History of water pore

Question 1

What did the first experiment show?

Answer 1

Identified that there were water pores (in various spherical cells) as Pf/Pd > 1, although some had less permeability (and trout not at all).

Question 2

What did the second?

Answer 2

An elegant set of experiments carried out by Solomon et al. (1957) showed that RBCs had water pores - Pf/Pd = 2.5. Estimated the size of pores to be 3.5A and 250,000 copies (turned out to be 2.5A and 200,000).

Question 3

What did the third show?

Answer 3

Peter Agre’s group elucidated the molecular structure. 28 kD protein co-precipitated with Rhesus protein, made ABs against it which didn’t stain 32kD Rh so wasn’t part of that family. From this staining found: higher MW protein (glyosylated), multimer staining (turned out to be tetramer). Also staining in PT and TDL.
I.e. it was a 28kD protein that could be glycosylated and existed as a multimer.

Question 4

Tell me a bit more about the high MW band?

Answer 4

This was the glycosylated band which is important for trafficking AQP’s to the PM from Golgi so can form functional pore - Hendriks et al. showed 25% of AQP2’s are glycosylated (and is not essential for tetrameric structure formation).

Question 5

What happened after 28kD staining and molecular structure elucidation by Agre?

Answer 5

Cloned ‘CHIP28’ with PCR and screened against library. Found 2.8kb transcript that had high homology with MIP26 (AQP0 in eye) with unknown function but all had NPA repeats. (essential role in water selective permeation. Later it was discovered that AQP11/12 don’t have NPA and aren’t expressed at cell surface suggesting another role in AQP trafficking). This transcript corresponded with RNA fraction from kidneys that induced maximum water permeability when injected into Xenopus oocytes.

i.e. Cloning, PCR and screening identified homologous proteins (MIP29 which is now AQP0) which all had NPA motifs, and found 2.9 kb transcript that correlated with RNA fraction from kidneys that induced permeability in oocytes.

Question 6

What was next regarding exploration of CHIP28’s function?

Answer 6

This correlation added to the mounting circumstantial evidence that CHIP28 was a water channel e.g….

1) Number of CHIP28 copies in RBC membrane almost = Soloman’s calculation (250,000)
2) CHIP28 resistant to enzymatic digestion as was RBC channel

Question 7

What are the facts so far about AQP1/CHIP28?

Answer 7

Identified water pores as Pf/Pd > 1, Molecular structure was 28kD which existed probably has multimers and can be glycosylated, the 2.9kb transcript had homology with other proteins (MIP28/AQP0) which all had NPA motifs and it correlated with permeability inducing kidney fraction.

It is probs a water channel as number of copies = similar to water pore estimation in RBCs and is resistant to digestion like these RBC pores.

Question 8

What was the experimental evidence for CHIP28 being a water channel?

Answer 8

Over-expressed CHIP28 in Xenopus oocytes and exposed to hypertonic shock.
Control = v. slow vol. change
CHIP28 = rapid change and lysis.
Water must have moved in so must be a water channel. (Now AQP1).

Question 9

What next?

Answer 9

Next, not only confirmed it was a water channel but also that it was a mercury sensitive water channel…
Same experiment but added mercury and volume change in response to hypotonic (this time) was much less i.e. water permeability decreased, similar to RBC water permeability mercury sensitivity.
When mercury binding reversed it returned to normal.

Question 10

How did the mercury sensitive mechanism get elucidated?

Answer 10

Agre’s group made mutants of all 4 AQP1 cysteine residues, found that C189S mutation prevented the sensitivity so C189 is the mercury sensitive residue.

Question 11

What did work on the structure of AQP1 reveal?

Answer 11

6TMBDs. 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. In the hourglass model these come together to form a pore with the mercury sensitive C189 in the middle.

When they looked at crystal structure of mercury binding to C183 in bacterial AQP1, it was seen that mercury indeed binds here in the middle of each monomer.

Question 12

What confirmed the structure ideas?

Answer 12

CyroElectron microscopy confirmed 4 hourglass monomers come toogether in tetramer (that was predicted by Agre). Saw a central pore - was this a water channel?…

Question 13

How did they elucidate the central pore role?

Answer 13

Tandem dimer of AQP1 and C189S. If central pore = channel, permeability not effected by Mercury, but it halved because half monomers were insensitive, so each monomer was a water channel. (central pore useful in gas and ion transport as AQPs can be multifunctional tetramers).

Question 14

What did we then learn about water transport through AQPs?

Answer 14

water molecule = 1.8A diameter, pore = 2A so single file, would expect protons to piggyback due to H bonds in water but there is no H+ transport. This is because NPA regions form bonds with H20 molecule instead making the pore very water selective.

Question 15

On to other AQPs… Tell me about aqua-glyceroporins.

Answer 15

AQP3 & 7. AQP3 permeable to glcyerol and urea.

Question 16

Tell me about unorthodox AQPs.

Answer 16

AQP6. Intracellular kidney co-localised with H+ pump in vesicles (not membrane). Transports both Cl- and H20 to neutralise charge (protons) brought in by pump. Stimulated by mercury (not inhibited) and by acidic pH, as more H+ ion influx = lower pH = more Cl- to neutralise.

Question 17

Link of AQP to diseases?

Answer 17

Diabetes insipidus - no functional AQP2 at PM of principle cells in inner medullar collecting duct IMCD so can’t reabsorb water - 20L urine a day.

