Lecture 13 - AQP continued 2 Flashcards
Small intestine membrane proteins
AQP on both apical and basolateral membranes
sodium, chloride and other solutes transported from the lumen to the blood
Small intestine osmolality
high in the lumen but isotonic in the blood, there are high levels of H2O absorption
Wet transport proteins
co-transporters that also transport water as part of their normal operation
Co-transport is…
against the osmotic gradient
Co-transport and osmosis used when..
the transport of solute changes the osmolality which int urn drives osmosis
Overton’s Law
the permeability of a membrane to a solute is proportional to the oil/water partition coefficient for that solute
natural extension fo Overton’s Law
because gases such as O2 or CO2 have a high solubility in oil, so all biological membranes were freely permeable to gases
Bilayer experiment with no cholesterol or proteins
show very high CO2 permeability
But it uses decaf which increases gas permeability, therefore may not be fully valid
NH4/NH3 ratio
19NH4 to 1NH3 in solution
NH4/NH3 experiment for pHi
expose cell to solution
both cross the membrane but NH3 has a higher permeability so enters cell and binds H+ which causes the cell to undergo alkalisation
Slower NH4 enters cell through proteins, dissociates and releases H so acidification occurs
CO2/HCO3 experiment on pHi
Co2 enters cell and combines with H2O but then dissociates to HCO3 and H+ causing acidification
HCO3 is charged so uses transporters to enter, binds H+ so alkalisation
thick ascending limb basolateral membrane permeability
high permeability to both NH4 and NH3
Thick ascending limb apical membrane permeability
high NH4 permeability but low NH3 permeability
as it is impermeable to H2O it is likely to be impermeable to NH3
Gastric gland basolateral membrane
with CO2 it shows small acidification therefore shows permeability to CO2
Gastric gland apical membrane
with high conc CO2 there is no effect, therefore CO2 impermeable
Low gas permeability is linked to
membrane cholesterol content as it reduces fluidity
Artificial liposome experiment
to increase cholesterol therefore decrease CO2 permeability
Gastric gland apical membrane cholesterol
75%
membranes with low cholesterol levels
CO2 permeability is high enough to support metabolic demands
Membranes with high cholesterol levels
provides barrier function so limits gas function
Co2 on pHi in xenopus oocytes
no background permeability for HCO3-
So CO2 rate not effected by HCO3- transport
acidification rate is proportional to CO2 permeability
AQP1 H2O and CO2 permeability
measuring cell burst to find increased water permeability
CO2 and H2O correlation
CO2 permeability is proportional to H2O permeability
More AQP1 channels means
faster rate of acidification
CO2 permeability is proportional to…
AQP1 expression
pCMBs
organic mercurial compound that binds to exposed cysteine residues
pCMBs and acidification
acidification rate decreases with pCMBs to baseline levels
Proof AQP1 is a CO2 channel
express C189S mutant so pCMBs can no longer bind
this shows high permeability proving AQP1 increases CO2 permeability
Colton Null
linked to lack of AQP1 expression or mutant AQP1 in RBCs
Colton null CO2 permeability
reduced
unaffected by pCMBs
DIDs and RBCs
inhibits CO2 permeability
proves there is an additional way CO2 enters the cell
CO2 movement in RBC (3 ways)
50% AQP1, 45% Rhesus-associated glycoproteins and 5% across lipid bilayer
Rhesus-associated proteins
permeable to NH3 and CO2
DIDS effect of AQP1
inhibits CO2 movement but H2O can still move through
CO2 movement through AQP1
moves through the 4 water pores and the central pore equally
O2 movement through AQP1
through central pore mainly
cGMP effect on AQO1
AQP1 is sensitive to cGMP binding and activates ion conductance
proves central pore can be used as a cation channel
knocks out CO2 permeability
