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
Heymann and Engel, 1999

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%)
84
97
99
95
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)
99
89
96
Aquaporins in fish
99
72
93
68
71
90
99
92
86
99
82
99
97
70
84
97
99
95
99
76
63
98
Solea a (DQ889223)
Gasterosteus a (DV005019)
Sparus a (AAV34610)
82
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)
96
Sparus b (AAV34609)
Takifugu b (Scaffold_68)
Tetraodon b (Scaffold SCAF14556)
Anguilla anguilla b (EF011738)
Ictalurus punctatus (CK418363)
99
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)
76
63
98
98
99
98
66
99
66
85
71
99
95
96
96
98
98
99
89
96
84
99
99
98
84
99
99
98
94
64
83
61
98
83
89
99
99
97
94
99
81
90
98
88
86
99
80
99
99
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)
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
DrAQP10c
DrAQP10b
DrAQP3a
Aquaglyceroporins
DrAQP10a
DrAQP3b
DrAQP9b
DrAQP9a
GlpF
DrAQP11
DrAQP8a
DrAQP12
DrAQP8b
DrAQP8c
DrAQP0b
DrAQP0a
AQPZ
DrAQP1b
DrAQP1a
0.2
DrAQP4
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
Xenopus laevis
Swelling measurement:
• 200mOsm → 20mOsm
• 12 pictures every 2s
• calculation of Pf
Injection of cRNA
Surgical recovery of
stage V and VI oocytes
Water permeability – Pf /
Hg2+ sensitivity
Dr AQP1a
Dr AQP8c
1200
700
1000
600
500
800
Pf [um/s] 600
Pf [um/s]
400
300
400
200
200
100
0
0
control
ZAQP1
Hg
BME
control
ZAQP8
Hg
BME
Dr AQP10a
Dr AQP9b
500
700
600
400
500
Pf [um/s]
300
400
?
Pf [um/s]
300
200
200
100
100
0
0
control
ZAQP9
Hg
BME
Conditions used: 1 ng of cRNA injetced
0,5 mM Hg2+
5mM BME
control
ZAQP10
Hg
BME
Dr AQP3b isoform
NO CYSTEINE PRESENT BEFORE THE SECOND NPA MOTIF
Dr AQP3b
NPAVTFALCLLGREKWRKFPVYFLFQTLGSFLGAAIIFAEYHDAIYDYAGESNELLVL
GEKETAGIFATYPSKYLTPLNGFFDQVIGTASLIVCILAIVDPYNNPIPQGLEAFTVGFS
VLIIGLSMGFNSGYAVNPA
ZAQP3 10ng/ 70mOSM, 120s
300
M
0,
5m
M
H
g
0,
3m
M
H
g
0,
1m
ZA
Q
P3
M
0,
5m
M
H
g
0,
3m
g
H
H
g
nt
ro
0,
1m
M
lP
f
0
g
50
H
150
100
Pf
Similarity to mammaliam
AQP6??
200
co
Pf [um/s]
250
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
AQP6M.musc
AQP6R.norv
AQP6H.sap
Dr AQP3b
---MEPGLCSRAYLLVGGLWTAISKALFAEFLATGLYVFFGVGSVLPWPVALPS---VLQ
---MEPGLCNRAYLLVGGLWTAISKALFAEFLATGLYVFFGVGSVLPWPVALPS---VLQ
MDAVEPGGRGWASMLACRLWKAISRALFAEFLATGLYVFFGVGSVMRWPTALPS---VLQ
--MGWQKSVLDKLAQTFQIRNKLLRQGLAECLGTLILVMFGCGSLAQLKLSEGSHGLFLT
. : . : : :** *.* : *:** **:
: *
.*
54
54
57
58
AQP6M.musc
AQP6R.norv
AQP6H.sap
Dr AQP3b
IAITFNLATATAVQISWKTSGAHANPAVTLAYLVGSHISLPRAMAYIAAQLAGATAGAAL
VAITFNLATATAVQISWKTSGAHANPAVTLAYLVGSHISLPRAVAYIAAQLAGATVGAAL
IAITFNLVTAMAVQVTWKASGAHANPAVTLAFLVGSHISLPRAVAYVAAQLVGATVGAAL
ANLAFGFGATLGILVCGQVSGGHLNPAVTFALCLLGREKWRKFPVYFLFQTLGSFLGAAI
::*.: :: .: : :.**.* *****:* : .: . : .*. * *: ***:
114
114
117
118
AQP6M.musc
AQP6R.norv
AQP6H.sap
Dr AQP3b
LYGVTPGGIRETLGVN---VVHNSTSTG--------------QAVAVELVLTLQLVLCVF
LYGVTPGGVRETLGVN---VVHNSTSTG--------------QAVAVELVLTLQLVLCVF
LYGVMPGDIRETLGIN---VVRNSVSTG--------------QAVAVELLLTLQLVLCVF
IFAEYHDAIYDYAGESNELLVLGEKETAGIFATYPSKYLTPLNGFFDQVIGTASLIVCIL
::.
