doi:10.1016/j.jmb.2005.10.081
J. Mol. Biol. (2006) 355, 628–639
Implications of the Aquaporin-4 Structure on Array
Formation and Cell Adhesion
Yoko Hiroaki1,2†, Kazutoshi Tani1†, Akiko Kamegawa1,2,
Nobuhiko Gyobu3, Kouki Nishikawa1, Hiroshi Suzuki1, Thomas Walz4,
Sei Sasaki5, Kaoru Mitsuoka3, Kazushi Kimura6, Akira Mizoguchi6
and Yoshinori Fujiyoshi1,2,3*
1
Department of Biophysics
Faculty of Science, Kyoto
University, Oiwake,
Kitashirakawa, Sakyo-ku
Kyoto 606-8502, Japan
2
Core Research for Evolution
Science and Technology
(CREST), Japan Science and
Technology Agency (JST)
Oiwake, Kitashirakawa, Sakyo-ku
Kyoto 606-8502, Japan
3
Information Research Center
(BIRC), National Institute of
Advanced Industrial Science and
Technology (AIST), 2-41-6, Aomi
Koto-ku, Tokyo 135-0064, Japan
Aquaporin-4 (AQP4) is the predominant water channel in the mammalian
brain and an important drug target for treatment of cerebral edema, bipolar
disorder and mesial temporal lobe epilepsy. We determined the AQP4
structure by electron crystallography of double-layered, two-dimensional
(2D) crystals. The structure allows us to discuss how the expression ratio
between the long and short AQP4 splicing variant can determine the size of
in vivo orthogonal arrays. Furthermore, AQP4 contains a short 310 helix in
an extracellular loop, which mediates weak but specific interactions
between AQP4 molecules in adjoining membranes. This finding suggests a
previously unexpected role for AQP4 in cell adhesion. This notion was
corroborated by expression of AQP4 in L-cells, which resulted in clustering
of the cells. Our AQP4 structure thus enables us to propose models for the
size regulation of orthogonal arrays and channel-mediated cell adhesion.
q 2005 Elsevier Ltd. All rights reserved.
4
Department of Cell Biology
Harvard Medical School, 240
Longwood Avenue, Boston, MA
02115, USA
5
Department of Nephrology
Graduate School, Tokyo Medical
and Dental University, 1-5-45
Yushima, Bunkyo-ku, Tokyo
113-8519, Japan
6
Department of Anatomy, Basic
Medicine, School of Medicine
Mie University, 2-174, Edobashi
Tsu 514-0001, Japan
*Corresponding author
Keywords: water channel; electron crystallography; cell adhesion; orthogonal array; glial lamellae
Introduction
† Y.H. & K.T. contributed equally to this work.
Abbreviations used: AQP, aquaporin; OG, n-octyl-b,Dglucopyranoside.
E-mail address of the corresponding author:
[email protected]
Members of the aquaporin (AQP) family fall into
two classes, aquaporins that only permit water
passage and aquaglyceroporins that also permit
passage of other small neutral solutes, such as
glycerol.1 Pure water channels, including AQP4, are
extremely permeable for water but prevent passage
0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
Implications of AQP4 Structure
of any other solutes, in particular protons. The first
structure of a water channel, AQP1, which was
determined by electron crystallography,2 revealed
the unusual AQP fold that consists of two tandem
repeats, each with three transmembrane helices
(helices 1–3 and helices 4–6) and a short pore helix
in loops B and E (termed HB and HE). The two
repeats are connected by the long loop C, which
spans the entire extracellular surface of the monomer. The water selectivity of aquaporins requires
the two pore helices HB and HE, which line one side
of the pore, to be in a specific configuration, which
is stabilized by interactions between the two AQP
tandem repeats. The AQP1 structure also showed
that the highly conserved Asn-Pro-Ala (NPA)
motifs together with the electrostatic field formed
by the two pore helices are responsible for the
prevention of proton conduction.2 The structures of
further family members (GlpF,3 AQPZ,4 AQP05,6)
established that all aquaporins share the same basic
AQP fold, although each water channel has specific
functional and physiological characteristics.7
629
Glial cells contain characteristic orthogonal
arrays in the plasma membrane, which are
especially prominent in glial end feet surrounding
vascular capillaries in the brain.8 Immunogold
labeling experiments showed that these arrays
consist of AQP4.8 While AQP4 and AQP1 both
function as very fast water-selective pores, AQP4
has distinctive biological characteristics, as it forms
orthogonal arrays in intact membranes and is not
sensitive to mercurials. Furthermore, AQP4 exists
in glial cells as a full-length protein starting with
Met1 (AQP4M1) and an alternative shorter splicing
isoform that starts with Met23 (AQP4M23)
(Figure 1).9 AQP4 resides in glial cells at the cell
surface opposite from where glutamate transporters are found, and it is expressed in glial lamellae
surrounding vasopressin-secretory neurons. In
addition, AQP4 is found in the sarcolemma of
fast-twitch fibers in skeletal muscle. The specific
localization of AQP4 molecules in glial cells and the
fast-twitch fibers can be attributed to a C-terminal
Ser-Ser-Val sequence, a characteristic binding motif
Figure 1. Comparison of the primary sequence of rAQP4 with those of hAQP1, bAQP1, AeaAQP, and hAQP0.
