1. Magdalena Calusinska Angèle Tingaud – Sequeira David Otero Joan Cerdà
Zebra Fish Aquaporins
Magdalena Calusinska
Angèle Tingaud – Sequeira
David Otero
Joan Cerdà

2. 1991 – discovery of water channels
2003 – Nobel Prize in Chemistry for Peter Agre for the discovery of aquaporins

3. Water – natural environment of fish

4. 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

5. Water and glycerol transport
de Groot and Grubmüller, 2005

6. 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

7. Aquaporins in fish

8. 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

9. Aquaporins in Zebra Fish
Aquaglyceroporins
Aquaporins
Neighbour – joining tree based on the
amino acid, full coding sequences of
Zebra Fish AQPs

10. Aquaporins and Aquaglyceroporins
Aquaglyceroporins contain two
Additional peptide spans located in
Loop C and E respectively

11. Location of AQPs on Zebra Fish chromosomes
Zebra Fish kariotype

12. Permeability properties – Xenopus laevis oocyte 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

13. Water permeability – Pf / Hg2+ sensitivity?
Conditions used: 1 ng of cRNA injetced
0,5 mM Hg2+
5mM BME

14. Similarity to mammaliam AQP6??
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+.

15. 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

16. Heterotetrameric composition of Dr AQP4??
DrAQP MTSCGALDTFRRCVSSCSCNNSIMAAFKGVWTQEFWRAVSGEFLAMIIFVLLSLGSTINW 60
ratAQP MSDGAAARRWGKCGPPCSRE-SIMVAFKGVWTQAFWKAVTAEFLAMLIFVLLSVGSTINW 59
*:. .* : :* ..** : ***.******** **:**:.*****:******:******
1 ng of cRNA injected *
Neely et al., 1999
* p=0,0062

17. 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

18. 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

19. 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