Lecture 11- History of the water pore

Question 1

Diffusional Water Permeability (Pd)

Answer 1

Permeability to water when there is no osmotic gradient.

Question 2

Cell in isotonic solution

Answer 2

Continuous exchange of water across them membrane
Influx= efflux
No volume change

Question 3

Osmotic Water Permeability (Pf)

Answer 3

Permeability to water when an osmotic gradient is applied.

Question 4

If the Pf/Pd ratio > 1

Answer 4

then a water pore is present in the cell membrane

Aquaporin

Question 5

Measured Pd and Pf

Answer 5

• Frog ovary
• Frog cavity
  etc

Question 6

Measuring Pd

Answer 6

cartesian diver balance

Question 7

Cartesian diver balance

Answer 7

If apply pressure – air bubble gets smaller – affect of bubble on buoyance lower so diver sinks
If apply suction – air bubble expands diver rises
Suction or pressure applied to keep it at steady state

Question 8

Cartesian diver balance- Experiment

Answer 8

Apply suction -
Cells equilibrated in H20
- cells placed into solution containing D20
- D20 exchanges with H20 in cells
- Cells become heavier - diver starts to sink
- apply suction to keep the diver at a constant height
- measure pressure needed to keep the diver at a constant height with time

Question 9

Cartesian diver balance- RESULTS

Answer 9

Rapid exchange of cell water in all cases with the t½ of exchange < 4.5 minutes for the cell types except for
Trout egg cells – after 5 hours there was no evidence of D2O moving into these cells – implies the trout egg cell membrane has no diffusional H2O permeability
- Must be something about lipid conc in trout cell

Question 10

Calculation of Pd

Answer 10

Change in equilibration pressure correlated with change in weight» allowed change in the cell measures - then it is known how much D20 has moved into the cell and the Pd can be calculated

Question 11

Measuring Pf

Answer 11

Much simpler approach, measure the change in cell volume over time when exposed to a hypertonic or hypotonic solution.
Use equation
∆V = Pf · SA · t · ∆C
Easy to apply to egg cells – spherical cells, measure cell diameter.

Question 12

Equation for a change in volume

Answer 12

Change in vol = osmotic water permeability times sA x time x water conc gradient

Question 13

Applied hypertonic solution

Answer 13

all cells were swelling with high osmotic water permeabilities
All Pf/Pd greater than one so all cell types have a water pore in the membrane
Physiological change in the cells during the process of being laid into water

Question 14

• Lower water permeability

Answer 14

– fresh water animal

Question 15

RBC

Answer 15

• High water permeability
• Pf/Pd = 2.5 – water tpores
• Predicted pores in RBC 3.5 antimers

Question 16

AQP1

Answer 16

• Protein responsible for pore in RBCs
• Identification
• Mercury sensitive
• Also known as CHIP28
• Tetrameric

Question 17

Identification of AQP1

Answer 17

Initially working on Rhesus proteins
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.
Doesn’t show up in traditional

Question 18

HMW band

Answer 18

appears to be glycosylated form of the 28kd

• Evidence the protein exist as a an oligomer in the membrane
• • suggests tetramer in cell membrane

Question 19

Where was it expressed ??

Answer 19

staining in proximal tubule and descending limb

Identified in areas with very high water permeability

Question 20

Cloning of aquaporin 1

Answer 20

combination of PCR and library screening was used to identify the message for CHIP28.
Sequence analysis predicted a protein with a molecular wt of 28kd
Predicted –
6 TM
N and C terminal
High homology of 2 proteins within a database MIP26 (Aquaporin 0)

Question 21

Is CHIP28 a water channel ?

Answer 21

• Actual copies of CHIP28 in RBC and biophysical calculations of channel number are in the same region
• 28.5 kd unit similar in size to the 30kd functional unit of the proximal tubule water channels
• CHIP28 transcript (2.9kb) corresponds to the RNA fraction from kidneys that produces greatest water channel activity.
• CHIP28 resistant to enzymatic digestion as is the RBC water channel.

Question 22

Experimental evidence for Chip28/AQP1 being a water channel ??

Answer 22

CHIP-28 was expressed in xenopus oocytes and then 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 with in a few minutes
CHIP28 confers high water permeability on RBC’s and the proximal tubule
- renamed Aquaporin 1 (AQP1)

Question 23

Mercurial Sensitivity –

Answer 23

The water permeability of red blood cells is very sensitivity to HgCl2 or the organic mercurial pCMBS

Question 24

Mercurial sensitivity -Experimental evidence -

SEE GRAPH

Answer 24

• Pre-incubation in HgCl2 has no effect upon control oocytes
• Pre-incubation in HgCl2 slows the volume change in oocytes expressing AQP1
• The reducing agent ß-mercatoethanol reversed the Hg-induced inhibition

Question 25

Mercury binds to what ?/

Answer 25

Exposed cysteine residues