Residues identical in the five sequences are shown in red and conserved residues in yellow. Helices, including the
additional 310 helix in loop C (HC) of rAQP4, are shown as rods above the sequences and are color coded as in
Figure 3(b). Residues important for lateral interactions between rAQP4 tetramers are indicated by dots and those for
vertical interactions by double-dots. The wavy line indicates highly conserved AQP4 residues Arg9 and Trp10. The C
symbol indicates positively charged residues in the C-terminal region that could interact with phosphorylated Ser180.
630
for PDZ domains found in scaffolding proteins such
as PSD-95 and a-syntrophin.
AQP4 is also expressed in glial lamellae of the
hypothalamus,10 where it may play a role in osmo-,
thermo- and glucose-sensing.11 In glial lamellae the
plasma membrane forms large junctions between
individual layers, which have been shown to
contain AQP4. Interestingly, another water channel,
lens-specific AQP0, forms the “thin junctions”
between fiber cells.12 A recent electron crystallographic structure analysis revealed the homophilic
interactions that underlie AQP0’s adhesive properties and showed that junction formation causes the
water channel to close.5
To fully appreciate the detailed biological function of a protein, it is essential to know its atomic
structure. The importance of AQP4 as the predominant water channel in brain7,11 and its
propensity to form ordered arrays made AQP4 an
attractive target for structure analysis by electron
crystallography. Although AQP4 is expressed in
many tissues, its expression level is limited, which
made it difficult to use native sources to purify
sufficient amounts of the protein for structural
studies. Another concern with purified AQP4 was
the structural heterogeneity in the protein preparation introduced by the two splicing variants,
since structural heterogeneity can deteriorate the
order of resulting crystals. We therefore decided to
express rat AQP4 (rAQP4) in insect cells using a
baculovirus system, knowing that previous
attempts to use expressed mammalian membrane
proteins for structure determination were usually
not successful.
Implications of AQP4 Structure
microscope13,14 and the carbon sandwich specimen
preparation technique, which significantly
increases the yield of good images,15 allowed us to
also take an image of each crystal that produced a
high-resolution diffraction pattern. Classification
based on the image data, which provided phase
information to better than 6 Å, identified one
predominant crystal type, which accounted for
about 70% of the analyzed crystals that yielded a
high-quality electron diffraction pattern. In this
crystal type, the two layers have a spacing of 45 Å
(molecule center to molecule center) and are related
to each other by a 21 symmetry. The excellent merge
of the corresponding diffraction patterns (Table 1)
and the high quality of the lattice lines (Figure 2(a))
confirmed that the classification of the AQP4
crystals based on the image data was successful.
The final 3.2 Å resolution intensity data set was
used to determine the AQP4 structure by molecular
replacement using the AQP1 structure analyzed by
X-ray crystallography16 as the search model. The
images (recorded after the corresponding electron
diffraction patterns and used for classification) were
also used to calculate a density map (Figure 2(b)).
While the resolution of this map is limited to 6 Å
due to radiation damage, it clearly resolves the
arrangement of the a-helices in AQP4 and thus
provides independent confirmation for the molecular replacement solution.
Figure 3(a) shows a stereo view of a representative area of the final 3.2 Å resolution map around
residues His201 and Met212 with the fit atomic
model. The rAQP4 molecule adopts the typical
AQP fold,2 but features an additional short 310 helix
Results
Expression of 6!His-tagged rAQP4 routinely
yielded more than 3 mg of purified protein from
one liter of cultured Sf9 insect cells. After purification by nickel affinity chromatography, the
n-octyl-b,D-glucopyranoside (OG) solubilized protein was reconstituted into lipid bilayers. The
smaller splicing isoform AQP4M23 produced
large, well-ordered, double-layered 2D arrays,
which gave rise to sharp high-resolution diffraction
spots and could therefore be used for electron
crystallographic structure determination.
