Lecture 13- Membranes and Gases Flashcards
Typical epithelium
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
Small Intestine
- 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
Wet transport proteins
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
Experiment in frog
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
Models of Water transport
AQP1 Osmosis KCC- Co transport SGLT1- Co transport and osmosis Conformational changes in protein Relies on small local gradients to drive the movement
Small intestine water reabsorption -
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
Overtons law-
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.
Experiment -
See Graph
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
CO2/HCO3 - See graph reverse of ammonia/ammonium
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
Acidification
Acidification- CO2 dominating
Alkalisation
Alkalisation – HCO3 dominating
Thick ascending limb; Permeability to NH4/NH3
- 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
2nd experiment – Gastric gland – permeability to CO2 and HCO3
- comparing luminal changes in PH and co2 with Bath pH and CO2 changes
Basolateral side – normal permeability to Co2 – weak acidification
Biological membrane impermeable to gas – no change in ph with different conc of dissolved Co2
What is the basis of the low permeability to gases ?
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
Experiment using liposomes - Artificial membrane
- Experiment using liposomes (artificial membrane)
Cholesterol content acting to reduce the fluidity of the membrane
Linear relationship between cholesterol content and CO2 permeability
Using MDK cells _ living Membrane
- Using MDK cells (living membrane)
Strip cholesterol from membrane – co2 permeability shoots up
Raise cholesterol levels- co2 goes down
Membrane with low cholesterol levels (30%)
Membrane with low cholesterol levels (30%)– for example many cancer cell lines. CO2 permeability is high enough to support metabolic demands
Membrane with high cholesterol levels –
Membrane with high cholesterol levels – for example apical membrane of the colonic crypt (77%). Provides barrier function limiting gas transport
RBC and apical membrane of the proximal tubule have cholesterol content of ?
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?
Effect of CO2 on intracellular pH in xenopus oocytes -
10mM HCO3-/1.5%CO2
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
Effect of CO2 on intracellular pH in xenopus oocytes - Experiment 2
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»_space; Mercury blocking the pore of AQP1
CO2-induced Acidification Rate (ΔpHi/ Δ t x10-4 pH.s-1) Vs Cell lysis time shows what ??
CO2 permeability is proportional to water permeability – the more AQP1 channels present the faster the rate of acidification.
Colton Null- Co2 permeability of RBC
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.
What forms Colton null blood group ?
AQP1 – forms colton blood group (LOF)
50% of RBC permeability due to AQP1
How does DIDs work/What is its effect ??
DIDs – blocks CO2 permeability but not water permeability
DIDs inhibits Rhesus protein 40% of permeability due to rhesus
10% - going through lipid bilayer
AQP1 and CGMP
CGMP binds to AQP1 and switches between water channel and Na channel
AQP1
Central pore - mainly hydrophobic, gated by hydrophobic residues
Aquapore- Hydrophilic and hydrophobic
