Lecture 11- History of the water pore Flashcards
Diffusional Water Permeability (Pd)
Permeability to water when there is no osmotic gradient.
Cell in isotonic solution
Continuous exchange of water across them membrane
Influx= efflux
No volume change
Osmotic Water Permeability (Pf)
Permeability to water when an osmotic gradient is applied.
If the Pf/Pd ratio > 1
then a water pore is present in the cell membrane
Aquaporin
Measured Pd and Pf
- Frog ovary
- Frog cavity
etc
Measuring Pd
cartesian diver balance
Cartesian diver balance
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
Cartesian diver balance- Experiment
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
Cartesian diver balance- RESULTS
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
Calculation of Pd
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
Measuring Pf
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.
Equation for a change in volume
Change in vol = osmotic water permeability times sA x time x water conc gradient
Applied hypertonic solution
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
- Lower water permeability
– fresh water animal
RBC
- High water permeability
- Pf/Pd = 2.5 – water tpores
- Predicted pores in RBC 3.5 antimers
AQP1
- Protein responsible for pore in RBCs
- Identification
- Mercury sensitive
- Also known as CHIP28
- Tetrameric
Identification of AQP1
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
HMW band
appears to be glycosylated form of the 28kd
- Evidence the protein exist as a an oligomer in the membrane
- suggests tetramer in cell membrane
Where was it expressed ??
staining in proximal tubule and descending limb
Identified in areas with very high water permeability
Cloning of aquaporin 1
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)
Is CHIP28 a water channel ?
- 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.
Experimental evidence for Chip28/AQP1 being a water channel ??
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)
Mercurial Sensitivity –
The water permeability of red blood cells is very sensitivity to HgCl2 or the organic mercurial pCMBS
Mercurial sensitivity -Experimental evidence -
SEE GRAPH
- 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
Mercury binds to what ?/
Exposed cysteine residues