Electron crystallographic structure
determination of AQP4
Analysis of the AQP4 structure was complicated
by variations in the double-layered 2D crystals in
terms of lateral alignment and distance between the
two layers. Despite the high resolution of 3.2 Å,
electron diffraction data proved not sufficiently
sensitive to distinguish between different crystal
variants. Phase data extracted from images, on the
other hand, were sensitive enough to discriminate
between crystals with different arrangements of the
two layers. The use of a helium-cooled electron
Table 1. Electron crystallographic data
Two-dimensional crystal
Space group
Lattice constants
Electron diffractions
Approximate tilt angle
08
208
458
608
Total
Resolution (Å)
(in membrane plane)a
(normal to membrane plane)
Completeness (%)
Number of merged intensities
Number of independent
reflections
Rfriedel
Rmerge
Rcrystb
Rfreec
a
P4212
aZbZ69.0 Å
cZ160.0 Å (assumed)
gZ90.08
No. of patterns
6
19
47
63
135
3.2 (3.35–3.20)
3.6
87.0 (84.7)
68,429
5992
0.114
0.223 (0.445)
0.283 (0.342)
0.338 (0.422)
Values in parentheses are for the outermost resolution shell.
Rcryst Z SjFobs KFcalc j=SjFobs j, where Fobs and Fcalc are the
observed and calculated structure factors, respectively.
c
The Rfree was calculated using 5% of all reflections.
b
631
Implications of AQP4 Structure
Figure 2. Lattice lines and density map. (a) Representative lattice lines (2, 7) and (10, 11) showing the good match
between experimental diffraction data and the fit curve and confirming the successful classification of the 2D crystals
based on image data. (b) Density map of end on and side views calculated with the images corresponding to the
diffraction patterns used for structure determination. The backbone of the AQP4 model is shown in yellow, revealing the
excellent match between electron diffraction and image data.
in extracellular loop C (helix HC; formed by
residues Ser140 to Gly143) (Figure 3(b)). Like all
known water channels to date, AQP4 forms a
homo-tetramer (Figure 3(c)), and as in AQP1, the
AQP4 subunits in the tetramer interact with each
other through cytoplasmic loop D, connecting
helices 4 and 5 (orange in Figure 3(c)). The AQP4
tetramer is, however, further stabilized by an
additional interaction between adjacent monomers
on the extracellular side, which is mediated by loop
A connecting helices 1 and 2. Figure 3(c) also
highlights Ser180 (in yellow). Ser180 is a known
phosphorylation site17 and is located close to the
cytoplasmic opening of the water channel.
Orthogonal arrays
All the tetramers in our AQP4 2D crystals have
the same orientation (Figure 4(a) and (b)), while
those in AQP1 2D crystals are oriented in an
antiparallel fashion.2 The extracellular side of the
interface between neighboring AQP4 tetramers is
formed by hydrophobic residues Gly157, Trp231,
and Ile239 (Figure 4(c)), which are conserved in
other aquaporins (Figure 1). AQP4 tetramers form,
however, two further interactions on the cytoplasmic side of the interface between neighboring
tetramers involving residues Arg108 and Tyr250
(Figure 4(c)), which are specific to AQP4. Computer
632
Implications of AQP4 Structure
Figure 3. The rAQP4 structure. (a) Stereo view of the density map around residues His201 and Met212 with the fit
model. The 2FoKFc map contoured at 1.2s is shown in blue and the FoKFc map for the two residues contoured at 3s in
red. (b) Ribbon diagram of AQP4; H1-H6, transmembrane helices; HB, HE, pore helices in loops B and E; HC, 310 helix in
loop C. (c) AQP4 tetramer showing the tetramer-stabilizing cytoplasmic loop D in orange and Ser180, a known
phosphorylation site, in yellow. One AQP4 monomer is shown in space-filling representation.
simulations based on our tetramer structure
showed that only tetramers in the parallel arrangement seen in our 2D crystals could form these
interactions. Because this arrangement also results
in the same lattice constants as those seen in in vivo
arrays, our AQP4 crystals appear to recapitulate the
structure of the orthogonal arrays found in native
membranes.
