerol facilitator proteins in bacteria that are responsible for glycerol uptake

Question 20

When expressed in Xenopus oocytes AQP3 increases water permeability. This permeability is inhibited by mercury. What can be inferred about AQP3

Answer 20

It must possess a cysteine residue close to 189 or that has a similar effect on the pore

Question 21

Other than water what other molecule does AQP3 expression confer permeability to and how was this shown

Answer 21

AQP3 also confers permeability to glycerol and to some extent urea. This was shown by incubating oocytes expressing AQP3 with radiolabelled glycerol and urea to see if they take it up. Indeed oocytes expressing AQP3 had a huge increase in glycerol and urea transport. This AQP3-mediated glycerol and urea permeability was also mercury sensitive

Question 22

Give some examples of other AQPs that belong to the same class as AQP3. Where are these expressed

Answer 22

AQP7 and AQP9 are also in the same class as AQP3 allowing small solutes through the pore. AQP7 is found in the PCT whereas AQP9 is expressed at high levels in the liver where it is seemingly involved with urea transport

Question 23

What are the three classes of AQPs and how do they differ

Answer 23

Classical aquaporins – primarily permeable to H2O. Aquaglyceroporins – H2O and small solute permeable (NH3 CO2 CO(NH2)2 and Na+ transport). Unorthodox aquaporins – have unusual properties and differences in the NPA motifs

Question 24

Below is a table with a list of known AQPs. Decide which class of AQP they each belong to

Answer 24

Seem completed table

Question 25

What is unusual about the expression of AQP6

Answer 25

It is expressed in the kidney but not at the cell membrane. AQP6 antibodies show co-localisation of the channel with the H+ATPase in intracellular vesicles in α-intercalated cells

Question 26

What is odd about the function of AQP6

Answer 26

AQP6 shows water permeability but not under baseline conditions. In addition AQP6 also acts as a chloride conductor. Even more significantly both its H2O permeability and Cl- conductance is stimulated by Hg

Question 27

What is the suspected function of AQP6

Answer 27

Interestingly AQP6 conductance of Cl- and indeed H2O is also stimulated by H+. This combined with the knowledge that it is found localised with the H+ATPase suggests a role of pH. In fact what is thought to happen is that the H+ATPase pumps H+ into the vesicles to acidify them. This then lowers the pH of the vesicle to around pH4 where there is an increase in water permeability and Cl- conductance. This AQP6 mediated Cl- conductance then acts as a charge balance for the movement of H+

Question 28

How much water reabsorption is the collecting duct responsible for

Answer 28

20L per day – involved in the fine tuning of water transport

Question 29

Briefly what happens in response to vasopressin in the CD

Answer 29

Under the control of vasopressin you insert AQP2 in the apical membrane of the CD cells allowing transcellular water transport

Question 30

Broadly what is nephrogenic diabetes insipidus

Answer 30

Where you have lost the sensitivity to vasopressin in the collecting duct

Question 31

Where can mutations be found that cause nephrogenic DI

Answer 31

AVPR2 AQP2 or urea transporters

Question 32

How can mutations in AQP2 that cause diabetes insipidus be classified

Answer 32

Mutations that disrupt the pore and that leads to an inhibition of water transport through that pore. Alternatively you can get mutations that disrupt the trafficking of the channel to the membrane

Question 33

Give an example of a reabsoprtive and a secretive epithelium

Answer 33

Reabsorptive – PCT Secretive – Salivary Gland

Question 34

What is unusual about the small intestine in terms of water transport

Answer 34

The small intestine doesn’t express AQPs and so there is an uphill movement of water

Question 35

The PCT undergoes isosmotic water reabsorption T or F

Answer 35

F - If the PCT was isosmotic this wouldn’t drive water reabsorption. Hence it is better to describe the PCT water reabsorption as near isosmotic

