Water transport

across mammalian

cell membranes

A. S. VERKMAN,
ALFRED
N. VAN HOEK, TONGHUI
MA, ANTONIO
FRIGERI,
W. R. SKACH, ALOK MITRA, B. K. TAMARAPPOO,
AND JAVIER FARINAS
Departments
of Medicine and Physiology,
Cardiovascular
Research Institute,
University
of California,
San Francisco
94143; Department of Cell Biology, Scripps Research
Institute,
La Jolla, California
92037; and Department
of Molecular
and Cellular Engineering,
University
of Pennsylvania,
Philadelphia,
Pennsylvania
19104
Verkman,
A. S., Alfred
N. van Hoek, Tonghui
Ma, Antonio
Frigeri,
W. R. Skach, Alok Mitra,
B. K. Tamarappoo,
and Javier
Farinas.
Water transport
across mammalian
cell membranes.
Am. J.
PhysioZ. 270 (CeZZ PhysioZ. 39): Cl2-C30,1996.-This
review summarizes
recent progress in water-transporting
mechanisms
across cell membranes. Modern biophysical concepts of water transport and new measurement strategies are evaluated. A family of water-transporting
proteins
(water channels,
aquaporins)
has been identified,
consisting
of small
hydrophobic
proteins expressed widely in epithelial
and nonepithelial
tissues. The functional
properties,
genetics, and cellular distributions
of
these proteins are summarized.
The majority of molecular-level
information about water-transporting
mechanisms comes from studies on CHIP28,
a 2%kDa glycoprotein
that forms tetramers
in membranes;
each monomer contains six putative helical domains surrounding
a central aqueous
pathway and functions independently
as a water-selective
channel. Only
mutations in the vasopressin-sensitive
water channel have been shown to
cause human
disease (non-X-linked
congenital
nephrogenic
diabetes
insipidus);
the physiological
significance of other water channels remains
unproven. One mercurial-insensitive
water channel has been identified,
which has the unique feature of multiple
overlapping
transcriptional
units. Systems for expression
of water channel proteins are described,
including
Xenopus oocy-tes, mammalian
and insect cells, and bacteria.
Further
work should be directed at elucidation
of the role of water
channels in normal physiology and disease, molecular analysis of regulatory mechanisms,
and water channel structure determination
at atomic
resolution.
water permeability;
aquaporins;
epithelia;
nel; CHIP28; kidney tubules; fluorescence

HAS BEEN GREAT

INTEREST
in the mechanisms by
which water moves across membranes, beginning with
early phenomenological studies of osmosis in porous
membranes over a century ago, to the recent appreciation that specific water-transporting
proteins play a
key role in organ physiology. The underpinnings for
much of the recent work were established in the 1950s.
It was found that human erythrocytes had a high water
permeability that was inhibited by 90% by mercurial
sulfhydryl reagents; biophysical analysis of the watertransporting pathway by measurements of temperature dependence and osmotic-to-diffusional water permeabilities suggested that the water-transporting
pathway consisted of an aqueous pore traversing the
membrane. Soon thereafter, it was discovered that the
kidney also contained facilitated water-transporting
pathways in epithelial cells of certain nephron segTHERE

Cl2

0363-6143/96

$5.00

Copyright

o 1996

protein

structure;

water

chan-

ments: in the proximal tubule, where water permeability is constitutively

high, and in the collecting duct,
where water permeability is regulated by the antidiuretic hormone vasopressin. The molecular nature of
these water-permeable pathways was unknown five
years ago.
When the topic Mechanisms and regulation of water
permeability in renal epithelia was reviewed in 1989
(131), the m ajority of available data concerned phenomenological measurements of water permeabilities in
various tissues. Several lines of evidence had implicated a vesicle-trafficking
mechanism for vasopressinregulated water permeability in kidney collecting duct
and amphibian urinary bladder. Central questions at
that time concerned the physiological significance of
water transporters, the cellular mechanisms by which
vasopressin induced the fusion of water-permeable
the American

Physiological

Society

INVITED

vesicles with the plasma membrane of collecting duct

epithelial cells, and the molecular identity of watertransporting units.Although
there remain major unresolved questions concerning the physiological significance of water channels and the cell biology of
vasopressin-stimulated water permeability, there have
been remarkable advances in the identification, molecular cloning, and structure-function
analysis of watertransporting proteins, also referred to as water channels or aquaporins.An unanticipated finding was the
existence of a family of water-transporting
proteins
that are expressed widely in mammalian tissues.
The purpose of this review is to summarize and
evaluate recent developments in the investigation of
water-transporting
mechanisms across cell membranes, focusing on the biophysics and physiology of
water channels. Modern aspects of water channel biophysics and new measurement methods are reviewed.
Current knowledge about the structure, function, and
tissue distribution of members of the water channel
family is summarized, together with a critical discussion of the role of water channels in mammalian
physiology. In addition, new cell culture models and
expression systems to study water channels are described.
BIOPHYSICS

OF WATER

TRANSPORT

Several biophysical parameters that characterize

water transport across membranes provide insight into
the geometric nature of the water-transporting
pathway (31, 132). The osmotic water permeability coefficient (P,; in cm/s) is defined as the net flow of volume
across a membrane in response to a hydrostatic or
osmotic driving force

ff = J,/(Sv,[(P,

- P,YRT + 2 cri(Kt2i- IIli)]}

(1)

where JV is volume flow (cm3/s), S is membrane surface

area (cm2), VW is the partial molar volume of water (18
cm3/mol), P is hydrostatic pressure (atmospheres), oi is
the reflection coefficient of the ith solute, I& is the
osmolality of the ith solute (mosM), R is the universal
gas constant, and T is absolute temperature in kelvin.
Although there is considerable variability in Pf values
in cell membranes and even in lipid bilayers of differing
compositions, Pf > 0.01 cm/s (for a single membrane) is
suggestive of water movement through channels. The
osmotic gradient term in Eq. 1 dominates over the
hydrostatic pressure in most biological situations. Pf is
generally independent of osmotic gradient size and
direction, except for some complex membrane barriers
(e.g., certain epithelia), in which the geometry of the
barrier is influenced by water flow.
If the number of water transporters per unit area of
membrane is known, then the single-channel water
permeability (pf; cm3/s) is determined by pf = Pfhln,
where n iS the membrane density of functional water
channels b umber per cm2). PFhrepres ents the ch annelmediated component of osmotic w ater permeability
(after subtraction of channel-indepen .dent water perme-

REVIEW

Cl3

ability from total Pf>. The parameter pf provides a

quantitative
measure of water flow through single
channels. For single-file transport of water through a
narrow cylindrical pore, pf has been related to pore
radius (r) and length (L) as follows: pf = m2Di/L,
where 0; is the diffusion coefficient of a water molecule
if it were the only molecule in the pore (31). For the case
of CHIP28 (see below), apf of 6 X lo-l4 cm3/s indicates a
pore of 2.2 A diameter that traverses a 4-nm-thick
membrane (154). However, this treatment is probably
too simplistic to provide realistic information about
pore geometry. In particular, equations for water flow
through narrow channels and wide pores are fundamentally incompatible (31). Several recent theoretical studies have attempted to relate pf to pore geometry for
more complex pore shapes (50-52); however, analysis
of the complex water-water and water-pore interactions
in a real pore will likely require real-time molecular
simulations based on atomic resolution structural data.
A second potentially useful parameter describing a
water-transporting
membrane is the activation energy
(E,; kcal/mol), obtained from the slope of an Arrhenius
plot (Itif vs. 1IRT). Based on limited empirical data in
erythrocytes and kidney, E, is generally X0 kcal/mol
for water movement by a channel-independent solubility-diffusion
mechanism and <6 kcal/mol for water
movement through aqueous pores (31, 132). The high
E, for water movement through lipid may be related to
the formation and breaking of hydrogen bonds as water
moves between aqueous and membrane phases. However, conclusions about channel-mediated water transport based on E, values must be viewed cautiously. E, is
low (-5 kcal/mol) if Pf is unstirred-layer
limited, even
in the absence of water channels. Complex lipid-phase
transitions in biological membranes and the presence
of multiple parallel pathways for water movement
often yield nonlinear Arrhenius plots that are not easily
interpreted. Finally, there is no a priori reason that E,
should be low for water movement through a channel;
E, will depend on the nature of the rate-limiting barrier
for water movement and the energetics of water-pore
interactions.
The ratio of osmotic-to-diffusional
water permeability (Pf/Pd) may provide useful information about the
presence of a facilitated water-transporting
pathway.
The parameter Pd (cm/s) quantifies diffusional water
movement in the absence of an osmotic gradient: Pd =
JHZo ( [H20] 1 - [H2012), where eJH+ is the diffusive
water flow and [H20] is the concentration of labeled
water (see MEASUREMENT METHODS). Pf /Pd should equal
unity for a simple membrane that does not contain
water channels. PflPd can be greater than unity when
water moves through a wide pore or narrow channel or
when measured Pd is lower than true membrane Pd
because of unstirred layers (4). For an idealized wide
right cylindrical pore, PflPd = 1 + RTr2/8qD,Vw, where
q is viscosity; for a narrow channel in which single-file
movement of water occurs, PflPd = N, the number of
water molecules in the channel. Quantitative interpretation of Pf/Pd ratios sh.ould be made with caution
because of the compl ex natu re of biological water

Cl4

INVITED

channels and the difficulties

associated with Pd measurement
related to rapid water transport
rates and
unstirred
layers (4).
An additional
parameter
describing
a waterpermeable membrane
is the solute selectivity
of the
water pathway.
The ability of small solutes (e.g., protons, formamide,
urea, NH3, COs) to move through the
water-transporting
pathway
may provide a lower limit
to effective pore size. A low reflection coefficient may
furnish
evidence for water-solute
interactions
in an
aqueous pathway
(31, 133); however, recent studies of
urea and NaCl reflection coefficients in various systems
(13, 70, 91, 109) suggest that low (TVvalues reported
previously
were compromised
by measurement
artifacts and that (-TVis probably
near unity for most
physiological
solutes and cell membranes.
In summary,
a membrane
with high Pf (>O.Ol cm),
low E, (~6 kcal/mol),
and a PJPd ratio >l is likely to
contain water
channels.
However,
these biophysical
criteria
suffer from measurement
difficulties
and/or
lack of rigorous theoretical justification.
Definition
of a
molecular
mechanism
for water
transport
requires
matching
the measured
osmotic water permeability
coefficient of a membrane with predictions
based on the
membrane
densities and single-channel
water permeabilities of cloned water channels.

