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Zebra Fish Aquaporins

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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  1. 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 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

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

  14. 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?? DrAQP4MTSCGALDTFRRCVSSCSCNNSIMAAFKGVWTQEFWRAVSGEFLAMIIFVLLSLGSTINW 60 ratAQP4MSDGAAARRWGKCGPPCSRE-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

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