Zebra fish aquaporins
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Zebra Fish Aquaporins. Magdalena Calusinska Ang è le Tingaud – Sequeira David Otero Joan Cerd à. 1991 – discovery of water channels. 2003 – Nobel Prize in Chemistry for Peter Agre for the discovery of aquaporins. Water – natural environment of fish.

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Zebra Fish Aquaporins

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Zebra fish aquaporins

Zebra Fish Aquaporins

Magdalena Calusinska

Angèle Tingaud – Sequeira

David Otero

Joan Cerdà


1991 discovery of water channels

1991 – discovery of water channels

2003 – Nobel Prize in Chemistry for Peter Agre for the discovery of aquaporins


Water natural environment of fish

Water – natural environment of fish


Major intrinsic proteins mips aquaporins

Major intrinsic proteins (MIPs) – Aquaporins

  • homotetramers consisting of four monomeric channels

  • composed of single polypeptide chain ~ 270aa

  • six transmembrane α helices, with three extracellular loops (A,C,E) and two intracellular (B,D)

  • N and C terminal ends reside in the cytoplasm

  • highly conserved NPA motifs are present in loop B and E

de Groot and Grubmüller, 2005


Water and glycerol transport

Water and glycerol transport

de Groot and Grubmüller, 2005


Aquaporins in metazoa

Aquaporins in Metazoa

  • 13 paralogs have been identified so far in mammals

  • they are present in many different cells and tissues

  • they have a unique cellular and subcellular distribution with little overlap between homologs

  • in mammals amino acid sequences of paralogs show 20 – 60% identity

  • they differ mainly at the intracellular N – and C – termini (<20%)

Heymann and Engel, 1999


Aquaporins in fish

Aquaporins in fish


Human aquaporin family gene cluster

Human aquaporin family gene cluster

Organization map of an aquaporin cluster in human

AQP2, 5 and 6 are absent from

the genome of Danio rerio

Distance between AQP0 and the AQP2, 5 and 6

gene cluster is approximately 500kb

Dr AQP0a, Dr AQP0b

Zebra Fish chromosome 23.

No sequences corresponding to

AQP2, 5 and 6 could be mapped


Aquaporins in zebra fish

Aquaporins in Zebra Fish

Aquaglyceroporins

Aquaporins

Neighbour – joining tree based on the

amino acid, full coding sequences of

Zebra Fish AQPs


Aquaporins and aquaglyceroporins

Aquaporins and Aquaglyceroporins

Aquaglyceroporins contain two

Additional peptide spans located in

Loop C and E respectively


Location of aqps on zebra fish chromosomes

Location of AQPs on Zebra Fish chromosomes

Zebra Fish kariotype


Permeability properties xenopus laevis oocyte swelling assay

Permeability properties – Xenopus laevisoocyte swelling assay

Injection of cRNA

Surgical recovery of

stage V and VI oocytes

Xenopus laevis

  • Swelling measurement:

  • 200mOsm → 20mOsm

  • 12 pictures every 2s

  • calculation of Pf


Water permeability p f hg 2 sensitivity

Water permeability – Pf / Hg2+ sensitivity

?

Conditions used: 1 ng of cRNA injetced

0,5 mM Hg2+

5mM BME


Dr aqp3b isoform

Dr AQP3b isoform

Similarity to mammaliam AQP6??

Dr AQP3a doesn’t show sensitivity

to Hg ions. Contrary, the water transport is stimulated after incubation with 0,5 mM Hg2+.


Dr aqp3b a possible ion channel

Dr AQP3b – a possible ion channel?

Unique residues implicated

in AQP6 ion conductance

Alignment of mammalian

AQP6 and Dr AQP3b

Structural model of highlighting

the crossing point between

TM2 and TM5

Liu et al., 2005


Heterotetrameric composition of dr aqp4

Heterotetrameric composition of Dr AQP4??

DrAQP4MTSCGALDTFRRCVSSCSCNNSIMAAFKGVWTQEFWRAVSGEFLAMIIFVLLSLGSTINW 60

ratAQP4MSDGAAARRWGKCGPPCSRE-SIMVAFKGVWTQAFWKAVTAEFLAMLIFVLLSVGSTINW 59

*:. .* : :* ..** : ***.******** **:**:.*****:******:******

1 ng of cRNA injected

*

Neely et al., 1999

* p=0,0062


Glycerol permeability p gly

Glycerol permeability - Pgly

?

Dr AQP1a used

as a negative control

Aquaglyceroporins used in this study:

Dr AQP1a – 1ng cRNA injected

Dr AQP3b – 10ng cRNA injected

Dr AQP9b – 1ng cRNA injected

Dr AQP10a – 1ng cRNA injected


Conclusions

Conclusions

  • 9 orthologs of AQPs out of 13 existing in mammals are present in Zebra Fish

  • Dr AQPs 0,1,9 represent two isoforms and Dr AQPs 8 and 10 three

  • No homologies to mammalian AQPs 2,5 and 6 have been found in fish


Future studies on zebra fish aqps

Future studies on Zebra Fish AQPs

  • Expression pattern of different AQPs isoforms in Zebra Fish tissues

  • Comparison of efficiency of water transport between different Dr AQPs – tagged proteins

  • ISH of AQPs in embryos

  • Permeability of selected AQPs to CPAs


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