While we could grow large and well-ordered 2D
crystals of AQP4M23, full-length AQP4M1 did not
formed significant crystalline arrays. This result is
consistent with the finding that AQP4M1 reduces the
size of arrays formed by AQP4M23.18 To understand
this effect of AQP4M1 on the size of AQP4M23 arrays,
we examined the influence of the N-terminal
sequence of full-length AQP4. Sequence comparison
Implications of AQP4 Structure
633
Figure 4. Interactions of AQP4 tetramers in a crystalline array. (a) Crystalline AQP4 in ball-and-stick representation
with one interacting AQP4 pair shown in ribbon representation. White outlines indicate tetramers in the adjoining layer.
(b) Double-layered AQP4 crystal in ball-and-stick representation with one pair of interacting molecules in ribbon
representation. (c) Stereo view of the interactions between adjacent tetramers that involve residues Arg108, Gly157,
Trp231, Ile239, and Tyr250.
revealed that Arg9 and Trp10 are highly conserved in
AQP4s from different species (indicated by a wavy
line in Figure 1). We therefore generated two 22
residue long peptides, one that corresponds to the
native rAQP4 N terminus and another one, in which
all the Arg residues were substituted by Lys residues
and Trp10 by an alanine. Addition of the native
peptide to AQP4M23 2D crystals at a molar peptide to
AQP4 ratio of 80:1 completely destroyed the order of
the arrays as shown by the Fourier transform of an
image taken from a sample in the presence of the
peptide (Figure 5(a), left half). Adding the modified
peptide had no effect on the crystal order, even at the
increased peptide to AQP4 ratio of 140:1 (Figure 5(a),
right half). The interactions between AQP4M23
tetramers and their destabilization by AQP4M1 are
represented schematically in Figure 5(b) and will be
discussed later.
Cell adhesion
Variations in the lateral alignment and distance
between the two membranes in the double-layered
crystals posed a difficult problem for the analysis of
the AQP4 structure. Nevertheless, 70% of the
crystals we analyzed formed specific interactions
between the two crystalline layers (Figure 4(b)).
These interactions are mediated by helical contacts
of a short 310 helix in extracellular loop C of AQP4
molecules in adjoining membranes (Figure 6). The
tetramers in an interacting pair are shifted relative
to each other, so that in the double-layered crystals
each AQP4 tetramer interacts with four tetramers in
the adjoining membrane (P42 12 symmetry)
(Figure 4(a)). The structure thus suggests that the
contacts between two interacting tetramers that are
not part of an array are restricted to a single subunit
in each tetramer (Figure 4(a) and (b)).
To establish whether the adhesive properties of
AQP4 are only sufficient to form membrane
junctions in the context of artificial 2D crystals or
whether adhesion is a physiological function of
AQP4, we prepared thin sections through the
hypothalamus. We observed large membrane
junctions in glial lamellae of the hypothalamus
broken by small areas where the membranes are
634
Implications of AQP4 Structure
Discussion
Structure analysis and possible gating
mechanism
Figure 5. Effect of the AQP4 N terminus on crystalline
arrays. (a) Fourier transforms of rAQP4M23 2D crystals
incubated with a peptide corresponding to the native
rAQP4 N terminus (left half) and a peptide where the Arg
residues were substituted by Lys residues (right half).
(b) Schematic drawings showing the major interactions
involved in orthogonal array formation and destabilization. In orthogonal arrays of AQP4M23 neighboring
tetramers interact through Arg108 and Tyr250 (left
panel). The conserved residue Arg9 in the AQP4M1 N
terminus can interact with Tyr250 in the same molecule,
thus competing for the formation of a bond with Arg108
in a neighboring AQP4 molecule (right panel).
separated (Figure 7(a)). As described before,10
labeling of glial lamellae with an antibody against
AQP4 localized the protein to areas where the
membranes are separated, but also all along
the junctional regions (Figure 7(b)), supporting the
notion that AQP4 has weak adhesive properties. To
show that we indeed imaged glial lamellae of the
hypothalamus, electron micrographs also include
part of the subarachnoid space (lower right corner
in Figure 7(a) and (b)). To further confirm that
AQP4 has adhesive properties, we stably
expressed AQP4M23 in L-cells, a fibroblast cell
line that does not express endogenous cell
adhesion molecules. Upon expression of
AQP4M23, L-cells reproducibly formed cell
clusters, whereas L-cells expressing AQP1 or flag
tag did not (Figure 7(c)).