AQP3 links to cancer (not just epithelial transport).

Question 18

Give examples of how different epithelia have different water transport.

Answer 18

Secretive Vs reabsorptive.
Reabsorptive = PT (AQP1 - near isoosmotic), Small int. (No AQPs).
Secretive = salivary glands (AQP5).

Question 19

Tell me about PT reabsorption.

Answer 19

AQP1.
AQP1 KO -/- = 80% Pf i.e. AQP1 critical in H2O transport here.
Near iso-osmotic i.e. small osmotic gradient generated.

How do we know this??…

Question 20

How do we know about proximal tubule small osmotic gradient generation?

Answer 20

Microperfusion of PT segment lumen (i.e. apical) with mineral oil surrounding baso.
As PT reabsorbs H20 and solutes forms droplet within oil.
Compared freshly generated absorbate with perfusate.
Pf was only 16mOsm difference between them, despite the PT reabsorbing 120L a day!!!

I.e. PT reabsorbs huge quantities with v small osmotic gradient (near iso-osmotic).

Question 21

Why is the osmotic gradient very small in PT?

Answer 21

Na+/aa and Na+/glucose co-transporters reabsorb solutes so lumen hypotonic (more water than solutes) so driving force for water reabsorption via AQP1. But because the PT is so permeable to water the gradient is small.

Flow rates influence the PT Pf…

Question 22

What experiments show that flow rates influence osmotic gradients and water reabsorption in the PT?

Answer 22

Experiment similar to PT perfusion one.
NaCl perfusion in lumen, fast flow rate had higher absorption because the small gradient thats driving reabsorption is neutralised slower (as less time to move out).

Question 23

Is the PT lumen hypo or hypertonic.

Answer 23

Hypo as more water than solutes due to solute reabsorption which generates small gradient.

Question 24

Experimental evidence to support hypotonic lumen?

Answer 24

AQP1 KO mice = more dilute i.e. hypotonic at end PT as water not moving out.

Question 25

Tell me about secretive epithelia.

Answer 25

E.g. salivary gland acinar cells.
AQP5.
Same idea but other way round as PT i.e. hypertonic lumen due to secreting salt so water follows, as baso uptake of Na+ and Cl- via NKCC1 and Na+ through tight junctions, H20 moves through AQP5 to be secreted.

Question 26

What is experimental evidence for secretory epithelial?

Answer 26

AQP5 KO mice = 1/2 volume of concentrated saliva produced when saliva production stimulated as H20 can’t move out.

Question 27

Tell me about a reabsorptive epithelia that doesn’t have AQP’s.

Answer 27

Small intestine.
No AQP’s on apical membrane.
This is a problem because:
1) apical and baso needed for transepithelial transport to reaborb
2) if AQP’s were present, the hypertonic nature of the lumen (digesting food = high solutes) it would drive secretion of water..

But SI does reabsorb water agianst a 200mOsm gradient. How? Co transport proteins.

Question 28

Which co-transport proteins does small intestine have?

Answer 28

NKCC1 (baso - 590 molecules of water each turnover), KCC4 (baso), SGLT (apical), GLUT4 (both).

Question 29

How many molecules of water does NKCC1 transport every turnover?

Answer 29

590.

Question 30

When and how is water moved against it’s osmotic potential?

Answer 30

Small intestine (reabsorptive epithelium) - uses NKCC1, SGLT, GLUT4 and KCC4 to co-transport it against a 200mOsm gradient.

Question 31

What is the difference in gradient from apical (lumen) to basolateral side in small intestine?

Answer 31

200mOsm.

Question 32

What is the experimental evidence to prove that the small intestine uses co-transport proteins to transport water against an osmotic potential?

Answer 32

Express KCC4 in frogs.
Expose to hypertonic shock.
NaCl - cells shrink (water moves out)
KCl - cells swell even though hypertonic so against osmotic potential as KCC4 brings H20 in.
Blocking with furosemide prevents this.

Question 33

What does Overton’s law state?

Answer 33

‘the permeability of a membrane to a solute is proportional to the oil/water separation coefficient for that solute’.
Originally (1800s) idea was that all membranes are permeable to gases that can freely diffuse in or out.

Question 34

What was wrong with the artificial membranes that seemed to confirm Overton’s law?

Answer 34

• contaminated with decane which made membrane more fluid and permeable to CO2
• no proteins or cholesterol

Question 35

What happens at pH 7.4 if a solution of amonia sulfate is added to water?

Answer 35

19 parts charged ammonium, 1 part uncharged ammonia.

NH3 + H+ –> NH4+. Alkalisation.
NH4+ –> NH3 + H+. Acidification (slower as charged)

Question 36

What happens at pH 6.1 if a solution of CO2 and HCO3- is added to water?

Answer 36

CO2 + H2O –> HCO3- + H+. Acidification.

HCO3- + H+ –> CO2 + H2O. Alkalisation (slower).

Question 37

Describe an experimental example of proof that a membrane is not always permeable to gases.

Answer 37

Gastric gland in stomach.
Perfused basolateral side, saw increase in acidification as expected.
Perfused apical side with high Co2 but no change in pH - impermeable to CO2 makes sense as prevents digestion of tissue in stomach DUE TO CHOLESTEROL.

Question 38

Experiments to prove cholesterol alters PCO2?

Answer 38

Liposomes (artificial membrane) and kidney cell line (MCDK cells) - increase in cholesterol = increase in PCO2.