Water Transport

Question 1

1. what is Pd?
2. what is Pf?
3. what is implied if Pf/Pd ratio is greater than 1?

Answer 1

1. diffusional water permeability - the permeability to water when there is no osmotic gradient
2. osmotic water permeability - the permeability to water when an osmotic gradient is applied.
3. that there is a water channel in the membrane.

Question 2

1. name 5 types of cells in which Pf/Pd was measured to look for the existence of water channels.
2. describe the cartesan diver balance, which was used to measure Pd
3. How was Pf measured? Give the equation used in the measurement.
4. what were the results of these experiments?

Answer 2

1. frog ovarian egg, frog body cavity egg, zebrafish ovarian egg, zebrafish body cavity egg, and trout egg
2. a cell is placed in a funel; a bubble sits at the opening of the funel. the height of the diver depends upon the size of the air bubble. applying pressure causes the diver to rise, and applying suction causes it to fall.

prior to the experiment, cells were equalibrated in a solution with normal H2O. In the tube, there is a solution of D2O. As H2O is exchanged for D2O, cells get heavier thus start to sink. The amount of suction required to keep the diver at a constant height is measured.

In a separate set of experiments, a change in equalibration (i.e. H2O/D2O exchange) is correlated to how much D2O has moved into the cell, thus Pd can be calculated from the change in cell weight.

3. cells were exposed to a hypertonic or hypotonic solution and cell volume was measured over time

Pf = ΔV/(SA.t.ΔC) where ΔV=change in cell volume, SA= surface area, t=time and ΔC= change in concentration

4. all cells except trout eggs had a Pf/Pd greater than 1.

Question 3

1. What was the initial focus of studies that identified AQP1?
2. what was co-precipitated with the Rhesus proteins?
3. what did antibodies against the co-precipitated protein recognise?
4. what did treatment that removed glycosylation reveal?
5. where did the antibody stain?

Answer 3

1. Rhesus proteins
2. a 28kDa protein
3. the 28kDa protein, as well as a higher MW band but not the 32kDa Rhesus polypeptide
4. that the higher MW band corresponded to the glycosylated form of the 28kDa protein
5. A and BL membranes and prox tubule and tDL which have high water permeabilities.

Question 4

1. why was it possible to use PCR to sequence the protein?
2. what did sequence analysis of CHIP28 predict? (2)
3. what did the predicted protein share 42% homology with?

Answer 4

1. because the N-terminal portion had already been sequenced
2. that the protein would have a molecular weight of 28kDathat the protein would have 6 TM domains
3. MIP26, a protein that is expressed abundantly in lens fibres.

Question 5

1. give 2 pieces of circumstantial evidence that CHIP28 is a water channel
2. how was it experimentally proven that CHIP28 is a water channel
3. what was CHIP28 renamed as?

Answer 5

1. • 28.5kDa subunit is similar in size to the 30kDa functional unit of the proximal tubule H2O water channels

CHIP transcript corresponds to RNA fraction from kidneys that confers greatest water channel activity

2. CHIP28 was expressed in xenopus oocytes (functional cloning); oocytes were exposed to hypotonic shock. the volume change was very slow in control oocytes. In oocytes expressing CHIP28, the volume change was very fast, and cells rapidly underwent lysis.
3. AQP1

Question 6

1. Name 2 mercurial agents used to test the mercural sensitivity of AQP1
2. what does preincubation of oocytes expressing AQP1 with a mercurial agent cause in terms of volume change?
3. what were the effects of beta-mecatoethanol on water permeability?
4. to which residues does Hg bind?
5. name 4 positions in which cysteine residues are found in AQP1?
6. How were the necessity of these residues for mercurial sensitivity investigated?
7. what were the findings?
8. Describe a second experiment using bacterial AQP1 that showed how Hg inhibits AQP1

Answer 6

1. HgCl or pCMBS
2. slows down the volume change
3. restores water permeability by reversing the effects of Mg
4. cysteine
5. 87, 102, 152 and 189
6. these residues were mutated to serine, and the effects on Hg sensitive water permeability observed
7. HgCl had no effect on C189S, but other mutants were still Hg sensitive
8. bacterial AQP1 contains a threonine at 189 rather than a cysteine, and is Hg insensitive. When this residue was changed to a cysteine, the channel became Hg sensitive. The protein was crystalised in the presence of Hg, which reveals that Hg lies in the middle of the pore, blocking the passage of water.

Question 7

1. describe the structure of AQP1
2. what type of dimers were used to investigate whether AQP1 functions as individual monomers (4 pores) or as a tetramer (1 central pore). What is the benefit of these dimers?
3. what would be expected if AQP1 functions as a) a monomer or b) a tetramer?
4. what were the findings and what does this imply?
5. What part of AQP1 prevents H+ from moving through the pore along with water?