REVIEW

index changes that produce anomalous

scattering
signals. Light scattering
is also useful to measure Pf in
some small cells in suspension
if scattering
from intracellular structures
and motion artifacts are minimal.
Fluorescence
quenching
is based on concentrationdependent self-quenching
of certain fluorophores
(e.g.,
carboxyfluorescein,
calcein) (12). Vesicles or liposomes
are loaded with a high concentration
of fluorophore
and
subjected to an inwardly
directed osmotic gradient. As
water efflux occurs, there is a time-dependent
increase
in fluorophore
concentration,
resulting
in self-quenching and decreased fluorescence
(Fig. 1A). Fluorescence
quenching has been used to measure water permeability in plasma membrane
vesicles (12) and endosomes
from toad urinary
bladder (43, 112) and kidney (136,
147) containing
the vasopressin-sensitive
water channel. In the latter studies, endosomes were labeled in
vivo by fluid-phase
endocytosis
of an intravenously
infused fluorescent
marker
that entered the tubule
lumen after filtration
by the kidney glomerulus
(67,
69) .

H2O

F
MEASUREMENT

METHODS

Measurements
of water
permeability
in vesicles,
cells, and tissues are essential to evaluate the role of
water channels in mammalian
physiology. The cloning
of various water
channel homologues
heightens
the
need for quantitative
water
permeability
measurements in cell culture and oocyte expression
systems
and in proteoliposomes
reconstituted
with putative
water channels.
Established
methods to measure Pf
and Pd are reviewed briefly here, and newer methods to
quantify the function of heterologously
expressed water
channels are described.
Pf in Liposomes

and Vesicles

Light-scattering
and fluorescence-quenching
methods are now well established
to measure Pfin liposomes
and membrane
vesicles derived from native tissues.
Light scattering
is based on the dependence of elastically scattered
light on vesicle volume. A transmembrane osmotic gradient is established
by rapid mixture
of a vesicle suspension
with an anisosmotic
solution in
a stopped-flow
apparatus.
Osmotically
induced water
flow causes vesicle swelling or shrinking,
resulting in a
corresponding
change in scattered light intensity. Pf is
determined from the light-scattering
time course, vesicle
size, and the calibration
relation between light scattering and vesicle volume (135, 138); single-channel
pf is
determined
from Pf and channel density
(124). The
light-scattering
method is technically
simple, and very
small samples are required; however, there are potential problems in quantitative
data interpretation,
including vesicle size heterogeneity,
motion artifacts
just
after mixing that occur before flow stops, and refractive

FF
FF
FF

FF+
FF

penetration
depth

290 mOsm

time

400 mOsm

400 mOsm
mOsm

,----+

325

360

375

+F/*
H2

Fig. 1. Methods
for measurement
of osmotic
water permeability.
A:
fluorescence-quenching
measurement
of permeability
coefficient
(Pi.).
Cells loaded
with
a membrane-impermeant
fluorophore
(F) that
undergoes
fluorescence
self-quenching
are subjected
to an osmotic
gradient.
As vesicle
shrinks,
concentrated
fluorophore
is selfquenched
and fluorescence
decreases.
B: measurement
of Pf, in
adherent
cells by total internal
reflection
fluorescence.
Cells are
loaded with a membrane-impermeant
volume marker.
A thin (50-200
nm) layer of cytosol (penetration
depth)
is illuminated
by a laser
beam directed
through
a glass prism at a subcritical
illumination
angle. As cell shrinks
in response
to an osmotic gradient,
fluorophore
concentration
in illuminated
region increases.
C: transepithelial
Pfin
a perfused
tubule
measured
by a luminal
fluorophore.
An isolated
tubule is perfused
with an isosmolar
solution
(290 mosM) containing
a membrane-impermeant
fluorescent
marker
(F) and bathed
in a
hyperosmolar
solution
(400 mosM).
As solute-free
water is extracted
from the tubule lumen, both luminal
osmolality
and concentration
of
F increase.

INVITED

Pf in Adherent

Cell Monolayers

Measurement
of water permeability
in adherent cells
in culture may be required in polarized epithelial cells
or when suspension
of cells is not feasible. In addition,
single cell measurements
are required when cell heterogeneity exists because of the presence of variable water
channel expression
of mixed cell populations
in a
transient
transfection
system. As in vesicle measurements, the experimental
strategy
is to measure
the
time course of cell volume in response to a rapid change
in extracellular
osmolality.
For some cells with favorable geometry (e.g., 5774 macrophages),
45 light scattering provides a volume-dependent
optical signal (23,
32, 34); however,
the signals are generally small (because of scattering from intracellular
structures),
sensitive to nonvolume factors (solution refractive
index and
cell geometry), and not easily amenable to quantitative
Pf determination.
Detection
of fluorescence
from a
cytoplasmically
entrapped fluorophore
has advantages
over light scattering.
Fluorescence
methods are based
on the change in fluorophore
concentration
that accompanies changes in cell volume. Confocal
optics (or
partial confocality, Ref. 85) can, in principle, be utilized
to monitor fluorophore
concentration
by detection of
fluorescence
from a slice of cytosol (l-3 urn thickness).
Recently, a technically
simple method to quantify fluorophore concentration
has been developed based on
fluorescence excitation by total internal reflection (TIR)
fluorescence
(5a), where fluorophores
in membraneadjacent cytosol near a high-to-low
refractive
index
interface are excited by a laser beam (Fig. 1B) (27). As
cell volume decreases
and fluorophore
concentration
increases,
the TIR fluorescence
signal increases.
Because the effective depth of TIR illumination
(25-200
nm) is much smaller than cell thickness,
TIR fluorescence permits
the continuous
quantitative
measurement of relative cell volume in adherent cells of arbitrary shape and size, without
significant
fluorescence
photobleaching.
Other more demanding
experimental
approaches
to assess cell volume, which may have
applications
in some systems, include cell shape reconstruction
by analysis
of differential
interference
contrast (35) or fluorescence
confocal images (27) and
three-dimensional
tracking
of beads adherent
to the
cell surface (58).
Pf in Xenopus

Oocytes

Measurement
of Pf in Xenopus Zaeuis oocytes (TV1.2mm-diameter
spheres) expressing heterologous
mRNAs
or cloned cDNAs provides useful information
on water
channel function. Native oocytes have low water permeability (156). The assay for Pf is based on the rate of
swelling of defolliculated
oocytes in response to dilution
of the extracellular
solution.
The original
swelling
assay employed estimation of oocyte volume by measurement of two orthogonal
oocyte diameters
on a video
monitor every l-5 min (33). An improved quantitative
imaging approach was subsequently
developed, in which
the shadow cast by an oocyte was recorded and digitized using transmission
light microscopy
(153, 156).

Cl5

REVIEW

Recently, we have further improved the accuracy of the

swelling
assay utilizing
a custom-designed
transmission light microscope
in which the oocyte is viewed
simultaneously
along two orthogonal
axes. To obtain
information
on the relative single-channel
pf of mutated water channels, oocyte Ps can be normalized
to
water channel plasma membrane expression by quantitative immunofluorescence
(ill),
assuming
that each
expressed water channel is equally functional.
Water nansport

in Epithelia

For a cylindrical
epithelial cell layer such as a kidney
tubule, Pf can be measured
by an in vitro perfusion
technique. An impermeable
tracer (e.g., [H]inulin)
is
perfused through the lumen at constant slow flow and a
transepithelial
osmotic gradient is established
by bathing the tubule in an anisosmotic
solution. Tracer concentration changes along the length of the tubule due to
transepithelial
osmotic water transport.
Pf is calculated from the tracer concentration,
lumen flow, lumen
and bath osmolalities,
and tubule length and surface
area (131). A fluorescence
method to quantify
tracer
concentration
(without
the need for collection of luminal fluid) has been developed based on replacement
of
the radiotracer
by a membrane-impermeant
fluorescent indicator (62). The change in fluorophore concentration, determined
from the luminal
fluorescence
of a
distal segment of tubule, provides a quantitative
measure of Pf (Fig. 1C). This approach was used to determine the kinetics
of vasopressin-stimulated
Pf in kidney collecting duct (62, 63, 65) and recently to measure
Pf in intact lung airways
(36). For Pf measurements
across flat epithelial sheets, the best current approach
is to measure net volume movement
by capacitance
changes (54); Pr measurements
in cell domes (e.g.,
MDCK cell culture) and individual
plasma membrane
Pf measurements
in tubules have been accomplished
effectively by image analysis methods (35,90>.
Our laboratory
has been developing novel strategies
to measure water permeability
in cell or tissue layers
(30). One method is based on laser interferometry.
A cell
layer in a perfusion chamber is positioned in the path of
one of two interfering
laser beams in a Michelson
interferometer.
In response
to osmotically
induced
changes in cell volume, there are changes in intracellular refractive
index and intracellular
vs. extracellular
beam transit
distances.
The difference in optical path
length produces a measurable
change in interferometric amplitude
that can be interpreted
in terms of cell
volume. Another
method is based on fluorescence
quenching
of an extracellularly
oriented membranebound fluorophore
by a large soluble quencher.
In
response to water flow normal to the plane of membrane, the quencher concentration
near the membrane
changes due to convection,
leading to a change in
fluorescence
signal. In contrast
to previous
methods
that rely on cell volume changes resulting
from timeintegrated
water flow, the fluorescence
signal provides
unique information
about instantaneous
water flow,

Cl6

INVITED

the equivalent
of electrical conductance.
These methods may be useful to measure water permeability
in flat
epithelial cell layers and tissue sections.
Diffusional

Water Permeability

Because of unstirred
layer effects in epithelia (4) and
oocytes (156), information
about water channel biophysics (e.g., Pf /Pd ratios) requires Pd measurement
in small
vesicles, liposomes, or cells. Conventional
strategies
to
measure Pd rely on the use of radioactively
(3HZO> or
magnetically
[nuclear magnetic resonance (NMR)]
labeled water (139). However,
3HZ0 uptake studies are
challenging
because of rapid water diffusional
transport rates, and NMR requires large amounts of concentrated sample and the use of potentially
toxic paramagnetic quenchers. An optical strategy to measure Pd has
been developed, which exploits the dependence of fluorescence quantum yield of certain fluorophores
to the
HZ0/2H20(D20)
ratio. It was found that the fluorescence of aminonapthalene
trisulfonic
acid increases by
more than threefold when H20 is replaced by D20; this
approach was utilized to measure Pd in liposomes, red
blood cell membranes
(1 48), and perfused kidney tubules (64). Recently,
we found- that several
cellpermeable and trappable carboxyseminapthorhodofluor
(SNARF)/SNAFL
fluorophores
exhibit H20/D20-dependent quantum
yields, providing
a noninvasive
approach to measure Pd in living cells.
THE

