Aquaporins

Question 1

Mercurial sensitivity - where does the mercury bind? How was this shown?

Answer 1

C189S residue on AQP1 – when different cysteine residues of AQP1 were mutated and then mercury added, no change in cell volume or Pf was seen in mutated C189S residue

Question 2

AQP1 - hourglass model - what does this refer to and what are the important features?

Answer 2

6TMDs, loops between tmds 2 and 3 and 4 and 5 –> middle of this NPA domain can be found

The amino and carboxy end of the protein are intracellular

Question 3

C189 residue - why important?

Answer 3

Crucial region of AQP1 lies close to opening of channel pore –> means AQP1 is sensitive from the inside of the cell

Question 4

AQP1 tetramer structure

Answer 4

AQP1 exists as tetramer (confirmed by cryo-electron microscopy)
Central pore structure
4 monomers to protein - each monomer has a water pore

Question 5

AQP1 AS A FUNCTIONAL UNIT - MONOMERS OR TETRAMERS? What experiment proved this

Answer 5

Tandem dimers created with WT-C189S, C189S-C189S and WT-WT structure
- Hg only impacted half and half by 50%
- Hg had no impact on both mutated monomers
»> each monomer is a functional unit

Question 6

Hg sensitivity - where does Hg bind?

How does it impact H20 movement in that region of the protein?

Answer 6

Hg binds to the middle of a crystal structure of AQP1 created –> prevents movement of H20 in that region

Question 7

Why is AQP1 an ideal water transporter – regarding hydrogen bonding?

Answer 7

AQP1 just slightly bigger than a water molecule so good size for single file movement of water molecules

• hydrogen bonding between water molecules means that they should be able to piggyback through the pore region
• AQP1 decouples water molecules by breaking hydroge bonds and reforming at the NPA motif
• Prevents H+ coming down the negatively charged back bone and makes pore very water selective

Question 8

AQP3 - where is it located? WHy is it different to AQP1?

Answer 8

Located in the skin and collecting duct

Aquaglycerolporin - can therefore transport small solutes such as urea and glycerol as well as water

Question 9

AQP3 structural differences to AQP1 –> what is shown with regards to mercurial sensitivity ?

Answer 9

Mercurial sensitivity of AQP3 increases in the presence of urea and glycerol –> allows for slower transport of solutes.
AQP3 also may be structurally different to AQP1

Question 10

AQP6 - where is it found and why is it unorthodox

Answer 10

Found in the kidney and colocalises with H+ ATPase pump - also acts as a Cl- transporter!!
Known as an unorthodox AQP as it ISNT FOUND IN THE MEMBRANE

Question 11

Mechanism of AQP6 - how is it PH sensitive?

Answer 11

Permeable to water and acts to balance the charges of vesicles –> Proton pump brings H+ into vesicles and then Cl- moves in via AQP6 to balance charge

Activity is PH sensitive!!!

Question 12

What disease in linked to AQP2 malfunctioning?

Answer 12

Diabetes insipidus
no response of cells to vasopressin - patients cannot properly concentrate their urine
Either caused by lack of response to vasopressin or a problem in AQP2

Question 13

AQP3 - what disease linked to?

Answer 13

Been linked to some cancers

Question 14

3 types of epithelia that involve water transport - examples?

Answer 14

Reabsorbtion epithelia - proximal tubule - AQP1
Secretive epithelia - salivary gland - AQP5
SMALL INTESTINE CHALLENGES THIS - reabsorption occurs here but no AQPs can be found!!

Question 15

PROXIMAL TUBULE - what conditions are present? why is this interesting?

Answer 15

High h20 permeability yet under nearly isotonic conditions (small gradient for reabsoprtion)

Question 16

PROXIMAL TUBULE - what conditions are present to drive h20 reabsorption?

Answer 16

Movement of Cl- and Na+ makes the lumen hypotonic in SMALL GRADIENTS –> this causes the drive for the movement of water into the epithelial cells.

Question 17

Proximal Tubule – Evidence for near iso-osmotic fluid reabsorption - use of micropuncture techniques?

Answer 17

Pipettes put into capillaries with perfused lumen of the tubule and the capillaries with a solution containing 154mM NaCl.
The rate of water rebsorption was measured

At a low flow in the proximal tubule there is less h20 absorption seen than at high flow - OSMOTIC EQUILIBRIUM IS REACHED MORE QUICKLY AT A SLOWER FLOW OF WATER

Question 18

Overall conclusion for transport by the proximal tubule

Answer 18

Transport by the proximal tubule generates a favourable osmotic gradient for water reabsorption. Because of the extremely high water permeability of the tubule this gradient is small, and is probably due to the generation of a hypotonic lumen.

Question 19

AQP1 mouse knockouts - observations

Answer 19

Null gradient - a more hypotonic lumen generated even though the Na+/Cl- transport still continues!!! Very very dilute lumen as water cannot follow solutes

Question 20

Salivary gland - what drives water transport? Mechanism? What occurs when AQP5 transport is lost?

Answer 20

Isotonic fluid secretion occurs –> Na+ and Cl- is secreted into the lumen and water follows to produce saliva!

Loss of AQP5 prevents the production of lumen hypertonicity. The result is the production of a small volume of saliva with a high salt content.