Lecture 10

Question 1

What is the diffusional water permeability?

Answer 1

Permeability to water when there is no osmotic gradient.

Question 2

what is osmotic water permeability?

Answer 2

Permeability to water when an osmotic gradient is applied

Question 3

what occurs in an isotonic solution?

Answer 3

water is constantly diffusing in and out of the cell
Continuous exchange – influx = efflux – no volume change
No net movement
diffusional water permeability

Question 4

what occurs in a hypertonic solution?

Answer 4

water leaves to balance out the osmotic gradient

move from isotonic cell – cell shrinks

Question 5

what occurs in a hypotonic solution?

Answer 5

cytoplasm is more concentrated than the outside water

water moves in and cell swells

Question 6

what does it mean if the Pf/Pd ratio is > 1?

Answer 6

means a water pore Is present in the cell membrane

Question 7

how can you measure Pd?

Answer 7

using a Cartesian diver balance

Question 8

how can you measure Pf?

Answer 8

Much simpler approach, measure the change in cell volume over time when exposed to a hypertonic or hypotonic solution. – measure diameter to work out volume of the cells.

Question 9

what are the features of RBC water permeability?

Answer 9

• Osmotic Permeability: 1.5 x 10-14 cm3/(rbc.cmH2O.sec)
• Diffusional Permeability: 5.3 x 10-3 cm/s
• Is the equivalent to:0.64 x 10-14 cm3/(rbc.cmH2O.sec)
• Pf/Pd ratio for RBC’s = 2.5
• Meaning RBC’s have water pores
o AQP1 is the primary water channel in RBC
• Prediction in this study the diameter of the water pore in RBC is 3.5Å

Question 10

what is CHIP28?

Answer 10

old terminology for AQP1
water channel
Mercurial Sensitivity of Aquaporin 1 – water permeability is blocked by mercury
tetramer

Question 11

How was AQP1 found?

Answer 11

• Peter Agre – noble prize
• Initially studies involved Rhesus proteins in RBCs.
• Kept finding a 28kd protein that co-precipitated with a 32kd Rh polypeptide.
• Isolated the 28kd protein and produced an antibody.
• The antibody recognises a 28kd protein, also a higher mw band – never found any labelling of a 32kd protein ->The 28kd and 32kd protein are not related.
• The HMW band appears to be glycosylated form of the 28kd.
• Also evidence that the protein exists as oligomers in the membrane.
• Did antibody staining on proximal tubule and descending thin limb – apical and basolateral membranes shows the unknown protein
• Find this protein in cells with high water permeability – could be water channel

Question 12

what did sequencing of AQP1 show?

Answer 12

The N-terminal portion of the 28kd protein was sequenced. With this information a combination of PCR and library screening was used to identify the message/DNA for CHIP28.
Sequence analysis predicted a protein with a molecular wt of 28kd

Question 13

what did structural analysis show?

Answer 13

Predicts 6 transmembrane spans
Predicted proteins has 42% homology to MIP 26 and high homology to several proteins with no known function
All these related clones have the tandem repeat of the amino acid sequence NPA - became calling signal for AQP

Question 14

what is CHIP28 resistant to?

Answer 14

enzymatic digestion - as is the RBC water channel

Question 15

what did experiments of CHIP28 in xenopus oocytes show?

Answer 15

the oocytes exposed to a hypotonic shock
In control oocytes the volume change is very slow.
In oocytes expressing CHIP-28 the volume change is rapid and the oocytes explode within a few minutes

Question 16

what is the mercurial sensitivity of AQP1 and what does this mean?

Answer 16

AQP1 is mercurial sensitive
Background: The water permeability of red blood cells is very sensitivity to HgCl2 or the organic mercurial pCMBS
Water permeability is reduced

Question 17

what is the basis of mercurial action?

Answer 17

• Mercurial agents exert an action by binding to cysteine residues (can also bind histidine)
• AQP1 contains 4 cysteine residues at amino acid positions 87,102,152 and 189
• Individually mutate each of the cysteine residues to serine. Test effect of HgCl2 on water permeability these mutants. – no mercurial binding
• The mutants have the same water permeability as wild type AQP1
• HgCl2 reduced water permeability of C87S, C102S and C152S mutants.
• HgCl2 has no effect on the C189S mutant - conclude that Hg binds to Cys 189.

Question 18

what is the hourglass model of AQP1

Answer 18

• Amino and Carboxy ends of the protein are both intracellular
• Six transmembrane spans
• Between TM 2-3 and TM 5-6 are loops B and E which dip into the membrane.
• In the middle of each of these loops is the NPA motif – calling sign for aquaporins
• As the protein folds in the membrane the two NPA motifs come to lie together – produces a channel pore – the hourglass model.
• The Cysteine residue at position 189 (C189) confers mercurial sensitivity – in the hourglass model this lies at the opening of the predicted pore.