FAMILY

OF

MAMMALIAN

WATER

CHANNELS

The historical
basis for the identification
of water
channel proteins has been summarized
in several recent reviews (2, 128, 133, 134, 137). Early biophysical
evidence implicated the existence of a facilitated watertransporting
pathway
in erythrocytes
and cell plasma
membranes
in kidney tubules and amphibian
urinary
bladder (77, 81, 131). However, it was unclear whether
water channels comprised distinct proteins or proteinlipid complexes or whether water moves through membrane proteins whose primary function is unrelated to
water
transport.
Indeed, several
membrane
trans-

Table 1. Properties
Original
Alternate

of mammalian

water

channel

family

REVIEW

porters were demonstrated

to have finite water permeability, including glucose transporters
(33,152) and the
cystic fibrosis transmembrane
conductance
regulator
chloride channel (47); however, the single-channel
water permeabilities
and/or membrane densities of these
proteins
are too low in native cell membranes
to
contribute
significantly
to total water
permeability.
Key observations
implicating
the existence of proteinaceous water
channels
were the increase
in Pf in
Xenopus oocytes expressing
heterologous
mRNAs from
kidney, reticulocytes,
and amphibian bladder (153,156)
and the determination
of an -30-kDa
water channel
target size in erythrocytes
and proximal tubule membranes by radiation inactivation
(122, 123). The subsequent identification
and characterization
of a family of
water-transporting
proteins
followed
the cloning of
human channel-forming
integral
protein of 28 kDa
(CHIP28)
(93) and demonstration
of increased water
permeability
on expression
of CHIP28
in Xenopus
oocytes (94).
Overview

of Water Channel

Family

Members

Table 1 summarizes
the properties
of mammalian
water channel family members
cloned to date. The
column headings are the original names of each protein
as reported; alternative
names that have been used are
also provided. Amino acid sequence alignment
of the
proteins shows high homology in hydrophobic
putative
membrane-spanning
segments
(Fig. 2, boxes), as well
as absolutely
conserved sequences including two NPA
motifs. The function of the prototype
molecule, major
intrinsic
protein (MIP) of lens fiber, is not clear. Early
studies suggested that MIP may have a role in formation of gap junctions between lens fibers or in transport
of glucose or ions (25, 26); recent data reveal that MIP
is slightly
water
permeable,
with a single-channel
-loo-fold
less than that of CHIP28
water permeability
(10, 125). Hu man CHIP28
(93) and a rat homologue
with 94% amino acid identity
(17, 154) are 28-kDa
hydrophobic
proteins that transport
water selectively
when expressed
in Xenopus
oocytes (94, 154) and
mammalian
cells (74) or when reconstituted
into proteo-

members

names
names

MIP26

CHIP28
AQP- 1

WCH-CD
AQP-2

WCH-3
hKID

MIWC
AQP-4

GLIP
AQP-3

AQP-5

Protein
size (amino acids)
mRNA size (kilobase)
N-linked
glycosylation
Mercurial
inhibition
Tissue expression
Transport
function
References

263
1.4
?
Lens
?
149

268
2.8
+
+
Wide
Water
17,154

265
1.9
+
+
Kidney
Water
40

271
2.2
?
Kidney
?
73

301
5.5
Wide
Water
46

285
5.5
+
+/Wide
Glycerol,
?urea/water
24, 53, 72

282
1.6
+
+
Wide
Water
99

Gene locus
Transcriptional

12q13
-

7p14
84

12q13
+
-

12q13
-

106

76

18q22
+
146

Alternative
References

regulation
mRNA

splicing

91

Major intrinsic
protein
(MIP) family members
expressed
in mammalian
tissues are listed (left to right> in order in which they were reported.
Data on mRNA and protein
are given for rats, and genetic information
is given for humans.
CHIP28,28-kDa
channel-forming
integral
protein;
WCH-CD,
water
channel
in kidney
collecting
duct; MIWC,
mercurial-insensitive
water
channel;
GLIP, glycerol
intrinsic
protein;
AQP,
aquaporin;
+, present;
-, absent; ?, uncertain.

INVITED

MIP
CHIP
CD
GLIP
MIWC
AQP5
WCH3 mepglcnr
YTIP -matrrys

- - -mwe lr
--mase fk
---mwe lr
----ml hi
mvafkgvwtqaFw$vtAEFLAmLIFVllsvGStib---wggsenp
--mkkevcsl
fAEFLATLIFVFFGLG
ayllvgglwt
fAEFLATglyVFFGvG
SlAEFasTfIFVFaGeGSg
fgrtdeathpdsmR@

icYmvAQLLGAvaGAAvLYsvT

Cl7

REVIEW

IgplhvLQVALAFGLAlATLVQAvGHiSGAHvNPAVTfAfLvGsq
1vqdnvkvsLAFGLsIATLaQsvGHiSGAHlNPAVTlglLlscq
sppsvLQIAvAFGLgIgiLVQA1GHVSGAHiNPAVTvAcLvGch
thggfLtInLAFGfAvtlgiliaGqVSGA.HlNPAVTfAmcflar
lpv*vlIsLcFGLsIATmVQcfGHiSGgHiNPAVTvAmvctrk
alptiLQIsiAFGLAIgTLaQAlGpVSGgHiNPAiTlAlLiGnq
,alpsvLQvAitFnLAtATaVQiswktSGAHaNPAVTlAyLvGsh
sagelLalALAhafALfaaVsAsmHVSGgHvNPAVsfgaLiGgr

MIP
CHIP
CD
GLIP
MIWC
AQP5
WCH3
')'TIP

mSl1
ISil
VSfl
epwi
ISia
IS11
ISlp
ISvi

MIP
CHIP
CD
GLIP
MIWC
AQP5
WCH3
YTIP

myyTGagMNPARS FaPAilT
lLfP&l ksvserlsilkgsrps
esngqpevtgepvelktqal
IdyTGCgiNPARS FGsAVlT
iLaP&s sdftdrmkvwtsgqve
eydldaddinsrvemkpk-IyFTGCSMNPARS
1aPAVvT
1LfPJsa .kslqerlavlkglepd
tdweerevrrrqsvelhspq
gfnsGyavNPARdFGPrlfT#ttgqhwwWvpiVsPLlGsiagvfvYqlmigchle~ppsnee
envklahvkhkeqi--------InyTGaSMNPARSFGPAVim-g-nwenHWiyWVGPiiGAvelkrrlkea
fskaaqqtkgsymevednrsqve
IyFTGCSMNPARSFGPAVvm-nrfspsHWvFWVGPivGAmLAaiLYfYlLfPsslslhdrvav
vkgtyepeedwedhreerkktie
IyFTGCSMNPARSFGPAViv-g-kfavHWiFWVGPLtGAvLAsLiYnfiLfP~tktvaqrlai
lvgttkvekwdlepqkkesqtn
atedy-----------------gpFdGacMNPAlaFGPslvg-w-qwhqHWiFWVGPLlGAaLAaLvYeYaviPieppphhhqpl

avrGnLalNt-Lhpgvs
$2QatiVEifLTlQfVLCiFAtl-yDeRRng-rl~SvALAvGfSltLGHLfG
sLgrNd-Largvns
QglgiEiigTlQLVLCvlAt-tDrRRrd-1
eirGdLaVNa-Lhnnata
QavtVELfLTmQLVLCiFAS-tDeRRgd-nl
SpALsIGfSVtLGHLlG
fadnql*hldmingffdq---1
fog' TasLivCvlAi~~np~~~~~~~~~~~~~,
gLgVtt-vhgnlta
hgllVELiiTfQLVftiFAS-cDskRtd-vt
SvALAIGfSVaiGpLfa
nLaVNa-Lnnttpg
amv-VELiLTfQLaLCiFsS-tDsRRts-p
tLgVNv-vhnstst
QavaVELvLTlQLVLCvFAS-mDsRq---tl
SpAsmIGtSVaLGHLiG
gfh---vspgvgv hmfi1EwmTfgLmytvygt
iDpkRga-vs
iapLAIGLiVganiLvG
*fvsapmdtagifatypsg

-------slprgska
--e---m-----

tedlilkpgvvIt-h-------sedtevsv----

#alagwgsavfagwgsavf
Fig. 2. Sequence
alignment
of water channel family members.
Amino acid sequences
rat, except for bovine major intrinsic
protein
and plant y-tonoplast
intrinsic
protein.
are capitalized
and absolutely
conserved
residues
are shown in bold. Boxes indicate
domains.

liposomes (124,150). As summarized in Table 2, CHIP28

is expressed widely in various epithelial and capillary
endothelia, including proximal tubule and thin descending limb in kidney (48, 88, 104, 154). A second water
channel expressed selectively in kidney collecting duct
apical membrane (WCH-CD) was identified by homology cloning (40) and subsequently shown to be the
vasopressin-inducible water channel by its mutation in
congenital nephrogenic diabetes insipidus (NDI) (18)
and its vasopressin-dependent membrane targeting
(22, 103). WCH-3 is a protein with 55% amino acid
identity to WCH-CD (73). WCH-3 is expressed only in
kidney, and, like WCH-CD, displays transcript upregulation in response to dehydration; however, the transporting function of WCH-3 is unclear. Recently, a
human counterpart of WCH-3 was cloned (hKID) with
80% overall amino acid identity to rat WCH-3 and
nonhomologous amino and carboxy termini (76).
A third mammalian water channel was the first
mercurial-insensitive water channel (MIWC) identified
(46) and found to b e expressed in multiple tissues,
including the basolateral membrane of kidney collecting duct (39). MIWC is most homologous to the big
brain protein of DrosophiZa (100). MIWC was shown to
be mercurial insensitive because of absence of a cysteine residue near its putative aqueous pore (113). An
unusual feature of MIWC was the tissue-specific expression of a nonfunctional spliced transcript containing an
exon deletion. Recently, another family member, given

are given for each protein

Highly
conserved-amino
putative
membrane-spanning

from
acids

the names glycerol intrinsic protein (GLIP) and AQP-3,

was cloned by three laboratories (24, 53, 72). GLIP is
most homologous to the bacterial glycerol facilitator
protein GlpF (55). Initial immunolocalization
studies
indicated expression in basolateral membrane of kidney collecting duct (53, 72); all groups reported that
GLIP (AQP-3) transported glycerol when expressed in
Xenopus oocytes; however, our laboratory (72) found
little or no increase in osmotic water and urea permeability. Another related water-transporting
protein
(AQP-5; Ref. 99) was identified with expression in
salivary gland, lacrimal gland, eye, and lung.
In addition to the mammalian proteins described
above, the MIP family includes homologous proteins in
amphibians, plants, Escherichia coli, yeast, and Drosophila (1, 14, 101, 144). An amphibian homologue of
the CHIP28 water channel has been cloned (1). The
glycerol facilitator protein of E. coli, GlpF, functions as
a bacterial glycerol transporter (3, 55); recent expression studies in Xenopus oocytes indicate that GlpF is
not water permeable (80). A portion of the big brain
protein of Drosophila of unknown function is highly
homologous to MIWC (100). In plants, y-tonoplast
intrinsic protein, a tonoplast membrane protein, functions as a water channel when expressed in oocytes
(79). A related turgor-responsive
homologue, RD28,
also increases water permeability when expressed in
oocytes (16). The induction of these proteins during
plant growth or in response to water deprivation sug-

Cl8

INVITED

Table 2. Tissue distribution

channel homologues

of mammalian

Nervous
system
Brain
Choroid
plexus: CHIP (4587)
Ependymal
cells: MIWC
(38,39),
Pia mater: MIWC
(38,39),
GLIP
Supraoptic
and paraventricular

water

other related MIP homologues remains to be elucidated.

