Lecture 11: Aquaporins Flashcards
When were AQPs discovered?
1992
What accounts for 5 litres of water absorption a day?
Na/glucose transporters as each sugar molecule is directly coupled to 260 water molecules
What direction do AQPs transport water in? What rate?
Bidirectional, 3 billion mol/sec
How many isoforms do mammals have and what are the two classes?
13 and AQPs and glyceroaquaporins
What is different about AQP 6 and 8?
6 lets in anions, 8 lets in urea.
What do E.coli have?
One AQP and one glyceroAQP
Explain how frog oocytes were modified with AQPs.
They don’t have AQP but were injected with AQP1 cRNA and transferred to hypotonic solution (no salt) and the frog oocytes swelled and exploded after 3 mins.
What size are monomers, how many TM alpha helices do they have (name them), how many half helices (which ones) and how many connecting loops?
28-30kDa, 6 (M1,2,4-7,8), 2 (M3,7) 5.
How was the tetrameric structure identified?
Freeze fracture
Where re the N and C domains?
Cytoplasm
Does it undergo conformational change? What does this mean?
No, transports water orders of magnitude faster than glucose or ions
What is key to the water selectivity?
M3 and M7
What is at the centre of each monomer?
A 2 nm long water selective channel or pore with a 0.28 nm diameter only slightly larger than water molecule
How does water move through?
One at a time
How is selectivity established in hAQP1?
conserved hydrophilic residue side chains turned towards the inside of the channel
What does the hydrophilic surface consist of?
alpha carbonyl groups from the polypeptide backbone (I191, C189, G188, G190)
What are the NPA motifs and how many are there?
Asn, Pro, Ala. are in the half helices and contain inward facing aspargine polar side chains (N76 and N192)
What are the NPA motifs important for?
hydrophilic surface of the amphipathic pore and are important for preventing proton conduction eg H3O+ so H2O specific.
What limits access to other molecules? Where is this and what is the diameter? How does it act?
Arg195 and His180. At the narrowest segment, 8A. Is a size excluder and electrostatic repulsion.
What do NPA motifs do to water?
cause a transient dipole reorientation of an isolated water molecule.
What do the alpha carbonyl groups and NPA motifs act as?
H-bond donors and acceptors that coordinate water transport through the pore.
What does water do as it moves through the channel?
Forms specific H-bonds with channel lining amino acids.
Describe the structure of AQP4
ampiphatic, hydrophobic residues along one side and carbonyls from 8 residues providing H-bonds for water. Passage of glycerol is prevented by the constriction of the channel diameter to 1.5A at His201 in the selectivity filter. Arg216 is H-bonded to the sterically excluded glycerol as well as 2 waters.
What is there no evidence for in AQPs?
Cooperation between monomers
Why are there 11 mammalian AQPs?
diverse requirements in different cells and tissues
Where is AQP10 found exclusively?
On intracellular vesicles.
What could AQPs also transport?
CO2, NH3, NO, sugars and ions.
What does a lack of inhibitors mean?
Info on AQPs has been confirmed by genetics and knock out mice
Which AQPs exist in the lung?
1,3,4
What are nephrons.
Functional units found in the kidney, about 1 million in each.
What do AQP facilitate water transport in response to?
Osmotic gradients created by the transport of ions and solutes.
What happens to AQP1 deficient mice?
Can’t concentrate urine due to lack of water reabsorption
What is inhibiting AQP1 predicted to do?
Cause water diuresis
Where is AQP1 normally located?
PM
Where is AQP2 found and what happens?
on intracellular vesicles and move to the apical PM of the endothelial cell triggered by vasopresin hormone
What is the AQP2 relocalisation similar to?
Glucose transporters which have to be localised to the PM.
Which AQPs are expressed in the kidney collecting duct ECs?
2,3,4
Which AQP are constitutively active on the basolateral PM?
3,4
What is NDI and what does it cause?
Nephrogenic diabetes insipidus and is hereditory which causes excretion of large volumes of dilute urine and cognition deficits. Causes autosomal dominant mutations in AQP2 causing there to be a mutant monomer which isn’t transported to the PM. Autosomal recessive leads to misfolding of mutant AQP2.
How rare are incidences of loss of function mutations in AQP2 and vasopresin receptors?
1/20 and 1/2 million
What is usually the therapy for NDI?
Fluid replacement
What is the misfolding of AQP2 similar to?
deltaF508 mutation in cystic fibrosis.
How could AQP inhibitors be used in oncology?
Could target infiltrating tumour cells and remodel a tumour to have distinct borders for surgical resection.
What are AQPs also involved with?
Tumour invasion, metastasis, growth and angiogenesis
Where is AQP1 highly expressed?
tumour-associated microvascular ECs and several different tumour cell types.
Where does AQP4 expression correlate with tumour severity?
Astrocytomas
How could targeting AQP1 help target cancer? What is the problem with this?
Mice lacking AQP1 have reduced growth of implanted and spontaneously generated tumours as a consequence of defective angiogenesis. However AQP1 is expressed in many tissues so would affect them all.
Explain how AQPs and malaria are connected.
Plasmodium berghei uses the host plasma glycerol for lipid biosynthesis and membrane biogenesis during asexual intraerythrocytic deveopment. This parasite’s AQPs expressed in frog oocytes were permeable to glycerol and water so is a glyceroaquaporin. Parasites lacking PbAQP show reduced proliferation and infected mice survive longer.