In the analysis of membrane protein structures it
is desirable to do so under conditions that are
similar to those in an intact membrane. X-ray
crystallography of type-I 3D crystals, which consist
of layers of crystalline membranes, and electron
crystallography of 2D crystals are therefore advantageous techniques for the structural analysis of
membrane proteins. However, multi-layered crystals can have variations in the lateral alignment
between layers, which causes a serious problem for
structure analysis. In the case of our AQP4 2D
crystals, we could overcome this problem by using
images to classify the crystals and to select only 2D
crystals of the same type for high-resolution
structure analysis. The success of this procedure
for our 2D crystals is reflected in the high quality of
the reciprocal lattice lines (Figure 2(a)) and the final
density map (Figure 3(a)). It would clearly be
difficult to adapt such an approach to 3D crystals
in X-ray crystallography. Our successful determination of the AQP4 structure from a sample of
heterogeneous double-layered 2D crystals thus
shows one of the strengths of electron crystallography for the structural study of membrane
proteins.
Phosphorylation of Ser180 has been implicated in
protein kinase C and dopamine-dependent
decrease in AQP4 water permeability.17 Because
Ser180 is located close to the cytoplasmic opening of
the AQP4 channel (yellow in Figure 3(c)), this
position makes it unlikely that phosphorylation
directly affects the structure of the water channel.
Phosphoserine residues are, however, well-known
binding sites for numerous proteins, such as 14-3-3
proteins.19 Phosphorylation of Ser180 may thus lead
to the recruitment of a protein that blocks the water
channel. One possible candidate for a protein
binding to phosphorylated Ser180 would be the
C-terminal domain of AQP4 itself, in which case the
interaction could be mediated by positively charged
amino acid residues present in the C terminus
(residues Lys259, Arg260 and Arg261, indicated by
C in Figure 1). Although such an interaction may
well block the cytoplasmic entrance of the water
channel, this model remains speculative at this
point and will require experimental testing. New
functions, including the gating mechanism of AQP4
expressed in the mammalian brain, require further
studies to understand the cause of serious diseases,
such as cerebral edema,20 bipolar disorder,21 mesial
temporal lobe epilepsy22 and a variant of multiple
sclerosis.23
Regulation of the size of orthogonal arrays
Our AQP4 structure provides an explanation of
why only AQP4M23, but not AQP4M1, can form
635
Implications of AQP4 Structure
Figure 6. Stereo view of the interactions between AQP4 monomers in adjoining membranes. AQP4 molecules are
shown in ribbon representation, while residues Pro139 and Val142 in the 310 helix, responsible for junction interactions,
are shown in ball-and-stick representation. These monomers correspond to the ribbon representation of interacting
monomers in the context of the two AQP4 layers as shown in Figure 4(b). The distance from molecule center to molecule
center is 45 Å.
orthogonal arrays. It has been suggested that the
mere bulk of the additional 22 N-terminal residues
in AQP4M1 would suffice to interfere with array
formation.18 However, the N terminus of our
crystal-forming AQP4M23 construct (consisting of
a 6!His tag and a linker sequence) has a length of
35 amino acid residues, which is longer than the
native AQP4M1 N terminus. Inhibition of array
formation thus appears to be a specific characteristic of the amino acid sequence of the native N
terminus. We therefore generated a 22 residue long
peptide corresponding to the native rAQP4 N
terminus as well as another peptide, in which we
substituted all the Arg residues by Lys residues.
While the native peptide completely obliterated the
order of AQP4M23 arrays (Figure 5(a), left half), the
modified peptide had no effect on the crystal order
(Figure 5(a), right half). We therefore propose that
conserved residue Arg9 (and/or Arg8 in rAQP4)
can form an intramolecular bond with Tyr250, thus
preventing the formation of the intermolecular
bond between Arg108 and Tyr250, which is
required to stabilize an orthogonal array
(Figure 5(b), left side). The repulsive force between
the positive charges of the Arg108 residues in
adjacent tetramers, no longer compensated by
bonding with the Tyr250 residues (as in the case
when they interact with the Arg9 residues in
AQP4M1) would further compromise the stability
of an orthogonal array (Figure 5(b), right side).