Tissue Distribution

AQP-4*
(56)
(72)
nuclei: MIWC

(38), AQP-4

(56)
Astrocytes:
MIWC
Spinal (astrocytes):

(38)
MIWC

(38)

EYe
Iris epithelium:
CHIP (45,87,
118), MIWC
(39)
Ciliary
body: CHIP (45,87),
MIWC
(39)
Retinal
(nuclear
layer):
MIWC
(39,46)
Cornea
Endothelium:
CHIP (45,87,118)
Epithelium:
AQP-5*
(99)
Conjunctiva:
GLIP (39)
Lens epithelium:
CHIP (45,87)
Lens fiber: MIP (130,149)
Lacrimal
gland
Excretory
duct: MIWC
(38)
Secretory
lobule: AQP-5*
(99)
Musculoskeletal
system
Skeletal
muscle sarcolemma:
MIWC
(38)
Respiratory
system
Tracheal
epithelium:
CHIP*
(45), AQP-5*
(99), GLIP (39),
MIWC
(39)
Bronchial
epithelium:
MIWC
(39)
Alveolar
endothelium
and epithelium:
CHIP (37,48*)
Cardiovascular
system
Myocardium:
CHIP (7,45f)
Capillary
endothelium:
CHIP (45,87)
Digestive
system
Salivary
gland
Excretory
duct: MIWC
(38)
Secretory
lobule: AQP-5*
(99)
Stomach
(gastric
parietal
cells): MIWC
(38)
Colon
Crypt epithelium:
CHIP (48,87)
Villus epithelium:
MIWC
(39), GLIP (39)
Pancreas:
CHIP (87)
Gallbladder:
CHIP (45,87)
Genitourinary
system
Kidney:
WCH-ST
(73)
Proximal
tubule and thin descending
limb of Henle: CHIP
(48,88,104,154)
Collecting
duct apical membrane:
WCH-CD
(40,75*)
Collecting
duct basolateral
membrane:
MIWC
(38,39),
GLIP
(24,38,39,53*)
Urinary
bladder
(transitional
epithelium):
GLIP (38)
Reproductive
system
Testis efferent
ductules,
seminal
vesicles,
prostate:
CHIP (9)
Uterus:
CHIP (71)
Placenta:
CHIP (45)
Other
Liver (interlobular
bile duct): CHIP (87)
Hematopoietic
Spleen red pulp: CHIP (7,48*)
Erythrocytes:
CHIP (93)
Skin
Epidermis:
GLIP (38)
Sweat gland: CHIP (45)
* In situ hybridization
only
ences are shown in parentheses.

-(-Northern

REVIEW

blot

analysis

only.

Refer-

gests a role in plant water uptake. Nodulin 26, a

peribacteroid protein expressed in the symbiosome
membrane of soybean root nodules (3), probably functions primarily as an ion channel. The functional role of

of Water Channel Family Members

Table 2 summarizes the tissue distributions of MIP

family members in mammalian tissues. MIP is expressed only in lens fibers. The vasopressin-inducible
water channel (AQP-CD) and the homologous protein
WCH-3 are expressed exclusively in kidney. CHIP28,
MIWC, GLIP, and AQP-5 are expressed in multiple
tissues. Except for colocalization of CHIP28 and MIWC
in ciliary body of eye, MIWC and GLIP were not
detected in cells that express CHIP28. In contrast,
MIWC and GLIP colocalized at the same membranes in
certain cells of kidney, colon, trachea, and brain. However MIWC, but not GLIP, was expressed in stomach,
small airways, astrocytes, and glandular epithelia,
whereas GLIP, but not MIWC, was expressed in epidermis and bladder epithelium.
Of particular interest was the colocalization of MIWC
and GLIP at the basolateral membrane of principal
cells in kidney collecting duct, demonstrated by immunofluorescence and immunogold electron microscopy
(38, 39). Figure 3 shows expression of MIWC (Fig. 3A)
in principal cells of kidney collecting duct that also
express WCH-CD at the apical membrane (Fig. 3B);
MIWC (Fig. 3C) and GLIP (Fig. 30) are seen to
colocalize by double immunofluorescence. The need for
two water channel homologues in this constitutively
water-permeable membrane, if indeed GLIP functions
as a water channel, is unclear: there may be differential
regulation of MIWC and GLIP expression and/or function, MIWC and GLIP may serve different functions, or
MIWC and GLIP may interact at the molecular level,
such as in the formation of heterooligomers. The
quantitative contributions of MIWC and GLIP to collecting duct water permeability need to be established.
In general, the specific sites of expression of water
channel family proteins is consistent with a role of
water channels in fluid transport. In brain, localization
of CHIP28 in choroid plexus suggests a role in cerebrospinal fluid production, and localization of MIWC and
GLIP in meningeal and ependymal cells suggests a role
in cerebrospinal fluid reabsorption (Fig. 3E). CHIP28
may also be involved in osmoreceptor function (102).
The functional significance is less clear for MIWC
expression in astrocytes in brain and gray matter of
spinal cord (Fig. 3F), in neural cells in retina, and in
skeletal myocytes. In eye, CHIP28 and/or MIWC may
be involved in aqueous humor secretion. In several
tissues of the pulmonary and gastrointestinal systems,
MIWC was localized to epithelial cell basolateral membranes. High basolateral membrane water permeability in gastric parietal cells (Fig. 3G) and airway epithelium (Fig. 31) may be important for maintenance of cell
volume. A water-permeable basolateral membrane and
impermeable apical membrane would stabilize cell
volume and intracellular
osmolality during rapid
changes in the osmolality of air space fluids or gastric
contents. Alternately, MIWC (and GLIP) may participate in transcellular fluid movement if the contralat-

Fig. 3. Cellular
localization
of water
channel
homologues
in rat. A and B: double-immunofluorescence
of
mercurial-insensitive
water channel
(MIWC;
A) and WCH-CD
(B) in collecting
duct of rat kidney
medulla.
Arrow
points to unstained
intercalated
cell. Bar: 7 pm. C and D: double-immunofluorescence
confocal
microscopy
showing
colocalization
of MIWC
(C, red) and glycerol
intrinsic
protein
(GLIP; D, green) in basolateral
membrane
of principal
cells in medullary
kidney
collecting
duct. E: double-immunofluorescence
confocal
microscopy
of brain ventricle
showing
localization
of CHIP28
to choroid plexus (red) and MIWC
to lining ependymal
cells (green).
Bar: 100 urn. F
and G: immunoperoxidase
localization
of MIWC
in gray matter
of spinal cord (F) (g, gray matter;
w, white matter)
and basolateral
membrane
of parietal
cells in stomach
(G) (s, surface;
o, glands).
Bars: 5 pm (F) and 35 urn (G). H:
expression
of GLIP in epidermis
(e). Bar: 20 pm. I and J: expression
of MIWC
in basolateral
membrane
of bronchial
epithelium
(I) (a, airway)
and lacrimal
glands (J). Bars: 20 pm (I) and 12 urn (J).

c20

INVITED

era1 apical membranes

of epithelial
cells expressing
these proteins
are water
permeable.
The functional
significance
of GLIP expression
in urinary
bladder
transitional
epithelium
and skin (Fig. 3H), tissues
thought to be water impermeable,
remains unclear. In
glandular
epithelia,
MIWC (Fig. 3J, salivary
gland)
and AQP-5 may play a role in secretory
fluid generation.
The tissue-specific
distribution
of MIWC raises the
interesting
possibility
that MIWC may be a component
of orthogonal
arrays of particles
(OAP), which are
square arrays of membrane-associated
particles visualized by freeze-fracture
electron microscopy
(38). Except
in lens, where OAP are comprised
of MIP, tissues
known to contain OAP (astrocytes,
sarcolemma,
trachea, ciliary body, intestine, basolateral
membranes
of
gastric parietal cells, kidney collecting duct principal
cells; Refs. 38, 121, 130) were found to express MIWC.
It is unclear whether
the crystalline
arrangement
of
intramembrane
particles in OAP has functional significance or whether
it is a consequence
of the natural
tendency of MIP family members to form two-dimensional crystals.
Recently, an antibody
raised against
muscle sarcolemmal
vesicles
enriched
in OAP was
shown to stain kidney collecting duct basolateral
membrane and astrocytes
(130). We recently
found that
MIWC forms OAP when expressed
heterologously
in
Chinese hamster ovary (CHO) cells (unpublished
observations). Definitive identification
of MIWC in OAP will
require label-fracture
electron microscopy.
Water Channel

Genetics

The water channels identified

to date belong to a
family of small hydrophobic
channel-forming
membrane proteins of which MIP is the prototype (89, 101,
144). Hydropathy
plots of these proteins are similar,
suggesting
up to six transmembrane
helical segments.
Homology in amino acid sequence between the first and
second halves of each protein suggests that they arose
from tandem intragenic
duplication
of a three-transmembrane
segment.
Gene structure
studies
demonstrated
that the sites of exon-intron
boundaries
in
human CHIP28 (84), WCH-CD
(120), MIWC (146), and
MIP (92) are localized at three identical points within
their coding sequences,
suggesting
evolution
from a
common ancestor gene.
The gene loci of the human water channels appear to
have been dispersed to different chromosomes
in single
copy during evolution.
The CHIP28
gene has been
localized to chromosome
7~14 (19, 60, 84), WCH-CD
to
chromosome
12q13 (106), and MIWC to chromosome
18q22 (146). Interestingly,
the WCH-CD
gene is located
near the MIP gene at chromosome
12q13 (91), along
with a newly cloned human water channel homologue
hKID (76). In addition, the sequences of these three
cDNAs are evolutionarily
more closely related than to
other MIP family members,
suggesting
a MIP family
gene cluster in chromosome region 12q13. The evolutionary events that formed this putative gene cluster and
presence of other MIP family members in the cluster
remain to be elucidated.