Question 36

Which AQP is vital for the osmotic water movement in the PCT

Answer 36

AQP1

Question 37

How much water reabsorption occurs in the PCT per day

Answer 37

~180L

Question 38

What evidence from animal models are there for the role of AQP1 in conferring water permeability in the PCT

Answer 38

AQP1 -/- mice have a drastically reduced Pf by 78%

Question 39

Why is there such a small osmotic gradient across the renal epithelium in the PCT

Answer 39

Solutes are being reabsorbed from the lumen at the apical membrane and then leave the basolateral membrane. This drives water reuptake and results in a hypotonic lumen and hypertonic blood. However because water reuptake is so rapid it equilibrates instantly and there is only a very small osmotic gradient across the epithelium of around 2mOsm

Question 40

Describe the first piece of evidence using microdissection that showed that near-isosmotic water reabsorption occurs in the PCT

Answer 40

Experimenters hand dissected out rabbit PCT segments which were set up with perfusion pipettes allow for the separate perfusion of the lumen and the basolateral side. These PCT segments being perfused were then placed under an oil droplet. So on the apical side of the PCT there was normal perfusate fluid moving through the lumen containing a normal saline solution on the basolateral side there was an oil droplet. They then allowed the PCT to absorb solutes and fluid from the lumen. After a while a fluid droplet had formed underneath the oil on the basolateral membrane this was known as the absorbate. They found that the absorbate was hypertonic to the perfusate roughly 10mOsm higher. This suggested that the reabsorption of solutes must have subsequently been driving water reabsorption

Question 41

Describe the second piece of evidence using micropuncture that showed that near-isosmotic water reabsorption occurs in the PCT

Answer 41

Rabbits were aneasthatised and micropunture pipettes were inserted into the blood vessels and lumen of the PCT. Either side of the epithelia were then perfused with a solution of known concentration before the osmolarity either side was measured. It was found that the capillary was only slightly hypertonic compared to the lumen (287.7mOsm in the lumen 289.4 in the capillary). This hence shows that the PCT is absorbing the large volume of fluid it is despite minute changes in the osmolarity (low osmotic gradient)

Question 42

What were the effects on the osmotic gradient of perfusing the PCT at high flow rates compared to low flow rates

Answer 42

There was a small osmotic gradient generated for both low and high flow rates however the osmotic gradient in the high flow rate was a little larger. This is likely because it doesn’t reach equilibrium and hence absorption continues all the way through the PCT

Question 43

Give an overview of how the salivary gland secretes solutes and fluid

Answer 43

NKCC1 is on the basolateral membrane of the salivary epithelium bringing Cl- Na+ and K+ into the cell. The subsequent rise in intracellular Cl- sets up a driving force for Cl- to leave at the apical membrane into the lumen. This occurs via ANO1 a voltage-gated Ca2+ activated Cl- channel. Na+ then moves from the basolateral side into the lumen via paracellular transport through tight junctions. This solute secretion acts to generate a hypertonic lumen that drives water transport through AQP5

Question 44

Below is some data showing the effect of AQP5 on pilocarpine-induced salivary secretion. Describe what this data shows about the role of AQP in the salivary glands

Answer 44

After 5 mins the salivary weight was massively decreased in the AQP5 knockout mice compared to both control wild type mice and AQP1/AQP4 knockouts. This suggests that AQP5 is essential for water secretion and saliva production

Question 45

What class of drug is pilocarpine

Answer 45

mAchR agonist

Question 46

Below is some data showing the effect of AQP5 on pilocarpine-induced salivary osmolarity. Describe what this data shows about the role of AQP in the salivary glands

Answer 46

Wild type mice generated a saliva that was nearly isotonic (~300mOsm) suggesting that the hypertonicity as a result of solute secretion into the lumen is dissipated by water movement. However the AQP5 knockout results in an increased osmolarity of the fluid in the lumen. This confirms that water secretion by the salivary gland requires AQP5 in order to dissipate the hypertonicity