New functions for AQP4
The two layers in our double-layered AQP4
crystals (Figure 4(b)) interact through helical
contacts mediated by the short 310 helix in
extracellular loop C of AQP4 molecules in adjoining
membranes (Figure 6). In the recently described
double-layered 2D crystals of AQP0,5 the tetramers
in the two adjoining membranes are precisely in
636
Figure 7. AQP4-mediated cell adhesion. (a) Section
through the hypothalamus of an adult rat showing
partially separated membrane areas of glial lamellae
(some separated areas are indicated by white arrowheads) in otherwise predominantly junctional membrane
areas. The subarachnoid space is in the lower right corner.
(b) Immunogold labeling of AQP4 in glial lamellae
localizing the protein to areas where the membranes are
separated but also all along junctional regions. The
subarachnoid space (indicated by SS) can be seen in the
lower right corner. Scale bars in (a) and (b) represent
200 nm. (c) Graph showing the reduction in the ratio of
isolated cells to total cells for AQP4M23 expressing L-cells
(filled circles). No significant reductions in the ratios were
observed for both AQP1 (open circles) and flag tag
expressing L-cells (open squares).
register (P422 symmetry), whereas the tetramers in
the two layers of our AQP4 crystals are shifted with
respect to each other by half a unit cell (P4212
Implications of AQP4 Structure
symmetry). In contrast to an AQP0 monomer,
which forms three interactions involving five
residues with a monomer in the adjoining membrane, an AQP4 monomer forms only one interaction involving two residues. Furthermore, all
subunits in an AQP0 tetramer make contact with
the corresponding subunits in the tetramer in the
opposite membrane, while only a single subunit in
an AQP4 tetramer can interact with a subunit in the
tetramer in the adjoining membrane as long as the
tetramer is not part of an orthogonal array
(Figure 4(a) and (b)). Furthermore, a 310 helix as
the one in loop C of AQP4 is less stable than an
a-helix. It is therefore likely that an isolated pair of
interacting tetramers would create only very weak
cell adhesion. In the double-layered crystals each
AQP4 tetramer interacts, however, with four
tetramers in the adjoining membrane (Figure 4(a)).
Formation of an orthogonal array would thus
enhance the adhesive properties of AQP4, although
the variations in the relative positions of the two
layers in our crystals suggest that even crystalline
AQP4 arrays promote only weak adhesion. Since
only AQP4M23 forms arrays,9,18 the expression
ratio between AQP4M1 and AQP4M23 must define
the size and abundance of AQP4 arrays, and thus
the membrane area involved in AQP4-mediated
junction formation.
In the images that we took of thin sections through
glial lamellae in the hypothalamus, we observed large
membrane junctions interrupted by small areas of
separated membranes (Figure 7(a)). Immunolabeling
showed that AQP4 localized to both junctional and
non-junctional membrane areas (Figure 7(b)). To
confirm the intrinsic adhesive properties of AQP4,
we stably expressed AQP4M23 in L-cells and
confirmed that the cells expressing AQP4M23
showed cell adhesion as opposed to L-cells expressing AQP1 or flag tag (Figure 7(c)). When compared
to the adhesive activity of nectin-3,24 the activity of
AQP4 is very weak, which is consistent with the
structural variability that we find in our doublelayered 2D crystals. These experiments thus corroborate that AQP4 functions as a weak cell adhesion
molecule, which acts through homophilic interactions between AQP4 molecules like those seen in
our double-layered 2D crystals.
AQP4 membrane junctions may reduce the water
permeability of glial cell plasma membranes,
because the tight tongue-into-groove packing of
the two crystalline layers results in a partial
blockage of the extracellular pore entrances
(Figure 4(a) and (b)). While the packing of the
AQP4 tetramers in the junctions must create
resistance for water flowing across the two
membranes, rapid water flow through the channels
may also reduce the adhesion between adjoining
membranes. This may establish the basis for a role
of AQP4 in osmo-sensing.11 For example, a high
AQP4M1/AQP4M23 expression ratio would produce small AQP4 arrays providing weak adhesion
between membranes, which could easily be separated and thus react to small water flows resulting
637
Implications of AQP4 Structure
from small osmotic differences. A low AQP4M1/
AQP4M23 expression ratio, on the other hand,
would result in extensive AQP4 arrays providing
relatively strong adhesion between membranes that
would withstand large water flows associated with
large osmotic differences. The consolidation of
water permeation and cell adhesion in single
membrane proteins, such as in AQP0, SoPIP2;125
and now AQP4, is intriguing. However, further
experiments will be needed to elucidate the interplay of the two functions in aquaporins and
potentially other membrane channels with adhesive
properties, which we propose to name “adhennels”
for “adhesive water and ion channels”.