REVIEW

Analyses of mRNA expression indicate tissue-specific

expression
and regulation
of water channel genes. The
WCH-CD
transcript
is expressed only in kidney, and its
level of expression
is strongly upregulated
in dehydration (40,49, 75). Sequencing of the Y-flanking
region of
the WCH-CD
gene revealed the existence of a adenosine 3,5-cyclic
monophosphate
(CAMP)-responsive
element and an AP-1 site (120). Because vasopressin
receptors are present in collecting duct principal cells,
elevations
in CAMP levels during dehydration
might
act on the CAMP response and AP-1 elements to enhance WCH-CD
gene expression.
The rat WCH-3 transcript is also expressed selectively in kidney and upregulated
in response
to dehydration
(73). MIWC
transcripts
are expressed widely in different organs in
rat (46, 146) and human (146). In human brain and
skeletal muscle, Northern
blot analysis indicated the
expression
of three different
mRNA
sizes. Reverse
transcriptase-polymerase
chain
reaction
analysis
showed expression
of nonfunctional
truncated
MIWC
transcripts
in rats (exon 2 deletion;
Ref. 46) and
humans (exon 3 deletion; Ref. 146), suggesting alternative splicing as a mechanism
of gene regulation. Analysis of the 5 region of the human MIWC gene indicated
overlapping
transcriptional
units, with distinct promoters producing proteins of 30 and 32 kDa by alternative
splicing (146). The specific regulatory
factors for MIWC
gene expression
are unknown.
Mutations
in water
channels
CHIP28
(97) and
WCH-CD
(18) have been detected in humans. Genomic
DNA analysis
on three rare individuals
who do not
express CHIP28-associated
Colton blood group antigens demonstrated
that two of them carried a CHIP28
pseudogene
with different
nonsense
mutations
and
another had a missense mutation encoding a nonfunctional CHIP28 molecule. Although red blood cells from
these individuals
had low osmotic water permeability,
the subjects were phenotypically
normal, raising questions about the physiological
importance
of CHIP28. It
is not known whether these individuals
manifest subtle
clinical abnormalities
under physiological
stress or
whether
upregulation
of other water
channels
can
functionally
compensate for the lack of CHIP28 expression in some tissues.
Mutations
in the WCH-CD
gene are associated with
the extremely rare non-X-linked
congenital NDI, manifested as inability of the kidney to concentrate
urine in
response to vasopressin.
The more common (but still
rare) congenital X-linked ND1 is caused by a mutation
in the vasopressin
Vg receptor. Two point mutations
were initially identified in exon 3 and exon 4 of genomic
WCH-CD
DNA from a patient with non-X-linked
ND1
(18). These nonconservative
substitutions
(Rl87C and
S216P) resulted in a nonfunctional
protein. More recently, three other individuals
were identified with ND1
caused by mutations
in WCH-CD
(G64R, Rl87C, deletion at base pair 369) (127). In vitro transcribed
cRNAs
did not increase water permeability
in Xenopus oocytes;
however, it is unclear whether
these mutations
interfere with WCH-CD
protein targeting and/or function.

INVITED
BIOCJXEMISTRY

OF

WATER

CHANNELS

The majority
of information
about water channel
biochemistry
comes from studies on isolated CHIP28
protein (21, 116, 125, 126). The erythrocyte membrane
is an excellent native tissue source to purify large
quantities of CHIP28 (2-5 mg/unit blood). Hemoglobinfree erythrocyte ghost membranes
prepared by hypotonic lysis are exposed to the anionic detergent Nlauroylsarcosine
(2-4%), which extracts virtually
all
proteins other than CHIP28
The stripped
membranes form osmotically active vesicles that are highly
water permeable (124). A similar procedure has been
used to obtain CHIP28containing
vesicles from renal
cortex (154). It is noted that insolubility
in N-lauroylsarcosine is not a general feature of water channels;
N-lauroylsarcosine
extracts MIP26 from lens fiber, as
well as MIWC and GLIP proteins expressed in Sf9 cells.
For further purification,
CHIP28-containing
vesicles
are solubilized with moderate-to-high
concentrations of
the nondenaturing
detergents Triton X-100 (4%) or
octyl-p-D-glucoside
(OG; 200 mM) (116, 125). Dissolved
CHIP28 has been delipidated
and purified by anionexchange chromatography
(1161, high-performance
sizeexclusion chromatography
(12.51, and phenylboronic
acid affinity chromatography
(126). Purified CHIP28 is
readily reconstituted
into proteoliposomes
by detergent
dilution or dialysis. Because liposome membranes have
substantial
background (lipid mediated) water permeability (Pf = 10m3 cm/s), relatively large amounts of the
CHIP28 protein are required to increase liposome Pf
above background. Protein-to-lipid
ratios of 1:20 to up
to 1:l (for crystallography
studies; Refs. 83, 142) give
reconstituted
proteoliposomes
with fully functional
CHIP28.
These high protein densities
are seldom
achieved in reconstitution
experiments;
the high efficiency of CHIP28 incorporation
is probably related to
its lipophilic character and small size.
An unusual feature of CHIP28 expression is that
both glycosylated and nonglycosylated
forms are present in native tissues as well as in CHIP28-expressing
CHO cells and Xenopus oocytes. Residue N42, which is
located in the first external loop after the first transmembrane
domain, is glycosylated in some CHIP28
molecules (1541, whereas a second consensus site for
N-linked
glycosylation
(N205) is not modified. On sodium dodecyl sulfate-polyacrylamide
gel electrophoresis and immunoblots
probed with anti-CHIP28
antibodies, CHIP28 migrates at two positions: a sharp band at
28 kDa, representing
nonglycosylated
CHIP28, and a
glycosylated broad band with an apparent size of 45-65
kDa (Fig. 4A) (104, 116). Monosaccharide
analysis
indicated a mean size of 5.4 kDa for the oligosaccharide
moiety with a polylactosamine
composition (126). Studies on the blood group antigens revealed that the
polylactosamine
oligosaccharide contains ABH determinants (117). Recent quantitative
image analysis of gels
and amino acid and sugar analyses indicated
that
approximately
one out of two CHIP28
molecules is
glycosylated (125,126), suggesting that two of the four

c21

REVIEW

a
69

46

30

inject

2i.5-

10 min
Fig. 4. SDS-polyacrylamide
gel electrophoresis (SDS-PAGE) and
size-exclusion high-performance
liquid chromatography (HPLC) of
CHIP23 A: Coomassie blue-stained SDS-PAGE of native human
erythrocyte CHIP28 (lane b) and after treatment with endo-pgalactosidase (lane a) and PNGase F (lane c). B: size-exclusion HPLC
(TSK 3000 column) of same samples run in 35 mM octyl+-Dglucoside. There was a right shift in peak position with endoglycosidase treatment (apparent molecular masses: 54 kDa for b, and 27
kDa for a and c), suggesting formation of monomers from dimers.

CHIP28 monomers in each membrane-associated

tetramer are glycosylated.
It has not been possible to separate nonglycosylated
and glycosylated
CHIP28 by conventional
chromatographic procedures using nondenaturing
detergents. In
OG, native CHIP28 forms dimers of nonglycosylated
and glycosylated CHIP28 (126). Interestingly,
the tight
association of nonglycosylated
and glycosylated CHIP28
disappears on enzymatic deglycosylation
by PNGase F
or oligosaccharide
trimming
by endo-@galactosidase
(Fig. 4B), suggesting that the oligosaccharide
moiety is
responsible for the association. However, functional,
structural,
and morphological
studies indicated that
the polylactosamine
oligosaccharide
is not essential for
CHIP28
function (155) and does not alter CHIP28
structure in membranes (126). It will be important
to
perform similar biochemical
analysis on other water
channels,
WATER

CHANNE!

Spectroscopic

L STRUCTURE

Studies

Several spectroscopic approaches were applied to

examine CHIP28 structure. Analysis of CHIP28 secondary structure by circular dichroism and Fourier transform infrared spectroscopy gave -40% o-helix and
-40% p-sheet and turn (125). The results indicated
multiple
membrane-spanning
o-helices or mixed o/pdomains but ruled out an all p-barrel structure like the
bacterial porins. The environment
of the four tryptophan residues in CHIP28
was studied by intrinsic
tryptophan fluorescence (28). A blue-shifted tryptophan
emission maximum
(324 nm) and insensitivity
to the
polar quenchers iodide and acrylamide
indicated that
all trytophans were located in a hydrophobic environment. Tryptophan
position mapping by n-anthroloxy
fatty acid probes suggested that the tryptophans
were
located just beneath the bilayer and deep in the bilayer.
A novel spectroscopic method, single-photon radiolumi-

c22

INVITED

nescence (SPR; Refs. 6, 107), was applied to demonstrate

an aqueous pathway traversing the CHIP28 protein. SPR
is based on the excitation of fluorescence from the energy
deposited by an electron produced by 3H beta decay. The
tryptophan
SPR signal in CHIP28
produced by 3HZ0
was greater than that produced by an equivalent
3H
amount of [3H]glucose (5), suggesting
that the interior
of CHIP28 is accessible to HZ0 but not to the larger
solute glucose. Recently, time-resolved
anisotropy
measurements
of reconstituted
fluorescein-labeled
CHIP28
indicated a mercurial-induced
change in the nanosecond segmental dynamics of CHIP28 (29).
CHIP28

Forms

Tetramers

in Membranes

Initial evidence that CHIP28

forms oligomers
in
detergent solution was obtained by cross-linking
and
sedimentation
studies (116). Freeze-fracture
electron
microscopy data of Verbavatz et al. (129) demonstrated
that CHIP28
formed tetramers
in native cell membranes and reconstituted
proteoliposomes.
By rotary
shadowing,
intramembrane
particles
were observed
with a mean diameter of 8.5 nm. The intramembrane
particles
consisted
of a distinct
arrangement
of four
smaller
subunits
surrounding
a central depression.
These characteristic
particles were observed in proteoliposomes containing purified CHIP28 CHIP2%expressing transfected
CHO cells, and plasma membranes
from proximal
tubule and thin descending
limb of
Henle. Assuming
that each CHIP28 monomer is a right
cylinder of length 5 nm, the monomer diameter would
be 3.2 nm; a symmetrical
arrangement
of four such
cylinders
would have a greatest diameter
of 7.2 nm,
which, after addition of the thickness
of the platinum
deposit in the shadowing
procedure
(l-l.5
nm), is
similar
to the measured
value of 8.5 nm. Similar
freeze-fracture
studies on enzymatically
deglycosylated
CHIP28 showed the formation
of tetramers
in membranes (126), indicating
that glycosylation
was not
required for the tetrameric
association.
Several low-resolution
structural
studies by negative
staining electron microscopy
showed that CHIP28 assembled as tetramers
in crystalline
membrane
arrays
obtained by reconstitution
at high protein-to-lipid
ratios (83, 141, 142). Transmission
electron micrographs
of sections stained with uranyl formate were analyzed
by Fourier
transform
techniques.
A 12-A resolution
reconstruction
showed a tetragonal
lattice with p4g
plane symmetry,
in which the unit cell contained four
CHIP28 dimers, each composed of two oblong-shaped
monomers
oriented in opposite directions
(83). It was
not possible with this limited resolution
to define the
location of the putative
aqueous pathway
through
CHIP28
which has been predicted to have a diameter
of -2.2 A (154).
CHIP28

