Membranes and Gases

Question 1

1. Define Overton’s Law?
2. What does Overton’s Law imply about the permeability of membranes to gases
3. why is changing the permeability of membranes to gases beneficial?

Answer 1

1. the permeability of a membrane to a solute is proportional to the oil/water coefficient for that solute
2. that membranes are freely permeable to gases
3. it enables the regulated transport of gasses across membranes.

Question 2

1. what is the effect of a mixed solution of NH₃/NH₄⁺ on pH_i and why?
2. what is the effect of a mixed solution of CO₂/HCO₃^- on pH_i and why?

Answer 2

1. rapid alkalisation as NH3 freely moves into the cell, and slow acidification as NH4 moves into cell via transporters
2. rapid acidification as CO2 freely diffuses across membrane, and slow alkalisation as HCO3 moves into the cell via transporters

Question 3

1. What is a benefit of using the microperfusion technique
2. describe how the microperfusion technique was used to show that membranes in the thick Ascending limb are not freely permeable to gases
3. describe how the microperfusion technique was used to show that membranes in the gastric gland are not freely permeable to gases
4. what is the physiological relevance of limiting the permeability to gases in the gastric gland?

Answer 3

1. it enables the A and BL sides of an epithelia to be pefused independently of each other
2. a NH3/NH4 mixture was added to either side of the limb. Adding NH3/NH4 to the BL side caused rapid alkalisation; Adding NH3/NH4 to the A side caused no alkalisation, but some acidification as NH4 entered the cell via transporters, meaning that the apical membrane is impermeable to gases
3. BL membrane was exposed to varying CO2 concentrations. Acidificiation was seen. When the A membrane was exposed to 100% CO2, no acidification was seen indicating that the apical membrane is impermeable to gases
4. the stomach has low pH, meaning that the CO2 concentration is high. It is therefore beneficial to have an apical membrane that is impermeable to CO2 to prevent acidification of gastric gland cells.

Question 4

1. What is the basis of the low permeability of membranes to gas
2. describe how liposome studies have shown this
3. describe how studies using mDCK cells have show this
4. why must other mechanisms for gas transport be present?

Answer 4

1. cholesterol content of the membrane
2. the cholesterol content was altered, and CO2 permeability was measured; increasing cholesterol content by 20% caused a 10 fold decrease in CO2 permeability
3. the cholesterol content of the cells was altered and CO2 permeability measured. Again, increasing the cholesterol content caused the CO2 permeability of the membrane to decrease and visa versa.
4. the cholesterol content of RBCs and proximal tubule is 45%, which would not support the level of CO2 transport which occurs here.

Question 5

1. what is the initial rate of acidification following the addition of CO2 proportional to?
2. how was this used to show that AQP1 is permeable to CO2?
3. What was done to investigate whether CO2 passed through the AQP1 pore, or whether the expression of the channel distorts the membrane to allow the passage of CO2? What was deduced from the findings?

Answer 5

1. the CO2 permeability of the membrane
2. when AQP1 was expressed in the cells, the rate of acidification negatively correlates with time to cell lysis (shorter time to cell lysis with increased AQP1 expression). This indicates that CO2 permeability is proportional with CO2 permeability
3. tested the effects of pCMBS on the CO2 permeability of AQP1 cells; pCMBS reduces the CO2 permeability to background levels of the cell

the same experiments were performed in the C189S mutant - CO2 permeability was unaffected by pCMBS

CO2 moves through the AQP1 pore.

Question 6

1. what does the cholesterol content of RBCs suggest their CO2 permeability should be?
2. what is the actual CO2 permeability of RBCs?
3. what people lack AQP1?
4. describe the CO2 permeability of RBCs of people lacking AQP1
5. describe the effects of DIDs and why this was seen.

Answer 6

1. 0.01cm/sec
2. 1 cm/sec
3. those with colton null blood group
4. reduced CO2 permeability that is unaffected with pCMBS (WT - v.high pCMBS sensitive CO2 permeability)
5. reduced the CO2 perm of the WT RBCs to a greater extent than DIDs and also had han effect on the colton null RBCs. This is because DIDs inhibits the rhesus protein, which is thought to confer the remaining 50% of CO2 permeability (AQP1 provides 50%); DIDs reduces the CO2 perm of colton null RBCs to 0.01cm/sec, the permeability suggested by the cholesterol content.

Question 7

1. what type of residues line the individual aquapores of AQP1?
2. what type of residues line the central pore of AQP1?
3. through which pores can CO2 move through?
4. how does cGMP alter the permeability of AQP1?

Answer 7

1. hydrophobic and hydrophilic
2. hydrophobic
3. central pores and aquapores
4. it induces a conformatioal change, meaning that water can now pass through the central pore, and AQP1 becomes a cation channel, whilst inhibiting CO2 permeability.