(Received for publication, December 8, 1995; and in revised form, January 8, 1996)
From the
The mercurial insensitive water channel (MIWC, AQP-4) is a
water-selective transporter expressed at the basolateral plasma
membrane of principal cells in kidney collecting duct, airway
epithelium, and gastric parietal cells, as well as in astrocytes and
skeletal muscle plasmalemma. Because these sites correspond to
membranes where orthogonal arrays of particles (OAPs) have been
observed by freeze-fracture electron microscopy, we tested the
hypothesis that MIWC forms OAPs. Chinese hamster ovary cells were
stably transfected with the coding sequence of rat MIWC under a
cytomegalovirus promoter. Immunostaining of clonal cell populations
showed MIWC expression at the plasma membrane. A single band at 31 kDa
was detected on immunoblot. Cell fractionation by sucrose gradient
centrifugation indicated strong MIWC expression in plasma membrane
fractions with lesser expression in Golgi. Functional analysis by
stopped-flow light scattering showed high mercurial insensitive water
permeability in plasma membrane vesicles. Freeze-fracture electron
microscopy showed distinct OAPs on the plasma membrane P-face of
MIWC-expressing cells with morphology indistinguishable from that in
basolateral membrane of kidney collecting duct; the E-face showed
corresponding linear grooves (spacing, 8 nm) in transfected cells
and collecting duct. OAPs were not observed in control (empty
vector-transfected) cells or CHIP28 (AQP1)-transfected cells in which
disorganized intramembrane particle aggregates were found. These
results provide direct evidence that a molecular water channel can
spontaneously assemble in regular arrays.
A family of water transporting proteins (``water
channels'' or ``aquaporins'') has been identified
recently, which are small (30 kDa) integral membrane proteins with
homology to the major intrinsic protein (MIP) (
)of lens
fiber (for review, see (1, 2, 3) ). Many
years prior to the molecular identification of water channels,
characteristic patterns of intramembrane particles (IMPs) observed by
freeze-fracture electron microscopy were proposed to represent a
morphological signature of water-transporting units. In amphibian skin,
regular square arrays of IMPs, called orthogonal arrays of particles
(OAPs), were found on the plasma membrane of water-permeable epithelial
cells after stimulation by vasopressin or isoproterenol(4) . In
toad bladder, vasopressin-induced IMP aggregates with a similar
morphology are induced by vasopressin(5, 6) ; in
kidney collecting duct, vasopressin induces the appearance of less
organized clusters of IMPs on the apical membrane of principal
cells(7, 8) . However, the basolateral plasma membrane
of the same collecting duct cell type contains large numbers of OAPs
that appear similar to the vasopressin-induced OAPs in amphibian
skin(9, 10) .
A series of morphological studies have indicated that OAPs are located in the plasma membranes of many different cell types in mammals: the basolateral membrane of principal cells in kidney collecting duct(10, 11) , epithelial cells in trachea(12) , intestine(13) , ciliary body (14) , and stomach(15) , brain astrocytes(16, 17) , skeletal muscle(18, 19) , and lens fiber. The OAPs in lens fibers were shown to contain MIP26, a protein expressed only in lens fiber that is homologous to all members of the aquaporin family but which has only limited water channel function(20, 21, 22, 23) . The identity and function of the OAPs in other tissues have remained enigmatic since their first discovery in astrocytes over two decades ago(17) .
We recently proposed, based on tissue distribution
studies and homology to MIP, that the mercurial insensitive water
channel (MIWC) may be the OAP protein(24) . MIWC was cloned
from rat lung(25) , and subsequently an isoform with an
extended NH terminus was cloned from rat brain (AQP-4, (26) ). MIWC is unique among the five cloned mammalian water
channels in that its water transport function is not inhibited by
mercurials because of the absence of a cysteine residue near its
putative aqueous pore(27) . Rat MIWC is a non-glycosylated,
301-amino acid protein that spans the membrane six times with cytosolic
NH
and COOH termini(28) . Immunolocalization showed
rat MIWC protein expression at the basolateral plasma membrane of
kidney collecting duct, astrocytes in brain and spinal cord, and
epithelial cells in stomach, trachea, bronchi, intestine, ciliary body,
and various exocrine glands(22, 29) . A human homolog
of MIWC is found as a single copy gene at chromosome locus 18q22 and
encodes distinct protein isoforms utilizing distinct transcripts
generated by alternative splicing(30) . The immunolocalization
data, taken together with functional studies in kidney (31) and
airways(32) , suggested a potential role for MIWC in the
urinary concentrating mechanism, cerebrospinal fluid reabsorption,
airway hydration, and glandular secretions.
The purpose of this study was to test the hypothesis that MIWC is able to form orthogonal arrays in a heterologous transfected cell model. CHO cells were stably transfected with rat MIWC utilizing procedures applied previously to generate cells that expressed functional and correctly targeted water channels CHIP28 (AQP-1) and AQP-2(33, 34) . The transfected cells expressed MIWC as a functional 31-kDa protein at the cell plasma membrane. Freeze-fracture electron microscopy showed prominent OAPs in MIWC-expressing cells that were morphologically indistinguishable from OAPs present in the basolateral membrane of kidney collecting duct.