bdenum grids and a final trehalose concentration of 7%
(w/v). Electron diffraction patterns and micrographs
were recorded with a JEM3000SFF electron microscope
equipped with a helium stage13,14 and operated at an
acceleration voltage of 300 kV. Low-dose electron diffraction patterns and micrographs at a magnification of
60,000! were collected from the same 2D crystals at tilt
angles of 08, 208, 458 and 608. Electron diffraction patterns
were recorded either with a 4 K or a 2 K slow-scan chargecoupled device (CCD) camera (Gatan). Electron diffraction patterns and images were processed with a modified
version of the MRC image processing programs.29
Electron diffraction patterns were analyzed using an
additional background correction step.30 Images were
computationally unbent and corrected for the contrast
transfer function.31
Materials and Methods
Model building
Constructs and expression of rAQP4M23
cDNA for rAQP4M2326 was subcloned into pBlueBacHis2B (Invitrogen). The construct was confirmed by
restriction enzyme digestion and DNA sequencing
(Hokkaido System Science), and plasmid DNA was
purified with the QIAGEN plasmid maxi kit. Recombinant rAQP4M23 baculovirus was prepared with the BacN-Blue transfection kit (Invitrogen). Sf9 cells were grown
in 3 l spinner flasks equipped with a Cell Master 1700
(Wakenyaku), keeping the temperature at 27 8C and the
dissolved oxygen concentration at 5.5 ppm. At a concentration of approximately 1.5!106 cells/ml, cells were
infected with 300 ml of high-titer virus stock (roughly 5!
107 pfu/ml) and harvested two days later.
Purification of rAQP4M23
Sf9 membranes were prepared as described,27 and
rAQP4M23 was purified as described28 with some
modifications. Briefly, membranes were washed with
2 M urea and alkaline buffer (pH 11) prior to solubilization with 5% (w/v) OG in 40 mM Tris–HCl (pH 7.5),
200 mM NaCl for 15 min at room temperature. After
centrifugation at 100,000 g for 45 min at 4 8C, the OG
concentration of the supernatant was lowered to 2% (w/
v) by dilution. Solubilized 6!His-tagged rAQP4M23 was
adsorbed overnight to Ni-NTA agarose at 4 8C. The resin
was washed with 10 mM histidine and the protein eluted
with 300 mM histidine in 20 mM Tris–HCl (pH 7),
300 mM NaCl, 2% OG for 2 h at 4 8C. One liter of Sf9
culture routinely yielded approximately 3 mg of AQP4.
2D crystallization of rAQP4M23
Purified protein was mixed with Escherichia coli total
lipid extract (Avanti) at room temperature using a lipid to
protein ratio of 1 (w/w). The mixture was dialyzed in a
dialysis button for three days against 10 mM Mes
(pH 6.0), 100 mM NaCl, 50 mM MgCl2, 2 mM DTT, 1%
(v/v) glycerol. During the first day of dialysis the
temperature was kept at 20 8C, increased to 37 8C on the
second day, and lowered to 20 8C on the third day.
Data collection and structure analysis
Specimens for cryo-electron microscopy were prepared
with the carbon sandwich technique15 using moly-
The amino acid sequences of rAQP4, bovine AQP1,
E. coli GlpF and human AQP0-9 were aligned with
CLUSTAL W 1.8232 and adjusted manually. Based on
this alignment MODELLER 6.133 was used to build a
model for rAQP4 starting with the bAQP116 and the
GlpF3 structures (PDB entries 1J4N and 1FX8). This model
was used as a search probe for molecular replacement
calculations in CNS 1.1,34 which was also used for further
refinements and calculation of potential maps. During
simulated annealing refinement, the dihedral angles (phi
and psi) in helical regions were restrained as
described.35,36 Model building was carried out with the
O molecular graphics package.37 The structure was
subjected to further rounds of refinement/rebuilding,
which included simulated annealing, positional and
B-factor refinement procedures. PROCHECK38 was used
to calculate a Ramachandran plot for the atomic model of
residues 31–254 (missing eight N-terminal and 69
C-terminal residues of rAQP4M23). A total of 81.5% of
the residues fell into the most favorable regions, whereas
only six residues fell into the generously allowed regions.
ALSCRIPT 39 was used for Figure 1, PyMOL‡ for
Figure 3(a), DINO§ for Figure 3(c), and MOLSCRIPT40
and Raster3D41 for the remaining Figures.
