What do square arrays and water
channels do in the brain?
Torgeir Holen, MNBTS9000
October 22nd, 2009
Square arrays in brain - studied since 1968 - function unknown
Principle of freeze-fracture EM
Endothelium
Freeze-fracture microscopy
Astrocyte endfoot
Square arrays - studied since 1968 - function unknown
Jared Diamond, "Collapse", 2005
John Stephens & Frederick Catherwood, 1839
Alfred Maudsley, Guatemala, 1880ies
Square arrays situated around blood vessels
in brain
Frigeri et al, 1995; Nielsen et al, 1997
Sørbø, Moe &
Holen, 2007
Square arrays are very dense
Furman...Rash, PNAS, 2003
500 - 600 sq. arrays / um2 - ca 50 % of membrane surface - Wolburg, 1995
Water channels – short intro
Hiroaki et al, J Mol Cell, 2006
Aquaporin 4 – the main brain water channel
Linking Square Arrays and Aquaporin-4
• Transfecting AQP4 into
CHO cells give pseudosqare arrays (Yang…
Verkman, 1996)
• Square arrays disappear
in AQP4-KO (Verbavatz...
Verkman, 1997)
• transfecting two Aqp4
isoforms into CHO cells
skew distribution of
square array size
(Furman...Rash, PNAS,
2003)
• but not to wild type
distribution
M1 and
AQP4
in M23 Transfected CHO
cells, Furman...Rash, 2003
CHO
cells
Aquaporins in the kidneys are well understood…
… but why this enormous
water transport capacity in
the brain? We don’t know
King, Kozuno & Agre, Nature Reviews, 2004
Water intoxication and brain swelling
in AQP4-KO mouse
Manley et al, Nat Medicine, 2000
• Peritonial water injection in mice…
• water intoxication in humans: diets, drinking contests,
college fraternity pledges and marathon runners…
Total length of capillaries in brain
• 640 km (total surface 93 m2), Hendrikse, 2007;
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400 miles (total surface 20 m2), Kratzer, 2007
If we use the lower value of 20 m2, there are 20 x
106 x 106 x 550 square arrays = 1.1 x 1016 …
In other words, if square arrays transport water,
they could empty the whole brain in less than a
second…
Part II: Our contributions to the
field
• Holen et al, NAR, 2005
• Sorbo, Moe & Holen, 2007
• Moe, Sorbo, Sogaard, Zeuthen, Ottersen &
•
•
Holen, 2008
Sorbo, Moe, Ottersen & Holen, 2008
Solbu, Moe, Strand, Vaadal & Holen, in revision
The
classical
AQP4
gene
model
New isoforms of Aquaporin-4
Moe et al, Genomics,
2008
Expression studies of AQP4 cDNA
isoforms in astrocytes: AQP4e
Water transport assay of new
isoforms in Xenopus ovocytes
Moe et al, Genomics, 2008
Regulation on mRNA level
• Isoform ratio ~30-fold discrepancy on the RNA
level vs protein level...
• ...this points to post-transcriptional regulation
of AQP4, at translational level or at the protein
stability level
Expression studies of AQP4 cDNA
isoforms in HeLa cells: AQP4d
AQP4d is colocalized with cis-Golgi marker GM130
Moe et al, Genomics, 2008
The new AQP4e is the largest
aquaporin isoform and has a novel
N-terminal domain
Biochemical analysis of square arrays
- since 1968 only studied by freeze-fracture electron
microscopy (FF-EM)
Furman...Rash, PNAS, 2003
Principle of blue-native gels
• Membrane proteins must be
stabilized and charged for
electrophoresis
Aquaporin
tetramer
Mild non-ionic detergent
Coomassie G-250
Analysis of square arrays using BlueNative PAGE
brain AQP4
vs
transfected
forms
Sorbo et al, Biochemistry, 2008
Aquaporin-1 and Aquaporin-9 do not
form square arrays in native gels
Sorbo et al, Biochemistry, 2008
Square arrays are present also in nasal
epithelium of rat and brain of mice
Sorbo et al, Biochemistry, 2008
Square arrays are rather stable under
biochemical purification
Sorbo et al, Biochemistry, 2008
Reminder: AQP4 transfection into celllines give pseudo-square arrays
M23
M1 and M23 Transfected CHO
cells, Furman...Rash, 2003
2D gels on BN-PAGE gels to analyze the
molecular composition of square arrays
Sorbo et al, Biochemistry, 2008
Biochemical isolation of AQP4 and mass
spectrometry identification of Mz
Sorbo, Moe & Holen, unpublished
The roles of AQP4 isoforms and
amino acids in square array
assembly
• A mutational analysis:
Strand, Moe, Solbu,
Vaadal & Holen,
Biochemistry, 2009
N-terminal blockage of aggregation
acts through C13 and C17
Replicated in astrocyte cell line CRL-2006
HeLa
cells
Solbu, Moe,
Strand, Vaadal,
Holen, unpublished
Isoform AQP4a interact with AQP4c,
and is imported into square arrays
Strand, Moe, Solbu,
Vaadal & Holen,
Biochemistry, 2009
The novel isoform AQP4e is also
incorporated into square arrays
Strand, Moe, Solbu,
Vaadal & Holen,
Biochemistry, 2009
Gradient ratio AQP4a-myc +
AQP4c
Summary
• square arrays are visible by FF-EM in high
•
•
•
•
•
•
concentrations around blood vessels in the brain
function unknown
AQP4 isoforms affect square array formation
we have isolated 4 new AQP4 isoforms (total 6)
we have demonstrated that square arrays can be
biochemically analyzed using BN-PAGE…
... and show square arrays contain Mz, a third
obligatory component
recently, we have analyzed the roles of AQP4
isoforms and amino acids in square array assembly
Acknowledgements
• Svein Erik Moe and Jan Gunnar Sørbø
• Line Strand (Forskerlinjen), Tom T. Solbu and
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•
•
•
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•
Marianne Vaadal (master student Aas)
FUGE - Norges Forskningsråd
CMBN & Ole Petter Ottersen
Rikke Sogaard & Thomas Zeuthen, Copenhagen
The Norwegian Cancer Society
You
..........the Heroes!
...................Thank you for your attention!