Lecture 12- Structure and Function of Aquaporins

Question 1

Aquaporin 4

Answer 1

Mercury insensitive

Question 2

Basis of mercury action similar to aquaporin

Answer 2

Location of mercury sensitive residues common in aquaporins

Question 3

How does mercury affect aquaporins ?

Answer 3

Binds to their cysteine residues

Question 4

AQ1 has how many cysteine residues ?

Answer 4

4

Question 5

Mutant study -

Answer 5

• 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

Question 6

Mutant study - Results

Answer 6

-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

Question 7

what is the mercurial sensitive residue in AQP1

Answer 7

Cysteine 189S

Question 8

Structure of AQP1 - Hourglass model

Answer 8

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

Question 9

What has Cryoelectron microscopy shown ?

Answer 9

Tetrameric structure of Aquaporin 1

Question 10

How did they test whether AQP1 functional unit a monomer or tetramer ?

Answer 10

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

Question 11

How did they test whether AQP1 functional unit a monomer or tetramer ? Preferred approach

Answer 11

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 **

Question 12

Is the functional unit in AQP1 a monomer or tetramer ??

Answer 12

monomer

Question 13

Water transport through AQP1 –

Answer 13

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

Question 14

Test basis of mercury inhibition –

Answer 14

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

Question 15

Aquaporin 3

Answer 15

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

Question 16

Aquaporin 6

Answer 16

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

Question 17

MIP-

Answer 17

superfamily 900 +Member

13 in mammals

Question 18

Classes of MIP

Answer 18

• Aquaporins
• Aqua-glyceroporins
• Unorthodox

Question 19

AQP2 and disease

Answer 19

Mutations - Diabetes insipidus
Reduces permeability to water – mutation near channel pore
Disrupting trafficking to the

Question 20

Reabsorptive Epithelia in PT - water transport

Answer 20

AQP1

Question 21

Secretive epithelia in salivary gland - water transport

Answer 21

AQP5

Question 22

Reabsorptive Epithelia in small intestines - water transport

Answer 22

NO AQPa

Question 23

Proximal tubule

Answer 23

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

Question 24

PT- Evidence for near iso-osmotic fluid reabsorption

Answer 24

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
?!

Question 25

PT- Evidence for near iso-osmotic fluid reabsorption 2

In vivo

Answer 25

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

Question 26

PT experimental evidence overall conclusion -

Answer 26

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

Question 27

Salivary Gland

Answer 27

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

Question 28

KO - AQP5

Answer 28

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