Clonal cell populations of stably transfected cells expressing MIWC had identical growth characteristics and morphology to control cells transfected with empty vector and selected identically. Cell clones were first analyzed for MIWC expression, membrane localization, and function. Immunofluorescence with a rabbit polyclonal anti-MIWC antibody showed MIWC distribution in a membrane pattern with staining often observed in a perinuclear location probably corresponding to Golgi (Fig. 1A). Staining of control (vector-transfected) cells with immune serum was minimal (Fig. 1B), as was staining of MIWC-transfected cells with preimmune serum (not shown). High magnification confocal microscopy (Fig. 1C) indicated a plasma membrane distribution for MIWC.
Figure 1:
Immunofluorescence of transfected CHO
cells with anti-MIWC antibody. MIWC-transfected (A) and
control (empty vector-transfected, B) cells were examined by
indirect immunofluorescence as described under ``Experimental
Procedures.'' Cells were viewed by wide-field fluorescence
microscopy with 20 dry objective. C, confocal
micrograph of MIWC-transfected cells with
60 oil immersion
objective.
Subcellular fractionation was carried out to confirm the tissue distribution of immunoreactive MIWC and to isolate vesicles suitable for functional analysis. Fractionation by sucrose density gradient centrifugation yielded fractions enriched in endoplasmic reticulum, Golgi membranes, and plasma membranes. The identification of fractions was carried out by marker enzyme assays as described previously(33) . Immunoblot analysis in Fig. 2A (left) showed prominent expression of MIWC in the cell homogenate and the plasma membrane fraction; there was in some blots a weak band detectable in the Golgi fraction. MIWC was seen as a single band on immunoblot without glycosylation, similar to immunoblot findings in native cell membranes(24) . No distinct bands were detected in immunoblots of membranes from control cells probed with immune serum (Fig. 2A, right) or membranes from MIWC-expressing cells probed with preimmune serum (not shown).
Figure 2:
Immunoblot and functional analysis of
subcellular fractions. A, immunoblot performed on cell
homogenates and isolated vesicles fractions as described under
``Experimental Procedures.'' Each lane contains
3 µg (homogenate) and
1 µg (vesicle fractions) of
total protein. B, stopped-flow light scattering measurement of
osmotic water permeability. Vesicles were subjected to a 100 mM inwardly directed sucrose gradient at 10 °C, and 90°
scattered light intensity was measured at 530 nm. Average vesicles
diameters were measured by quasi-elastic light scattering to be 225 nm
(endoplasmic reticulum (ER)), 157 nm (Golgi), and 160 nm
(plasma membranes). See text for P
.
Functional analysis of water permeability was conducted on
the isolated membrane fractions. Fig. 2B shows
stopped-flow light scattering measurements of osmotic water
permeability in which vesicle suspensions were subjected to a 100
mM inwardly directed gradient of sucrose as described under
``Experimental Procedures.'' The gradient caused vesicle
shrinkage, which produced a change in scattered light intensity. The
time course of light scattering was similar in endoplasmic reticulum
from the control and MIWC-expressing cells. Shrinkage was slightly
faster in Golgi vesicles but much faster in plasma membrane vesicles
from MIWC-expressing cells than from control cells. Calculated osmotic
water permeability (P) for the plasma membrane
vesicles was 0.018 cm/s at 10 °C. There was no effect of 0.3 mM HgCl
on P
(not shown), a
concentration of HgCl
that strongly inhibited water
permeability mediated by CHIP28(36) . Stopped-flow light
scattering studies performed with urea and glycerol as solute (100
mM inwardly directed gradients) showed that MIWC is selective
for passage of water. These results are consistent with functional
measurements in Xenopus oocytes expressing MIWC(25) .
Freeze-fracture electron microscopy of cells transfected with MIWC
cDNA revealed aggregates of intramembrane particles scattered on the
plasma membrane P-face. In each membrane fragment from 11 separate
cells examined from two different cultures, the particles comprising
these aggregates were arranged into orthogonal arrays (Fig. 3a) that closely resemble the OAPs seen in other
cell types, including the basolateral plasma membrane of collecting
duct principal cells (Fig. 3b). In addition to large
OAPs, the cell membrane in Fig. 3a also contained many
large IMPs that appeared more numerous than those present in control
cells. These large IMPs could represent MIWC proteins that are not
assembled into characteristic OAPs. On the plasma membrane E-face of
MIWC-expressing cells, corresponding linear arrays of grooves were
frequently observed (Fig. 3c). The precise appearance
of the E-face grooves depended on the direction of platinum shadowing
relative to the groove array. In this case, the imprint appeared as a
series of linear grooves (arrows), each formed by a series of
small closely apposed depressions in the membrane leaflet. These arrays
of grooves were similar to those seen on the basolateral membrane
E-face of collecting duct principal cells (Fig. 3d). In
some areas, parallel arrays of grooves are seen (arrows) that
were indistinguishable from the arrays of grooves in the
MIWC-transfected CHO cells. These organized arrays were never seen on
control (vector-transfected) cells (Fig. 3e) nor were
they found in cells transfected with CHIP28 (Fig. 3f).
