Zebra Fish Aquaporins

Zebra Fish Aquaporins

Magdalena CalusinskaAngèle Tingaud – Sequeira

David OteroJoan 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

Solea a (DQ889223) Gasterosteus a (DV005019) Sparus a (AAV34610) Centropristis (AAV34607) Oryzias (BJ489731) Dicentrarchus (AAV34611) Takifugu a (NEWSINFRUP000001302258) Tetraodon a (CAF98423) Oncorhynchus (CA378544) Anguilla anguilla a (CAD92027) Anguilla japonica (BAC82110) Danio a (NP_996942) Pimephales (DT351671) Danio b (XP_687705) Sparus b (AAV34609) Takifugu b (Scaffold_68) Tetraodon b (Scaffold SCAF14556) Anguilla anguilla b (EF011738) Ictalurus punctatus (CK418363) Solea b (AAV34612) Gasterosteus b (BROADS1:groupIII) Homo (NP_932766) Homo AQP2 (CAG46821) Homo AQP6 (NP_001643) Homo AQP5 (CAG46819) Homo (NP_036196) Tetraodon a (CAG04065) Takifugu a (NEWSINFRUP00000127844) Danio a (AAY21805) Danio b (AAH77129) Fundulus (AAF04146) Tetraodon b (CAG07459) Takifugu b (NEWSINFRUP00000161827) Eptatretus (BAE93686) Homo (NP_001641) Danio (AAH78213) Gasterosteus (DW593588) Tetraodon (CAG07606) Takifugu (NEWSINFRUP00000162705) AqpZ (NP_415396) GlpF (NP_756733) Tetraodon a (CAF99586) Takifugu a (NEWSINFRUP00000142951) Danio a (AAI00052) Danio b (XM_689233) Tetraodon b (CAG05555) Takifugu b (NEWSINFRUP00000129418) Homo (CAG46824) Homo AQP7 (NP_001161) Takifugu (NEWSINFRUP00000154197) Gasterosteus (DW597988) Oreochromis (BAD20708) Tribolodon (BAB83082) Danio b (Q803U6) Anguilla anguilla (CAC85286) Danio a (CAE50608) Pimephales (DT136147) Homo (CAG46822) Ictalurus furcatus (CK402696) Danio a (NP_001002349) Tetraodon a (CAG11881) Tetraodon c (GSCT00023754001) Takifugu c (NEWSINFRUP00000151009) Danio c (AAH56802) Homo (CAH70483) Anguilla anguilla (CAH04573) Danio b (FGENESH00000052491) Tetraodon b (CAG10846) Takifugu b (NEWSINFRUP00000160942) Sparus (AAR13054) Dicentrarchus (DQ889224) Danio c (AAH81511) Ictalurus furcatus (CK402738) Oncorhynchus b (BX881112) Salmo (DW573347) Homo (NP_001160) Tetraodon (CAG11438) Takifugu (GENSCANSLICE00000006719) Sparus (DQ889225) Danio a (XP_683241) Oryzias (BAC20300) Oncorhynchus a (CU071568) Danio b (XP_691585) Pimephales (DT342244) Tetraodon (CAG10931) Ictalurus furcatus (CK408292) Danio (AAH95564) Homo (NP_945349) Homo (AAH40443) Danio (AAH95775) Takifugu (GENSCANSLICE00000028964) Tetraodon (GIDT00006243001)99

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9863

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6183

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6698

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9863

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Aquaporins in fish


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8886

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6183

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Human aquaporin family gene clusterOrganization map of an aquaporin cluster in human

Distance between AQP0 and the AQP2, 5 and 6 gene cluster is approximately 500kb

Zebra Fish chromosome 23. No sequences corresponding to AQP2, 5 and 6 could be mapped

