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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
98
99
8886
80
90
99
97
81
94
99
99
83
89
99
96
96
96
98
89
98
84
95
98
6183
99
99
99
98
64
99
94
66
85
71
99
99
96
6698
99
9863
76
99
99
95
82 98
70
84
97
97
8299
99
86
92
99
90
71 68
93
72
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
98
99
8886
80
90
99
97
81
94
99
99
83
89
99
96
96
96
98
89
98
84
95
98
6183
99
99
99
98
64
99
94
66
85
71
99
99
96
6698
99
9863
76
99
99
95
82 98
70
84
97
97
8299
99
86
92
99
90
71 68
93
72
Aquaporins in fish
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
98
99
8886
80
90
99
97
81
94
99
99
83
89
99
96
96
96
98
89
98
84
95
98
6183
99
99
99
98
64
99
94
66
85
71
99
99
96
6698
99
9863
76
99
99
95
82 98
70
84
97
97
8299
99
86
92
99
90
71 68
93
72
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
control ZAQP8 Hg BME
Pf [um/s]
Dr AQP9b
0
100
200
300
400
500
600
700
control ZAQP9 Hg BME
Pf [um/s]
Dr AQP10a
0
100
200
300
400
500
control ZAQP10 Hg BME
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
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