Monomers

Function

Independently

Although
CHIP28
forms tetramers
in membranes,
several lines of evidence initially suggested that CHIP28
monomers function independently.
Radiation inactivation studies in native kidney vesicles performed before

REVIEW

the cloning of CHIP28 showed a 30-kDa target size for

the functional unit of osmotic water permeability
(122,
123). A second line of evidence comes from coinjection of
Xenopus oocytes with cRNA encoding wild-type
CHIP28
and mutated CHIP28
monomers
that are either nonfunctional
(Cl89W)
or functional
but not inhibited by
mercurials
(Cl89S). Although
the wild-type
and mutant monomers
appeared not to interact
(95, 155), it
was not possible to rule out the formation
of homotetramers
consisting
of all wild-type
and all mutant
monomers.
Strong evidence came from expression
of
chimeric cDNA dimers that contained wild-type
CHIP28
in series with either the Cl89W
or Cl89S mutants
(111). Transcribed
cRNAs were injected in Xenopus
oocytes, and plasma membrane expression was assayed
by quantitative
immunofluorescence.
Oocyte water permeability was proportional
to the number of CHIP28
wild-type
monomeric
units expressed
at the plasma
membrane,
independent
of the expression
of nonfunctional Cl89W units. In addition, mercurial inhibition of
water permeability
in oocytes expressing
the CHIP28Cl89S dimer was intermediate
between that in CHIP28expressing
oocytes and C 189S-expressing
oocytes. The
results
indicated
that, despite their assembly
into
tetramers,
monomeric CHIP28 subunits function independently as water channels.
Biogenesis

and Dansmembrane

Topology of CHIP28

Hydropathy analysis of CHIP28 indicates up to seven

hydrophobic regions (HR; Fig. 5A), which initially
suggested a six-spanning topology model (Fig. 5B) (93,
154). As found experimentally,
the sites for N-linked
glycosylation (N42) and mercurial sensitivity (C 189)
are predicted to be externally oriented (95, 155). Two
studies of CHIP28 topology have been reported, one
examining transmembrane topology of mature CHIP28
at the plasma membrane ofXenopus oocytes (96) and a
second investigating early events of CHIP28 biogenesis
which give rise to CHIP28 topology as it is synthesized
at the endoplasmic reticulum (ER) (114). The complementary approaches have yielded surprisingly different results, suggesting a potentially complex mechanism of CHIP28 biogenesis.
Preston et al. (96) inserted peptide-derived epitope
tags (El epitope of the avian carona virus capsid
protein) at six different locations in full-length CHIP28.
A principal finding was that insertion of the El epitope
at position T120 permitted chymotrypsin digestion of
CHIP28 at the surface of intact oocytes, supporting the
topology model in Fig. 5B. Skach et al. (114) studied the
process by which CHIP28 is assembled at the ER
membrane. Experiments were designed to identify
specific translocation initiation and termination events
that give rise to early CHIP28 topology. Nine CHIP28
fusion proteins containing a reporter of translocation
(derived from the secretory protein bovine prolactin)
ligated onto sequentially truncated CHIP28 fragments
were expressed in Xenopus oocytes. Reporter topology
determined by protease sensitivity indicated a fourspanning topology of the nascent chain as it emerged
from the ribosome (Fig. SC). This conclusion was

INVITED

C23

REVIEW

i:
:
i
i
I.
.
I...................................~..*........*......................o....................................j....................................9...................................~............
*
.
1
............

.....................~...................................~....................................:

::

pJ

::::::

. . . . . ~~~~~

::::

. . . : : : i pJ..

............

............

fg...;

.....
.B..

..................

+J.

..........................
. . . . . . fg..

..i...................................4
..........

t..

. . . . . . . . . fyJ

...........

..........
.
...........
..a.+ . .
. ............
..* . . . . . . . . . . . . .
...........
1 .. .. . .. .. . .. ...~..............4...................................~....................................~
.. . .. .. . .. .. . .. .. . .. .. . .. .. . .. .. ..~...............~..................~.............
:
:
:
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ~ . . . . . . . . . . . . . . ~ . . . . . . . . . . . . . . . . . . . . ~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ~ . . . . . . . . . . . . :. . ~ . . . . . . . . . . . . . . . . . . . . ~ . . . . ~ . . . . . . .
.
:
:.
:
E
I
I
I
I

ami no acid

50

100

150

200

Cl89

............
.w..........+

; ...........

..+ . . . . . . . . . . . .
...+ . . . . . . . . . . . .
:
i
I

250

N
COOH

Fig. 5. Topology
and structure
of the CHIP28
water channel. A: Kyte-Doolittle
hydropathy
plot of CHIP28
showing
7 hydrophobic
regions (HRs) that are potential
membrane-spanning
domains.
B: proposed
transmembrane
topology
of mature
CHIP28
with
6 membrane-spanning
domains
with
the amino
and carboxy
termini
in a cytosolic
orientation.
Locations
of N-linked
glycosylation
site (N42) and mercurial-sensitive
cysteine
(ClS9)
are shown.
C:
proposed
intermediate
transmembrane
topology
of CHIP28
as it is synthesized
at the endoplasmic
reticulum.
D:
projection
structure
of CHIP28
in membranes
at 6 A resolution
by cryoelectron
crystallography
(82). Dotted
curve
represents
1 CHIP28
monomer
in a tetramer.
Putative
transmembrane
o-helical
domains
are numbered
l-6. Bar:
10 & See text for details.

corroborated by independent techniques, including proteolysis of sequentially truncated native protein, characterization
of internal stop-transfer sequences, Nlinked glycosylation at engineered internal consensus
sites, and insertion and protease digestion of a short
epitope flag. Two internal signal sequences for initiating translocation of the nascent chain into the ER
lumen and two stop-transfer sequences that terminate
ongoing translocation were identified. It was found that
HR2 alone was unable to terminate translocation or
direct a membrane-spanning orientation, whereas the
fragment HR2-HR4 efficiently terminated translocation and spanned the membrane. Thus, immediately
after synthesis, HR4 spans the membrane with its
carboxy terminus in the cytosol.
Additional studies have begun to explain apparent
differences in CHIP28 transmembrane topology observed at the ER vs. the plasma membrane. Preliminary data suggest that CHIP28 utilizes a novel mechanism of biogenesis in which two distinct topological
isoforms are initially generated at the ER. Pulse-chase
analysis of CHIP28 fusion proteins showed that the
ratio of isoforms changes as a function of time: at early

time points, topology was consistent with four membrane-spanning segments, whereas, at later time points,
a gradual change toward a six-membrane spanning
topology was observed (115). The mechanism underlying this topological maturation appears to involve
interconversion of folding intermediates during or after
synthesis and possibly selective degradation of specific
isoforms.
To explore whether the unexpected mechanism of
CHIP28 biogenesis was characteristic of other water
channels, a parallel analysis of MIWC biogenesis was
carried out (110). In contrast to CHIP28, MIWC exhibited a conventional biogenesis mechanism. Six membrane-spanning segments were observed at the ER. In
contrast to CHIP28, the second HR of MIWC terminated translocation and spanned the membrane. Membrane integration of MIWC occurred after synthesis of
a single hydrophobic region (HRl), whereas CHIP28
integration was delayed until synthesis of four HR.
Consistent with these findings, MIWC and CHIP28
exhibited opposite orientations of HR4 at the ER membrane. These results raise interesting questions about

C24

INVITED

how and why closely related water-transporting

teins utilize very different biogenesis pathways.
Structure

of the Putative

pro-

CHIP28 Water Pore

As described above, Kyte-Doolittle hydropathy analysis of CHIP28 predicts six bilayer-spanning hydrophobic cx-helices (93, 125). The Garnier-osguthorpeRobson and Thornton algorithms predict turns for the
hydrophilic regions between the six hydrophobic segments. These algorithms predict that each of the conserved NPAmotifs (residues 76-79 and 192-195) forms
a turn in a region that is predicted to contain P-sheet.
One interpretation
of these results is that CHIP28
contains six transmembrane hydrophobic cx-helices and
that the loop regions containing the NPA motifs form
anti-parallel P-sheets. Based on mutagenesis results, it
has been suggested that these P-sheets dip into the
bilayer, parallel to the membrane normal, and line the
aqueous channel through which water transport occurs
(57). Other models of CHIP28 structure place the
aqueous pathway at the center of membrane-spanning
amphiphilic u-helical domains in which polar residues
line the pore (114).
A recent study of frozen-hydrated
CHIP28 crystals
utilized electron crystallography
to obtain a highresolution projection map in the membrane plane (82).
As shown in Fig. 50, the projection map shows clear
definition of monomers within tetramers. Each monomer contains six peaks characteristic of cx-helices oriented nearly normal to the plane of the membrane. This
bundle of putative antiparallel cx-helices appears to
enclose a vestibule leading to the hypothesized waterselective pore. Taken together with the information
above, these results suggest that CHIP28 contains six
transbilayer ar-helices that pack together to form the
aqueous channel. Loops containing the NPA motifs
cannot be visualized in the projection structure. These
loops may form P-sheets; however, their orientations
with respect to the bilayer have not been defined.
Atomic resolution structural
information
on threedimensional crystals will be required to furnish further
details about the structure of the aqueous pathway.
EXPRESSION
CHANNEL

SYSTEMS
PROTEINS

FOR

WATER

The expression of water channel proteins in heterologous systems is important for analysis of water channel
function and regulatory mechanisms and, when suitable native tissue sources are not av,ailable, for protein
isolation. The Xenopus oocyte has been useful to study
water channel function in oocytes microinjected with
heterologous unfractionated and fractionated mRNAs
(119, 152, 153, 156), cRNAs from cloned proteins (46,
94, 154), and cRNAs from mutant and chimeric constructs (57, 95, 111, 155). Although the oocyte expression system is not suitable to generate large amounts of
protein, protein expression can be quantified by immunoblotting (94) and quantitative
immunofluorescence
methods (111). CHO cells have been stably transfected
with CHIP28 cDNAunder the control of a cytomegalovi-