. : : * .
:* .. .*.
:.. ::: * .*::*::
157
157
160
178
AQP6M.musc
AQP6R.norv
AQP6H.sap
Dr AQP3b
ASMDGRQ---TLASPAAMIGTSVALGHLIGIYFTGCSMNPARSFGPAVIVGKFAVHWIFW
ASMDSRQ---TLGSPAAMIGTSVALGHLIGIYFTGCSMNPARSFGPAVIVGKFAVHWIFW
ASTDSRQ---TSGSPATMIGISVALGHLIGIHFTGCSMNPARSFGPAIIIGKFTVHWVFW
AIVDPYNNPIPQGLEAFTVGFSVLIIGLSMGFNSGYAVNPARDFGPRLFT--AMAGWGSE
* * :
. . * :* ** : *
. :* ::****.*** ::
. *
214
214
217
236
AQP6M.musc
AQP6R.norv
AQP6H.sap
Dr AQP3b
VGPLTGAVLASLIYNFILFPDTKTVAQRLAILVGTTKVEK--VVDLEPQKKESQTNSEDT
VGPLTGAVLASLIYNFILFPDTKTVAQRLAILVGTTKVEK--VVDLEPQKKESQTNSEDT
VGPLMGALLASLIYNFVLFPDTKTLAQRLAILTGTVEVGTGAGAGAEPLKKESQPGSGAV
VFTARDYWFLVPIFAPFIGAVIGVIVYQLMVGWHVEGEAR----DKKAKAREEVMNLNDV
* . . :
*: .: .
.:. :* :
.
. :. :*. .
.
272
272
277
292
AQP6M.musc
AQP6R.norv
AQP6H.sap
Dr AQP3b
EVSVEVSVEMESV
ASKE.
Structural model of highlighting
the crossing point between
TM2 and TM5
276
276
282
296
Liu et al., 2005
Heterotetrameric composition of
Dr AQP4??
DrAQP4
ratAQP4
1 ng of cRNA injected
MTSCGALDTFRRCVSSCSCNNSIMAAFKGVWTQEFWRAVSGEFLAMIIFVLLSLGSTINW 60
MSDGAAARRWGKCGPPCSRE-SIMVAFKGVWTQAFWKAVTAEFLAMLIFVLLSVGSTINW 59
*:. .*
: :* ..** : ***.******** **:**:.*****:******:******
Dr AQP4 Pf values
800
700
Pf [um/s]
600
500
400
300
200
100
0
control
M1
M24
M1+M24
800
700
600
500
400
300
200
100
0
*
M
1+
M
24
/H
g
1+
M
24
M
24
/H
g
M
24
M
M
1/
Hg
M
co
n
1
Neely et al., 1999
tr
ol
Pf [um/s]
Dr AQP4 Hg sensitivity
* p=0,0062
Glycerol permeability - Pgly
120
100
80
E
BM
Hg
Q
P9
b
Dr
A
E
BM
Hg
Q
P1
0a
Dr
A
E
BM
Q
P3
b
Dr
A
E
Hg
BM
Q
P1
a
Dr
A
Hg
BM
E
Dr AQP1a used
as a negative control
Hg
?
60
40
20
0
Co
nt
ro
l
Pgly [um/s]
Glycerol permeability
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