Many large IMPs, previously shown to correspond to CHIP28
tetramers(37, 38) , are present on the plasma
membrane. While they are sometimes concentrated into dense clusters of
IMPs, no organized geometric arrangement of the IMPs was seen. OAPs
were also never seen in freeze-fracture micrographs of LLC-PK1 and
Madin-Darby canine kidney cells as well as CHO cells transfected with
the AQP-2 protein (not shown).
Figure 3: Freeze-fracture electron micrographs of CHO cell plasma membranes and collecting duct principal cell basolateral plasma membranes. a, large OAPs on the P-face of a MIWC-expressing CHO cell. The particles are arranged in an orthogonal, geometric pattern characteristic of OAPs from other tissues. b, OAPs in the basolateral membrane P-face of a collecting duct principal cell. c, plasma membrane E-face of a MIWC-transfected CHO cell showing an array of parallel, linear grooves that represent the imprints of P-face OAPs shown in a. d, E-face of the basolateral plasma membrane of a collecting duct principal cell showing the appearance of imprints left in this membrane leaflet by the P-face OAP arrays. e, plasma membrane P-face from a non-transfected CHO cell showing small intramembrane particles, randomly distributed on the membrane. No large IMP clusters or arrays were detected. f, plasma membrane P-face from CHO cells expressing CHIP28 (AQP-1). Bars: 70 nm (a, b, e, f) and 50 nm (c, d).
In Fig. 3, c and d, the center-to-center spacing of the E-face grooves was
8 nm, as was the center-to-center spacing of the indentations
along the wall of each groove. On the P-face (Fig. 3, a and b), each large square IMP was
16
16 nm
in size and in many cases appeared to be composed of four subunits
(center-to-center spacing,
8 nm) when examined at higher
magnification. Thus, the grooves probably correspond to impressions
produced by the four subunits of the larger IMP. Based on previous
analysis of CHIP28 IMP size(37) , each 8-nm
``subunit'' might consist of a MIWC tetramer.
Taken together with previous data(20, 21, 37, 38, 39) , the results here indicate that MIWC and MIP spontaneously assemble into OAPs in a heterologous expression system and/or in proteoliposomes, whereas CHIP28 and AQP-2 generally do not. Highly purified CHIP28 does assembly into two-dimensional crystalline arrays when reconstituted into liposomes at very high protein-to-lipid ratios under conditions of slow detergent removal(40, 41) ; however, the CHIP28 orientation is very different from that in real membranes in that adjacent CHIP28 molecules in the crystal are oriented in opposite directions.
The physical interactions that drive the formation of OAPs are not known. Because of the close proximity between adjacent protein units, protein-protein interactions may provide the driving force for OAP formation such as interhelix dipole-dipole coupling and attraction between polar residues. We carried out hydropathy(22) , helical wheel (42) , and helix dipole (43) calculations for MIWC and MIP (proteins that form OAPs) and CHIP28 and AQP-2 (proteins that do not). The sequences for each putative helical domain were aligned, hydropathy values were compared before and with arbitrary helix rotation, and dipole moments were computed. However, no significant difference was found that distinguished MIWC and MIP from their homologs. The driving force for OAP assembly may involve protein and/or lipid interactions that were not considered, or the free energy for individual helix-helix interactions may be too small to detect computationally.
Is there
functional significance to the assembly of water channel proteins in
OAPs? Biophysical analysis has indicated that the single channel water
permeability of CHIP28 is low (<10 cm
/s; (36) ) so that water channel density
must be very high (>1000 channels
µm
) to
increase water permeability significantly above channel-independent
water permeability through lipids(1) . For CHIP28, each monomer
functions as an independent water channel(44, 45) ,
giving no advantage to specific supermolecular assemblies. In contrast,
MIWC was found recently to display subunit-subunit interactions,
whereby an adjacent mutant subunit affected the function of wild-type
MIWC(27) . Such interactions, which might occur in adjacent
units in OAPs, might enhance MIWC water permeability. Theoretically, it
is possible that parallel movement of water through closely spaced
channels might augment net osmosis by a bulk flow
mechanism(46) ; experimental verification of this hypothesis
will be needed utilizing membrane systems having different MIWC
assemblies. Another potential advantage for regular OAPs is to maximize
water channel surface density while minimizing protein-protein contact.
Our data suggest that OAPs in plasma membranes of many different cell types are composed of the water-selective transporting protein MIWC. Interestingly, modification of the membrane content and/or the morphological appearance of these OAPs has been reported for various physiological stimuli and pathological states, including stimulation of acid secretion by pentagastrin in parietal cells(47) , brain ischemia (48, 49) , aluminum-associated epilepsy in astrocytes(50) , and muscular dystrophy(51) . Further studies are required to determine the significance of these observations.