Answer 7

1. Intracellular N and C termini; 6 TM domains, B and E loops between TM2&3 and 5&6 respectively. B and E loops contain NPA motifs, and insert into the membrane to form a pore (hourglass model). C189 is found in E loop
2. tandem dimers of WT and C189S AQP1. Advantageous, as if AQP1 and C189S were expressed separately, only 1/6 of tetramers would be 50/50 mixes. This way, all tetramers are 50/50 mixes
   3a. Hg would inhibit water permeability by 50%
   3b. Hg would inhibit water permeability completely
3. Hg only inhibited water permeability by 50% indicating that AQP1 functions as a monomer.
4. NPA motifs, by breaking H bonding between the water molecules.

Question 8

1. what is AQP0 important for? In what condition is it cleaved? Name another possible role of AQP0
2. to what hormone/chemical is AQP2 sensitive to? where is it expressed? In what condition is it mutated?
3. where is AQP3 expressed? Name 2 other substances it is permeable to. What mechanism is it important in?
4. where is AQP4 expressed? Why do KOs only have a mild urine concentrating defect?
5. name 2 places in which AQP5 is expressed. Name a symptom caused by AQP5 mutations?
6. where is AQP6 expressed? what does it co-localise with? What is unusual about this AQP? What is the role of this channel?

Answer 8

1. maintaining lens transparency. cataracts. delivery of oxygen and nutrients to lens which is relatively devoid of blood vessels
2. vasopressin. expressed in the apical membrane of the prinicpal cells of the collecting duct. Diabetes insipidus
3. BL membrane of principal cells in the CD. also permeable to urea and glycerol. important in urine concentration
4. BL membrane of principal cells in the collecting duct. mainly expressed in medullary collecting duct; most urine concentration occurs in the cortex.
5. type 1 pneumocytes of the lung (gas exchange) and in exocrine glands. Xerostoma
6. IC vesicles. colocalises with H+ ATPase in IC vesicles in alpha-intercalated cells. water perm and Cl conductance are stimulated by Hg. acts to balance the movement of protons into IC vesicles.

Question 9

1. In the proximal tubule, under what conditions does water reabsorption occur?
2. How was this examined (2 experiments)? What was the resulting evidence?
3. What sets up the driving force for water reabsorption?
4. why is the osmotic gradient for water reabsorption only small?
5. What did the perfused tubule technique show about the Pf of AQP1 KOs?
6. Explain why the osmotic gradient is amplified in AQP1 KOs.

Answer 9

1. near isosmotic conditions
2. proximal tubule segments were microperfused - the A side with saline ans the BL side with mineral oil. As fluid is reabsorpbed, a smal volume of fluid appears in the oil

the osmolarities of the perfusate and absorbate were not very different

the lumen of the tubule and capillaries were perfused with 145mM NaCl solution using micropuncture in vivo. Water reabsorption was higher when the flow rate was faster, because it is unlikely that osmotic equilibrium has been reached.

3. the reabsorption of Na, Cl and other solutes makes the lumen hypotonic, thus driving water reabsorption
4. because of the very high water permeability of the proximal tubule
5. that is is decreased by 78%
6. because NaCl reabsorption increases the hypotonicity, however, there is no water movement to equalibrate this

Question 10

1. what type of water movement occurs in the salivary gland? Under what conditions does this occur?
2. what drives this movement?
3. via which AQP does movement occur?
4. Explain the findngs when WT and KOs of this AQP were stimulated with pilocarpine.

Answer 10

1. isosmotic water secretion
2. the secretion of NaCl, which makes the lumen hypertonic
3. AQP5
4. WT mice secrete a large volume of relatively isotonic saliva

AQP4 KO mice secrete a small volume of concentrated saliva

Question 11

1. what is unusual about water reabsorption in the small intestine? (2 points)
2. via which proteins is water transported? Name 4 examples
3. what happens when mannitol and NaCl are added to the lumen and why?
4. what happens wen KCl is added to the lumen and why?
5. what happens when KCl and furosemide are added to the lumen and why?
6. what are the physiological implications of wat water transporters in terms of isotonic drinks and oral rehydration therapies

Answer 11

1. it is not facilitated by AQPsit occurs against the osmotic gradient (the BL side is relatively isotonic, whilst the A side is highly hypertonic following a meal)
2. wet transport proteins - co-transporters which also transport water as part of their normal operation

GLUT2, SGLT1, NKCC1 and KCC4

3. cells shrink because water moves along its osmotic gradient out of the cell
4. cells swell, because water is transported against its osmotic gradient as K is absorbed via KCC4
5. cells shrink, because KCC4 is injibited, thus water moves along its osmotic gradient
6. isotonic drinks and oral rehydration therapies contain glucose and other solutes. They may aid rehydration by providing more lumenal solutes to increase the action of wet water transporters thus more water can be reabsorbed.