Lecture 13- Membranes and Gases

Question 1

Typical epithelium

Answer 1

Reabsorption across the epithelial membrane
Na, CL and solute reabsorbed from lumen to blood
Low osmolarity on lumen side to high osmolarity in blood > reabsorption of fluid

Question 2

Small Intestine

Answer 2

• Reabsorbs large vol of water
• Lacks aquaporins on the apical side
• Faced with hypertonic luminal environment
• Lumen – High osmolarity
• Blood- isotonic
• Small intestine able to reabsorb water in uphill movement

Question 3

Wet transport proteins

Answer 3

Some co transporters also transport water as part of their normal function
e.g KCC4- 500 molecules of water
NKCC1 – 590 molecules of water
Water transported against osmotic gradient

Question 4

Experiment in frog

Answer 4

Looking at NKCC1 co transporter
Look at vol in the cells
1st exposed to hypertonic sol – shrink
KCL – opposite – cell swells > against 100 msm gradient
Control KCL and furosemide > cell shrinkage

Question 5

Models of Water transport

Answer 5

AQP1 Osmosis 
KCC- Co transport 
SGLT1- Co transport and osmosis 
Conformational changes in protein 
Relies on small local gradients to drive the movement

Question 6

Small intestine water reabsorption -

Answer 6

GLUT2 in apical when stimulated by digestion
- Movement of water against the osmotic gradient
Apically-
SGLT1 - Na/GLucose/H20
GLUT2- Glucose/ H20

Basolateral- 
ATPase
KCC-K/CL/H20 
GLUT2 - Glucose/ H20 
500mOsm apical to 300 mOsm basolateraly 
NA and glucose goes from apical to basolateral

Question 7

Overtons law-

Answer 7

The permeability of a membrane to a solute is proportional to the oil/water partition coefficient for that solute.

Gases such as oxygen and carbon dioxide have a high solubility in oil, so a natural extension of this law was that all biological membranes were freely permeable to gases.

Question 8

Experiment -

See Graph

Answer 8

Effect of NH4/NH3 on pHi
Dissolved in solution ammonium and ammonia gas
Weak base
-Ammonium isn’t permeable across the bilayer so moved in by transporters
NkCC2 – ammonium will substitute for K
Can also move through k channels eg ROMK
Uptake of ammonium into cell gives you acidification of the cell
Use this to determine which species is predominating across the membrane

Question 9

CO2/HCO3 - See graph reverse of ammonia/ammonium

Answer 9

CO2 effectively a weak acid 
Bicarbonate pH 6.1 – will dominate 
Uptake of CO2 into a cell cause acidification – 
Acidification- CO2 dominating 
Alkalisation – HCO3 dominating

Question 10

Acidification

Answer 10

Acidification- CO2 dominating

Question 11

Alkalisation

Answer 11

Alkalisation – HCO3 dominating

Question 12

Thick ascending limb; Permeability to NH4/NH3

Answer 12

• Low permeability to a gas
• Basolateral membrane- high permeability to NH4 and NH3
• Nacl to Basolateral side
• Apical membrane – no alkalisation at all – high permeability to NH4 but extremely low permeability to NH3
• Shown limited permeability to a gas

Question 13

2nd experiment – Gastric gland – permeability to CO2 and HCO3
- comparing luminal changes in PH and co2 with Bath pH and CO2 changes

Answer 13

Basolateral side – normal permeability to Co2 – weak acidification
Biological membrane impermeable to gas – no change in ph with different conc of dissolved Co2

Question 14

What is the basis of the low permeability to gases ?

Answer 14

Linked to cholesterol content of the membrane
1. Experiment using liposomes (artificial membrane)
Cholesterol content acting to reduce the fluidity of the membrane
Linear relationship between cholesterol content and CO2 permeability
2. Using MDK cells (living membrane)
Strip cholesterol from membrane – co2 permeability shoots up
Raise cholesterol levels- co2 goes down

Question 15

Experiment using liposomes - Artificial membrane

Answer 15

1. Experiment using liposomes (artificial membrane)
   Cholesterol content acting to reduce the fluidity of the membrane
   Linear relationship between cholesterol content and CO2 permeability

Question 16

Using MDK cells _ living Membrane

Answer 16

2. Using MDK cells (living membrane)
   Strip cholesterol from membrane – co2 permeability shoots up
   Raise cholesterol levels- co2 goes down

Question 17

Membrane with low cholesterol levels (30%)

Answer 17

Membrane with low cholesterol levels (30%)– for example many cancer cell lines. CO2 permeability is high enough to support metabolic demands

Question 18

Membrane with high cholesterol levels –

Answer 18

Membrane with high cholesterol levels – for example apical membrane of the colonic crypt (77%). Provides barrier function limiting gas transport

Question 19

RBC and apical membrane of the proximal tubule have cholesterol content of ?

Answer 19

The RBC and apical membrane of the proximal tubule have cholesterol content of 45%. This would not support the level of CO2 transport that has been measured.
How is CO2 transported in these cells?

Question 20

Effect of CO2 on intracellular pH in xenopus oocytes -

10mM HCO3-/1.5%CO2

Answer 20

Co2/hco3 solution – acidification proportional to CO2 permeability
Correlation between H20 and CO2 permeability
CO2 permeability is proportional to water permeability – the more AQP1 channels present the faster the rate of acidification

Question 21

Effect of CO2 on intracellular pH in xenopus oocytes - Experiment 2

Answer 21

repeated using a mercurial compound that binds to exposed cysteine residues – pCMBS
Oocytes expressing AQP1- high CO2 permeability but with addition of mercurial agent it goes down&raquo_space; Mercury blocking the pore of AQP1

Question 22

CO2-induced 
Acidification Rate
(ΔpHi/ Δ t
x10-4 pH.s-1)
 Vs Cell lysis time shows what 
??

Answer 22

CO2 permeability is proportional to water permeability – the more AQP1 channels present the faster the rate of acidification.

Question 23

Colton Null- Co2 permeability of RBC

Answer 23

Colton Null - Red blood cell CO2 permeability is greatly reduced compared to normal RBCs.
Colton Null - CO2 permeability is unaltered by pCMBS.
CO2 permeability in Colton null and WT is inhibited by DIDS.

Question 24

What forms Colton null blood group ?

Answer 24

AQP1 – forms colton blood group (LOF)

50% of RBC permeability due to AQP1

Question 25

How does DIDs work/What is its effect ??

Answer 25

DIDs – blocks CO2 permeability but not water permeability
DIDs inhibits Rhesus protein 40% of permeability due to rhesus
10% - going through lipid bilayer

Question 26

AQP1 and CGMP

Answer 26

CGMP binds to AQP1 and switches between water channel and Na channel

Question 27

AQP1

Answer 27

Central pore - mainly hydrophobic, gated by hydrophobic residues

Aquapore- Hydrophilic and hydrophobic