Lecture 12- Structure and Function of Aquaporins Flashcards
Aquaporin 4
Mercury insensitive
Basis of mercury action similar to aquaporin
Location of mercury sensitive residues common in aquaporins
How does mercury affect aquaporins ?
Binds to their cysteine residues
AQ1 has how many cysteine residues ?
4
Mutant study -
- Made 4 mutants Individually mutated each of the 4 residues to serine and tested the effect of HgCl2 on water permeability in these mutants. – little effect on protein similar size and similar charger
Mutant study - Results
-Inject oocytes with cRNA – measured water permeability
-Mutants have the same water permeability as the WT AQP1
-All mutants basic function isn’t changed – water pores still
Effect is shown in the action of mercury
3 mutants reduction in water permeability – no effect on C189s mutant
C198s mutant changing cysteine to serine > protein no longer sensitive to mercury
-Cysteine 189S is the mercurial sensitive residue in AQP1
what is the mercurial sensitive residue in AQP1
Cysteine 189S
Structure of AQP1 - Hourglass model
E loop and B loop loop back into the membrane
B and E loop overlap which gives the water selective pore
Amino and carboxy ends of the protein are both intracellular
6 TM
Between TM2-3 and TM5-6 are loops B and E which dip into the membrane- in the middle of these loops is the NPA motif
Mercury binds to cysteine and blocks water pore
What has Cryoelectron microscopy shown ?
Tetrameric structure of Aquaporin 1
How did they test whether AQP1 functional unit a monomer or tetramer ?
Approach - Construct tandem dimers of WT AQP1 and C189s mutant channel and express in oocytes
Advantage- force each tetramer to contain 2 WT and 2 mutant channels but no guarantee of how tetramers form
Mix of AQP1+C189s
How did they test whether AQP1 functional unit a monomer or tetramer ? Preferred approach
Instead use tandem dimer ½ and ½
Iject into oocyte and look at water function
Express AQP1-AQP1 or AQP1-C189s or C189s -C189s
Dimers all produce water channels
Adding mercury to C189s- Cl89s no effect on water permeability
Wt mercury – water permeability goes down – all 4 pores blocked
C189s- AQP1- water permeability goes down by 50% so 2 pores blocked
*** each AQP1 monomer is a functional water channel **
Is the functional unit in AQP1 a monomer or tetramer ??
monomer
Water transport through AQP1 –
NPA motifs
Single file movement through pore – Basic model
Suggest a proton channel as well – but not the case
Hydrogen bond between molecules as it moves through the pore cause them to bond with the NPA motif on the back bone making the poor selective
Test basis of mercury inhibition –
Use a bacterial AQP which isn’t usually sensitive to mercury
Mutate and introduce a cysteine
Mercury binds in the middle of the pore -prevents water movement
Aquaporin 3
When expressed in oocytes mercury sensitive increase in glycerol and urea permeability
Aqua glycerol porins
Basolateral membrane of the collecting duct
Can also transport glycerol and urea as well as water
Aquaporin 6
Isn’t found in cell membrane
Expressed in the kidney and co- localise with proton pump in intracellular vesicles in alpha intercalated cells
AQP6 - shows water permeability an also acts as a chloride conductance
Also stimulated by mercury
Increase in CL current
Also pH sensitive
Acidic pH stimulates the Cl conductance
AQP6- working to balance charge in the vesicles
As more protons into vesicle > more acidic which activates AQP6 which returns the balance
MIP-
superfamily 900 +Member
13 in mammals
Classes of MIP
- Aquaporins
- Aqua-glyceroporins
- Unorthodox
AQP2 and disease
Mutations - Diabetes insipidus
Reduces permeability to water – mutation near channel pore
Disrupting trafficking to the
Reabsorptive Epithelia in PT - water transport
AQP1
Secretive epithelia in salivary gland - water transport
AQP5
Reabsorptive Epithelia in small intestines - water transport
NO AQPa
Proximal tubule
AQP1
- High water permeability
- Using perfuse tubules KO of AQP1 cause a 78% decrease in PF
- AQP1 essential for water transport across PT
- Reabsorption occurs under near isotonic conditions
- Transport from lumen to blood of Na, CL, solute
PT- Evidence for near iso-osmotic fluid reabsorption
using a microperfused PT segments
Absorbate forms arund perfusate - small droplet of water form around the oil – suck it off and measure conditions within it
In vitro
?!
PT- Evidence for near iso-osmotic fluid reabsorption 2
In vivo
Using Micropuncture techniques
Perfused the lumen of the tubule and the capillaries with a solution containing 154mM NaCl
-Slightly unreal conditions- normal Na solution lacking bicarbonate but show small osmotic gradients drive water movement in PT
-High flow rate reabsorption is taking place but doesn’t reach equilibrium
-Low flow rate – equilibrium met much quicker
-High water movement in PT driven by small concentration gradients
PT experimental evidence overall conclusion -
Transport by the PT generates a favourable osmotic gradient for water reabsorption.
Because of extremely hight water permeability of the tubule this gradient is mall and is probably due to the generation of a hypotonic lumen
Salivary Gland
iso-osmotic fluid secretion
Secretin Nacl into lumen making it hypertonic
Rpvodes osmotic gradient for water movement
AQP5 drives water movement
Acinar cell
Secreting water to balance out the gradient
Uptake at basolateral mem through Nkcc1
Na and cl secreted with water following
KO - AQP5
Loss of AQP5 prevents the complete dissipation of the lumen hypertonicity
Results in the production of a small volume of saliva with high salt content