Crystal destabilization assay
Peptides 22 amino acid residues long were synthesized
by the Peptide Institute, Inc. (Japan, Osaka). Various
amounts of each peptide were added to rAQP4M23 2D
crystals and the mixtures incubated at 20 8C for 16 h. The
effect on crystal order was evaluated by inspection of
negatively stained specimens with a JEM-1010 electron
microscope equipped with a 2 K slow-scan CCD camera
(Gatan).
Ultrathin sectioning and immunolabeling
Brains of adult SD rats were perfusion fixed with 4%
paraformaldehyde (PFA) and 0.1% glutaraldehyde (GA) in
100 mM phosphate buffer (pH 7.2) at 36 8C and post-fixed
for 3 h at 4 8C. Brains were immersed overnight first in 15%
and then in 30% (w/v) sucrose in 100 mM phosphate buffer
(pH 7.2) at 4 8C. Brains were frozen in Tissue-Tek O.C.T.
Compound (Sakura Finetechnical Co., Ltd.) at K80 8C and
‡ http://www.pymol.org
§ http://www.dino3d.org
638
cryo-sectioned at K20 8C adjusting the thickness of the
sections to 10 mm. Sections were stained with 0.5% OsO4 in
100 mM phosphate buffer (pH 7.2) at 4 8C for 90 min and
then with 2% uranyl acetate (UA) at 4 8C for 20 min. Stained
sections were dehydrated with anhydrous ethanol,
embedded in epoxy resin and hardened at 60 8C overnight.
Ultrathin sections were cut with a thickness of 70–80 nm.
For immunogold labeling, rat brains, which were perfusion
fixed with 2% PFA and 0.1% GA in 100 mM phosphate
buffer (pH 7.2), were sectioned into 400 mm thick slices
using a Vibratome. After additional fixation at 4 8C overnight, slices were soaked with 2.3 M sucrose in 100 mM
phosphate buffer (pH 7.2) at 4 8C for two days. After
freezing the slices in liquid nitrogen, cryo-dehydration was
performed by immersing the samples for 48 h in OsO4/
methanol at K80 8C and then in 2% UA/methanol. After
changing the solution, the temperature was gradually
raised to K40 8C, where it was kept for 24 h. The solution
was changed to methanol, and the temperature gradually
raised to K30 8C and kept there for 72 h. The cryodehydrated slices were cryo-substituted from methanol to
HM20 overnight at K30 8C and polymerized at K30 8C for
48 h under ultraviolet light. The slices embedded in HM20
were micro-sectioned into 60–70 nm thick sections. The
sections were incubated with anti-AQP4 antibody (SANTA
CRUZ) at 4 8C for 72 h followed by incubation with 10 nm
gold conjugated goat anti-rabbit IgG (Amersham Biosciences) at 4 8C overnight. After immunolabeling, the
sections were fixed with 1% GA for 10 min, stained with
2% UA for 10 min and 3% lead solution for 10 min.
Cell adhesion assay
The adhesive function of AQP4 was examined by cell
aggregation measurements using fibroblasts (L-cells),
which have no endogenous adhesive properties, following
a published protocol.42 Stable L-cell lines exogenously
expressing AQP4M23, AQP1 and flag tag were suspended
in Hanks’ balanced salt solution as single-cell suspensions.
Suspensions were placed in 12-well plates precoated with
bovine serum albumin, and rotated on a gyratory shaker at
37 8C for 30 min and 60 min. Cell aggregation was stopped
with the addition of 2% GA. The extent of aggregated cells
in the three stable cell lines after various time periods was
judged by the ratio of total particle numbers to the initial
particle numbers. The results were averaged over three
independent experiments.
Protein Data Bank accession codes
The coordinates of the AQP4 structure reported here
have been deposited in the RCSB Protein Data Bank, with
accession code 2D57.
Acknowledgements
The authors thank Drs Lorenz Hasler, Michio
Kuwahara, Kenichi Ishibashi, Tomoko Doi and
Masato Yasui for scientific advice. This research
was supported by Grants-in Aid for Specially
Promoted Research, Grant-in-Aid for 21st Century
COE Research Kyoto University (A2), the Japan
Science and Technology Agency (JST), and the
Implications of AQP4 Structure
Japan New Energy and Industrial Technology
Development Organization (NEDO).
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Edited by W. Baumeister
(Received 16 September 2005; received in revised form 26 October 2005; accepted 28 October 2005)
Available online 17 November 2005