Dr AQP0a, Dr AQP0b

AQP2, 5 and 6 are absent from

the genome of Danio rerio

Aquaporins in Zebra Fish

DrAQP0b

DrAQP0a

DrAQP4 DrAQP1aDrAQP1b

AQPZ

DrAQP8c

DrAQP8b

DrAQP8a

GlpF

DrAQP3b

DrAQP3aDrAQP10b

DrAQP10c

DrAQP10a

DrAQP9b

DrAQP9a

DrAQP11

DrAQP12

0.2

Aquaglyceroporins

Aquaporins

Neighbour – joining tree based on theamino acid, full coding sequences of

Zebra Fish AQPs

Aquaporins and Aquaglyceroporins

Aquaglyceroporins contain two Additional peptide spans located inLoop C and E respectively

Location of AQPs on Zebra Fish chromosomes

Zebra Fish kariotype

Permeability properties – Xenopus laevis oocyte swelling assay

Xenopus laevis

Swelling measurement:• 200mOsm → 20mOsm• 12 pictures every 2s• calculation of Pf

Injectio

n o

f cR

NA

Surgical recovery of stage V and VI oocytes

Water permeability – Pf / Hg2+ sensitivity

Dr AQP1a

0

200

400

600

800

1000

1200

control ZAQP1 Hg BME

Pf [um/s]

Dr AQP8c

0

100

200

300

400

500

600

700


Pf [um/s]

Dr AQP9b

0

100

200

300

400

500

600

700


Pf [um/s]

Dr AQP10a

0

100

200

300

400

500


Pf [um/s]

Conditions used: 1 ng of cRNA injetced 0,5 mM Hg2+

5mM BME

?

Dr AQP3b isoform

ZAQP3 10ng/ 70mOSM, 120s

0

50

100

150

200

250

300

cont

rolP

f

Hg 0,

1mM

Hg 0,

3mM

Hg 0,

5mM

ZAQ

P3 Pf

Hg 0,

1mM

Hg 0,

3mM

Hg 0,

5mM

Pf

[um

/s]

NO CYSTEINE PRESENT BEFORE THE SECOND NPA MOTIF Dr AQP3b NPAVTFALCLLGREKWRKFPVYFLFQTLGSFLGAAIIFAEYHDAIYDYAGESNELLVLGEKETAGIFATYPSKYLTPLNGFFDQVIGTASLIVCILAIVDPYNNPIPQGLEAFTVGFSVLIIGLSMGFNSGYAVNPA

Dr AQP3a doesn’t show sensitivity to Hg ions. Contrary, the water transport is

stimulated after incubation with 0,5 mM Hg2+.

Similarity to mammaliam AQP6??

Dr AQP3b – a possible ion channel?