REVIEW

rus promoter (74). Immunolocalization

and functional
assays revealed the presence of fully functional protein
at the plasma membrane and in the Golgi vesicle
fraction. Quantitative
immunoblotting of cell homogenates indicated -8 X lo6 CHIP28 monomers per cell.
As described below, transfected LLC-PK1 strongly
express CHIP28 and WCH-CD proteins and demonstrate vasopressin-dependent membrane targeting of
WCH-CD (59).
To generate larger quantities of protein for purification, bacterial and insect cell expression systems have
been evaluated recently. Nodulin 26, a water channel
homologue expressed in plants, has been expressed as a
polyhistidine-tagged
fusion protein in E. coli (66). The
affinity-purified
fusion protein (yield - 100 pg/l culture)
was reconstituted into planar lipid bilayers and shown
to have similar electrical properties to native nodulin
26. A similar bacterial expression strategy was successful for expression of small amounts of CHIP28, WCHCD, and MIWC proteins (unpublished observations).
WCH-CD and GLIP proteins have been expressed
using the baculovirus expression system (27). Infection
of Sf9 insect cells with baculovirus containing GLIP
cDNA under the control of the strong polyhedrin promoter conferred increased glycerol permeability in Sf9
cells, confirming protein expression in functional form.
The proteins were localized to the plasma membrane by
immunostaining. Protein purification and reconstitution will be required to assesssingle-channel properties
of the expressed proteins.
CELLULAR
MECHANISMS
VASOPRESSIN-STIMULATED

OF
WATER

TRANSPORT

Vasopressin stimulates apical membrane water permeability in principal cells of mammalian kidney collecting duct and granular cells of amphibian bladder.
Kinetic studies indicate that transepithelial Pf in collecting duct begins to increase within 20 s of vasopressin
exposure and increases by lo- to ZO-fold with a halftime of -5 min (65). Vasopressin acts primarily by
binding to basolateral membrane V2 receptors and
stimulation of adenylate cyclase; however, interactions
with the phosphoinositide and/or Ca2+ second messenger systems may also be important. Activation
of
protein kinase A by CAMP is then believed to promote
phosphorylation
of one or more as yet unidentified
protein(s), leading to the insertion of intracellular
vesicles containing functional water channels into the
apical plasma membrane. When the vasopressin stimulus is withdrawn, water channels are retrieved from
the apical membrane by endocytosis. This membrane
shuttle hypothesis was developed in the early 198Os,
based on electron microscopic evidence that the appearance of intramembrane
particles, thought to be the
morphological signature of water channels, paralleled
vasopressin-stimulated water permeability (for review,
see Refs. 8, 41, 44, 132, 140). More recent measurements of water permeability in endosomes formed in
the presence of vasopressin support the notion that
vasopressin-stimulated
water permeability
is regulated by the apical membrane insertion and retrieval of

INVITED

functional

water channels (42, 43, 67, 108, 112, 136,

151).
The membrane shuttle hypothesis was confirmed
directly after the cloning of WCH-CD. Several laboratories have now demonstrated the vasopressin-dependent redistribution
of WCH-CD protein in fixed sections of kidney medulla by immunofluorescence and
immunogold electron microscopy (22, 85a, 86, 103).
However, a number of interesting questions about
WCH-CD trafficking
mechanisms remain to be answered. WCH-CD appeared to be localized to a novel
nonacidic or mildly acidic endosomal compartment
after endocytic retrieval from the apical plasma membrane (15, 68, 105, 143). The molecular factors that
target WCH-CD water channels to this compartment
are not known, and the ability of internalized WCH-CD
protein to reenter the apical membrane has not been
demonstrated. A similar recycling mechanism for the
glucose transporter GLUT-4 has been shown in muscle,
where it appears that the signal for GLUT-4 membrane
targeting is encoded in the carboxy-terminal
sequence
(15a). Furth er studies are required to determine the
epitope(s) that target wild-type and mutant WCH-CD
proteins.
An interesting cell culture model of vasopressinstimulated water permeability was developed recently
(59), which may be useful to st*udy the questions posed
above. LLC-PKi cells were stably transfected with
WCH-CD. In the absence of vasopressin, immunofluorescence of transfected cells showed an intracellular
membrane-staining pattern for WCH-CD; after addition of vasopressin or forskolin, WCH-CD was present
in a plasma membrane pattern. Cell osmotic water
permeability was also increased after vasopressin treatment. In contrast, LLC-PK1 cells stably transfected
with CHIP28 had constitutively high water permeability and showed a plasma membrane staining pattern
that did not change with vasopressin treatment. It is
remarkable that LLC-PK1 cells possess the complex
machinery to permit vasopressin-dependent WCH-CD
targeting.
PHYSIOLOGICAL

ROLE

OF

WATER

CHANNELS

Water movement across cell membranes occurs passively in response to osmotic gradients that are produced by primary and secondary active transport of
ions and neutral solutes. Most cell membranes probably have adequate water permeability through membrane lipids to support volume regulation and other
housekeeping functions. Certain cell plasma membranes, such as those in secretory and absorptive
epithelial and endothelial cells, may require a high or
regulated water permeability to facilitate the vectorial
transport of fluids across cell layers, often in response
to very small osmotic gradients. There is remarkable
axial heterogeneity in water permeability along the
mammalian nephron (44, 61, 131). High water permeability in proximal tubule is required for the nearisosmotic reabsorption of glomerular filtrate. The renal
concentrating mechanism relies on high water permeability in the thin descending limb of Henle and vasa

REVIEW

C25

recta, low water permeability in the ascending limb of

Henle, and vasopressin-regulated water permeability
in the collecting duct. Vectorial fluid transport also
occurs in extrarenal tissues that secrete fluid into or
absorb fluid from closed compartments (e.g., cerebrospinal space, aqueous humor in the anterior chamber of
eye) and open compartments (e.g., secretory and absorptive epithelia in the respiratory, gastrointestinal,
and
glandular tissues).
The data in Table 2 indicate a wide distribution of
water channel proteins in mammalian tissues. These
results suggest but do not prove a physiological role for
water channels in some tissues. A physiologically important water channel should confer increased cellular
water permeability
and, when absent or mutated,
should result in abnormal physiology. Both criteria are
satisfied for the WCH-CD protein (18). However,
CHIP28 confers high water permeability in erythrocytes and various kidney tubule segments (78), but its
absence in erythrocytes
of rare individuals
is not
associated with clinically apparent disease (97). Similar information is not available for other mammalian
water channels. Transgenic knockout animal models
should be useful to resolve questions about the physiological importance of water channels.
There is an increasing body of data on water permeability in extrarenal tissues. Vesicle studies have shown
low water permeability in isolated apical membranes
from trachea (145), small intestine (20), stomach (98),
and urinary bladder (11). However, these studies must
be viewed cautiously because of heterogeneity in vesicle
preparations and loss of factor(s) during preparation,
which may confer high membrane water permeability.
Few measurements of water permeability
in intact
tissues have been reported. In the in situ perfused
sheep lung, osmotic water transport was measured in
response to an imposed osmotic gradient (37). Lungs
were perfused continuously with an isosmotic dilute
blood solution. Hypertonic fluid (900 mosM) was instilled bronchoscopically into the air spaces, and the
time course of water movement from capillary to air
space was followed from the dilution of instilled radiolabeled albumin and from the osmolality of air space fluid
samples. In control lungs, osmotically induced water
movement was rapid (equilibration half time -45 s)
and had an apparent water permeability similar to that
in erythrocytes. Water permeability in the contralatera1 lung was inhibited reversibly by -70% by HgCIZ.
The results indicated that mercurial-sensitive
water
channels participated in the transcellular movement of
water between the air space and capillary compartments in lung. High osmotic water permeability in lung
may be important for air space fluid replacement to
offset insensible respiratory losses and for reabsorption
of fluid in alveolar edema and in the neonatal lung. In
intact small airways, water permeability was measured recently using a perfusion technique utilized
initially
for studies in kidney tubules (36). Airway
water permeability was relatively high, consistent with
the expression of MIWC in the airway basolateral cell
membrane.

C26

INVITED

SUMMARY

AND

PERSPECTIVE

The understanding
of water-transporting
mechanisms has advanced dramatically
over the past four
years, yet major questions
remain. Why are several
related water channel homologues
expressed
in the
same or different tissues, each with apparently
similar
function? Are the water channel family proteins (other
than the vasopressin-sensitive
water channel) important in normal physiology
and clinical disease? New
measurement
methods and cell and animal models will
be needed to address these questions.
Although
the
vesicle-shuttling
mechanism
is well established
for
vasopressin-regulated
water permeability,
the molecular targeting
mechanisms
remain unknown.
At a time
when gene therapy
is being actively
evaluated
for
hematopoietic
diseases and cystic fibrosis, the possibility of gene replacement
in some forms of ND1 will be
considered. Finally, high-resolution
structural
information is needed to visualize the aqueous pathway traversing molecular water channels; resolution to ~2.5 A will
require X-ray crystallography
on suitable three-dimensional water channel crystals. The availability
of structural data might permit the development
of novel water
channel inhibitors
(aquaretics)
for in vivo therapy of
human disease associated with abnormalities
in fluid
homeostasis.
NOTE

ADDED

Two structural

IN

PROOF

studies

(B. K. Jap and H. Li. J. Mol. Biol.

P. Agre,
and A. Engel. %t. Struct. Biol. 2: 730-732, 1995) in addition
to that cited (Ref. 82) have presented similar projection maps
generated
from two-dimensional
crystals of partially
glycosylated CHIP28 preserved in glucose. The genomic cloning of
AQP-3 indicated a different gene structure from other family
members (N. Inase, K. Fushimi, K. Ishibashi,
S. Uchida, M.
Ichioka, S. Sasaki, and F. Marumo. J. Biol. Chem. 270: 1791317916, 1995). Two studies have identified
possible components involved in AQP-2 trafficking:
a VAMP-2 like protein (S.
Nielsen, D. Marples, 1111.
Mohtashami,
N. 0. Dalby, W. Trimble,
and M. Knepper. J. Clin. Invest. 96: 1834-1844,
1995) and
synaptobrevin
(I. H. Jo, H. W. Harris, A. M. Amendtraduege,
R. R. Majewski,
and T. G. Hammond.
Proc. Natl. Acad. Sci.
USA92: 1876-1880,1995).

