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

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

Water – natural environment of fish

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 de Groot and Grubmüller, 2005

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

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

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

Location of AQPs on Zebra Fish chromosomes Zebra Fish kariotype

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

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

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+.

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?? DrAQP4 MTSCGALDTFRRCVSSCSCNNSIMAAFKGVWTQEFWRAVSGEFLAMIIFVLLSLGSTINW 60 ratAQP4 MSDGAAARRWGKCGPPCSRE-SIMVAFKGVWTQAFWKAVTAEFLAMLIFVLLSVGSTINW 59 *:. .* : :* ..** : ***.******** **:**:.*****:******:****** 1 ng of cRNA injected * Neely et al., 1999 * p=0,0062

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 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 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