AQP6M.musc ---MEPGLCSRAYLLVGGLWTAISKALFAEFLATGLYVFFGVGSVLPWPVALPS---VLQ 54 AQP6R.norv ---MEPGLCNRAYLLVGGLWTAISKALFAEFLATGLYVFFGVGSVLPWPVALPS---VLQ 54 AQP6H.sap MDAVEPGGRGWASMLACRLWKAISRALFAEFLATGLYVFFGVGSVMRWPTALPS---VLQ 57 Dr AQP3b --MGWQKSVLDKLAQTFQIRNKLLRQGLAECLGTLILVMFGCGSLAQLKLSEGSHGLFLT 58 . : . : : :** *.* : *:** **: : * .* AQP6M.musc IAITFNLATATAVQISWKTSGAHANPAVTLAYLVGSHISLPRAMAYIAAQLAGATAGAAL 114 AQP6R.norv VAITFNLATATAVQISWKTSGAHANPAVTLAYLVGSHISLPRAVAYIAAQLAGATVGAAL 114 AQP6H.sap IAITFNLVTAMAVQVTWKASGAHANPAVTLAFLVGSHISLPRAVAYVAAQLVGATVGAAL 117 Dr AQP3b ANLAFGFGATLGILVCGQVSGGHLNPAVTFALCLLGREKWRKFPVYFLFQTLGSFLGAAI 118 ::*.: :: .: : :.**.* *****:* : .: . : .*. * *: ***: AQP6M.musc LYGVTPGGIRETLGVN---VVHNSTSTG--------------QAVAVELVLTLQLVLCVF 157 AQP6R.norv LYGVTPGGVRETLGVN---VVHNSTSTG--------------QAVAVELVLTLQLVLCVF 157 AQP6H.sap LYGVMPGDIRETLGIN---VVRNSVSTG--------------QAVAVELLLTLQLVLCVF 160 Dr AQP3b IFAEYHDAIYDYAGESNELLVLGEKETAGIFATYPSKYLTPLNGFFDQVIGTASLIVCIL 178 ::. . : : * . :* .. .*. :.. ::: * .*::*:: AQP6M.musc ASMDGRQ---TLASPAAMIGTSVALGHLIGIYFTGCSMNPARSFGPAVIVGKFAVHWIFW 214 AQP6R.norv ASMDSRQ---TLGSPAAMIGTSVALGHLIGIYFTGCSMNPARSFGPAVIVGKFAVHWIFW 214 AQP6H.sap ASTDSRQ---TSGSPATMIGISVALGHLIGIHFTGCSMNPARSFGPAIIIGKFTVHWVFW 217 Dr AQP3b AIVDPYNNPIPQGLEAFTVGFSVLIIGLSMGFNSGYAVNPARDFGPRLFT--AMAGWGSE 236 * * : . . * :* ** : * . :* ::****.*** :: . * AQP6M.musc VGPLTGAVLASLIYNFILFPDTKTVAQRLAILVGTTKVEK--VVDLEPQKKESQTNSEDT 272 AQP6R.norv VGPLTGAVLASLIYNFILFPDTKTVAQRLAILVGTTKVEK--VVDLEPQKKESQTNSEDT 272 AQP6H.sap VGPLMGALLASLIYNFVLFPDTKTLAQRLAILTGTVEVGTGAGAGAEPLKKESQPGSGAV 277 Dr AQP3b VFTARDYWFLVPIFAPFIGAVIGVIVYQLMVGWHVEGEAR----DKKAKAREEVMNLNDV 292 * . . : *: .: . .:. :* : . . :. :*. . . AQP6M.musc EVSV- 276 AQP6R.norv EVSV- 276 AQP6H.sap EMESV 282 Dr AQP3b ASKE- 296 .

Alignment of mammalian AQP6 and Dr AQP3b

Unique residues implicatedin AQP6 ion conductance

Structural model of highlighting the crossing point between

TM2 and TM5

Liu et al., 2005

Heterotetrameric composition of Dr AQP4??

Dr AQP4 Hg sensitivity

0100200300400500600700800

contro

lM

1

M1/

HgM

24

M24

/Hg

M1+

M24

M1+

M24/Hg

Pf

[um

/s]

Neely et al., 1999

DrAQP4 MTSCGALDTFRRCVSSCSCNNSIMAAFKGVWTQEFWRAVSGEFLAMIIFVLLSLGSTINW 60ratAQP4 MSDGAAARRWGKCGPPCSRE-SIMVAFKGVWTQAFWKAVTAEFLAMLIFVLLSVGSTINW 59 *:. .* : :* ..** : ***.******** **:**:.*****:******:******

1 ng of cRNA injected

Dr AQP4 Pf values

0

100

200

300

400

500

600

700

800

control M1 M24 M1+M24

Pf

[um

/s]

*

* p=0,0062

Glycerol permeability - Pgly

Dr AQP1a used as a negative control

Glycerol permeability

0

20

40

60

80

100

120

Contro

lHg

BME

Dr AQP1a

Hg

BME

Dr AQP3b Hg

BME

Dr AQP10

a Hg

BME

Dr AQP9b Hg

BME

Pg

ly [

um

/s]

?

Aquaglyceroporins used in this study:Dr AQP1a – 1ng cRNA injectedDr AQP3b – 10ng cRNA injectedDr AQP9b – 1ng cRNA injectedDr 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

Documents

Zebra Fish Aquaporins