251: 413-420, 1995; T. Walz, D. Typke, B. L. Smith,

We thank
Drs. Michael
Gropper,
Michael
Matthay,
Michael
Wiener, Lan-bo
Shi, Baxoue
Yang, and Fuminori
Umenishi
for critical
reading
of this manuscript.
This work was supported
by National
Institutes
of Health
Grants
DK-35124,
HL-42368,
HL-65654,
and DK-43840
and by grants
from
the National
Cystic Fibrosis Foundation
and American
Heart Association.
Address
for reprint
requests:
A. S. Verkman,
1246 Health
Sciences
East Tower, Cardiovascular
Research
Institute,
Univ. of California,
San Francisco,
CA 94143-0521
(E-mail:
verkman@itsa.ucsf.edu).
REFERENCES
1. Abrami,
L., M. Simon,
G. Rousselet,
V. Berthonaud,
J. M.
Buhler,
and P. Ripoche.
Sequence
and functional
expression
of an amphibial
water channel,
FA-CHIP:
a new member
of the
MIP family. Biochim.
Biophys.
Acta 1192: 147-151,1994.
2. Agre, P., G. M. Preston,
B. L. Smith,
J. S. Jung,
S. Raina,
C.
Moon,
W. B. Guggino,
and S. Nielsen.
Aquaporin
CHIP: the
archetypal
molecular
water channel. Am. J. Physiol.
265 (RenaZ
Fluid Electrolyte
Physiol.
34): F463-F476,
1993.

REVIEW
3. Baker,
M. E., and M. H. Saier. A common
ancestor
for bovine
lens fiber major intrinsic
protein,
soybean
nodulin-26
protein,
and E. coZi glycerol facilitator.
Cell 60: 185-186,
1990.
4. Barry,
P. H., and J. M. Diamond.
Effects of unstirred
layers
on membrane
phenomena.
Physiol. Rev. 64: 763-872,1984.
5. Bicknese,
S., J. Park,
D. Zimet,
A. N. van Hoek,
S. B.
Shohet,
and A. S. Verkman.
Detection
of water proximity
to
tryptophan
residues
in proteins
by single photon radioluminescence. Biophys.
Chem. 54: 279-290,1995.
5a.Bicknese,
S., N. Periasamy,
S. B. Shohet,
and A. S. Verkman.
Cytoplasmic
viscosity
near the cell plasma
membrane:
measurement
by evanescent
field frequency-domain
microfluorimetry.
Biophys.
J. 65: 1272-1282,
1993.
6. Bicknese,
S., Z. Shahrohk,
S. B. Shohet,
and A. S. Verkman. Single photon radioluminescence.
I. Theory
and spectroscopic properties.
Biophys.
J. 63: 1256-1266,1992.
7. Bondy,
C., E. Chin, B. L. Smith,
G. M. Preston,
and P.Agre.
Developmental
gene expression
and tissue distribution
of the
CHIP28
water-channel
protein.
Proc. NatZ. Acad. Sci. USA 90:
4500-4504,1993.
8. Brown,
D. Membrane
recycling
and epithelial
cell function.
Am. J. Physiol.
256 (Renal
Fluid
Electrolyte
PhysioZ.
25):
Fl-F12,1989.
9. Brown,
D., J.-M.
Verbavatz,
G. Valenti,
B. Lui,
and I.
Sabolic.
Localization
of CHIP28
water
channel
in the absorptive segments
of the rat male reproductive
tract. Eur. J. CeZZ
BioZ. 61: 264-273,
1993.
10. Chandy,
G., M. Kreman,
D. L. Laidlaw,
G. A. Zampigi,
and
J. E. Hall. The water permeability
per molecule
of MIP is less
than that of CHIP (Abstract).
Biophys.
J. 68: A353,1995.
11. Chang,
A., T. G. Hammond,
T. T. Sun, and M. L. Zeidel.
Permeability
properties
of the mammalian
bladder
apical membrane.
Am. J. Physiol.
275 (CeZZ Physiol.
44): C1483-C1492,
1994.
12. Chen,
P-Y., D. Pearce,
and A. S. Verkman.
Membrane
water
and solute
permeability
determined
quantitatively
by selfquenching
of an entrapped
fluorophore.
Biochemistry
27: 57135719,1988.
13. Chou,
C.-L., J. M. Sands,
H. Nonoguchi,
and M. A. Knepper. Urea gradient-associated
fluid absorption
with uurea = 1 in
rat terminal
collecting
duct. Am. J. Physiol.
258 (RenaZ Fluid
Electrolyte
Physiol.
27): F1173-F1180,
1990.
14. Chrispeels,
M. J., and C. Maurel.
Aquaporins-the
molecular basis of facilitated
water
movement
through
living
plant
cells. PZant Physiol.
105: 9-13, 1994.
15. Coleman,
R. A., and J. B. Wade. Role of nonacidic
endosomes
in recycling
of ADH-sensitive
water channel.
Eur. J. Cell BioZ.
58: 44-56,1992.
-iF
laa.Corvera,
S., A. Chawla,
R. Chakrabarti,
M. Joly, J. Buxton, and M. P. Czech.
A double
leucine
within
the GLUT4
glucose
transporter
COOH-terminal
domain
functions
as an
endocytosis
signal. J. Cell BioZ. 126: 979-989,
1994.
16. Daniels,
M. J., T. E. Mirkov,
and M. J. Chrispeels.
The
plasma membrane
of Arabidopsis
thaZiana
contains
a mercuryinsensitive
aquaporin
that is a homolog
of the tonoplast
water
channel protein
TIP Plant Physiol.
106: 1325-1333,
1994.
17. Deen,
P. M. T., J. A. Dempster,
B. Wieringa,
and C. H. van
OS. Isolation
of a cDNA for rat CHIP28
water
channel:
high
mRNA expression
in kidney
cortex and inner medulla.
Biochim.
Biophys.
Res. Commun.
188: 1267-1273,1992.
18. Deen,
P. M., M. A. Verkijk,
N. V. Knoers,
B. Wieringa,
L. A.
Monnens,
C. H. van OS, and B. A. van Oost. Requirement
of
human
renal
water
channel
aquaporin-2
for vasopressindependent
concentration
of urine. Science Wash. DC 264: 9295, 1994.
19. Deen,
P. M. T., D. 0. Weghuis,
A. G. van
Kessel,
B.
Wierigna,
and C. H. van
OS. The human
gene for water
channel
CHIP28
is localized
on chromosome
7~14-15.
Cell
Genet. 65: 243-246,1993.
20. Dempster,
J. A., A. N. van Hoek,
M. D. de Jong,
and C. H.
van OS. Glucose transporters
do not serve as water channels
in
renal and intestinal
epithelia.
PfZuegers
Arch. 419: 249-255,
1991.
21. Denker,
B. M., B. L. Smith,
F. P. Kuhajda,
and P. Agre.
Identification,
purification,
and partial
characterization
of a

INVITED

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

novel &$. 28,000 integral

membrane
protein
from erythrocytes
and renal tubules.
J. Biol. Chem. 263: 15634-15642,1988.
DiGiovanni,
S. R., S. Nielsen,
E. I. Christensen,
and M. A.
Knepper.
Regulation
of collecting
duct water channel
expression by vasopressin
in Brattleboro
rat. Proc. Natl. Acad. Sci.
USA 91: 8984-8988,1994.
Echevarria,
M., and A. S. Verkman.
Optical
measurement
of
osmotic water transport
in cultured
cells: evaluation
of the role
of glucose transporters.
J. Gen. Physiol.
99: 573-589,
1992.
Echevarria,
M., E. E. Windhager,
S. S. Tate, and G. Frindt.
Cloning
and expression
of AQP3,
a water
channel
from the
medullary
collecting
duct of rat kidney.
Proc. Natl. Acad. Sci.
USA 91: 10997-11001,1994.
Ehring,
G. R., G.A. Zamphigi,
and J. E. Hall. Does MIP play
a role in cell-cell communication?
In: Gap Junctions,
Progress
in
Cell Research,
edited by J. E. Hall, G. A. Zamphigi,
and R. M.
Davis. Amsterdam:
Elsevier,
1993, vol. III, p.143-152.
Ehring,
G. R., G. A. Zamphigi,
J. Horowitz,
D. Bok, and
J. E. Hall. Properties
of channels
reconstituted
from the major
intrinsic
protein
of lens fiber membranes.
J. Gen. Physiol.
96:
631-664,199O.
Farinas,
J., V. Simenak,
and A. S. Verkman.
Cell volume
measured
in adherent
cells by total internal
reflection
microfluorimetry:
application
to permeability
in cells transfected
with
water channel homologs.
Biophys.
J. 68: 1613-1620,1995.
Farinas,
J., A. N. van Hoek,
L.-B. Shi, C. Erickson,
and
A. S. Verkman.
Non-polar
environment
of tryptophans
in
erythrocyte
water channel
CHIP28
determined
by fluorescence
quenching.
Biochemistry
32: 11857-11864,1993.
Farinas,
J., and A. S. Verkman.
Fluorescence
depolarization
of fluorescein-labeled
erythrocyte
water
channel
CHIP28
(Abstract). Biophys.
J. 66: A166, 1994.
Farinas,
J., and A. S. Verkman.
Laser interferometric
measurement
of cell volume:
determination
of epithelial
cell membrane water permeability
(Abstract).
Biophys.
J. In press.
Finkelstein,
A. Water Movement
Through
Lipid
BiZayers,
Pores, and Plasma Membranes:
Theory and Reality.
New York:
Wiley, 1987.
Fischbarg,
J., K. Kunyan,
J. Hirsch,
S. Lecuona,
L.
Rogozinski,
S. Silverstein,
and J. Loike.
Evidence
that the
glucose transporter
serves as a water
channel
in 5774 macrophages. Proc. NatZ. Acad. Sci. USA 86: 8397-8401,1989.
Fischbarg,
J., K. Kunyan,
J. C. Vera,
S. Arant,
S. Silverstein,
J. Loike,
and 0. M. Rosen.
Glucose transporters
serve
as water channels.
Proc. NatZ. Acad. Sci. USA 87: 3244-3247,
1990.
Fischbarg,
J., J. Li, K. Kuang,
M. Echevarria,
and P.
Iserovich.
Determination
of volume
and water permeability
of
plated
cells from measurements
of light
scattering.
Am. J.
Physiol.
265 (Cell Physiol.
34): C1412-C1423,
1993.
Flamion,
B., and K. R. Spring.
Water permeability
of apical
and basolateral
cell membranes
of rat inner medullary
collecting duct. Am. J. Physiol.
259 (Renal FZuid Electrolyte
Physiol.
28): F986-F999,1990.
Folkesson,
H., M. Matthay,
A. Frigeri,
and A. S. Verkman.
High transcellular
water permeability
in microperfused
distal
airways:
evidence
for channel-mediated
water
transport.
J.
CZin. Invest. In press.
Folkesson,
H. G., M. A. Matthay,
H. Hasegawa,
F. Kheradmand,
and A. S. Verkman.
Transcellular
water transport
in
lung alveolar
epithelium
through
mercurial-sensitive
water
channels.
Proc. NatZ. Acad. Sci. USA 91: 4970-4974,
1994.
Frigeri,
A., M. Gropper,
D. Brown,
and A. S. Verkman.
Localization
of MIWC
and GLIP water
channel
homologs
in
neuromuscular,
epithelial
and glandular
tissues.
J. Cell Sci.
108: 2993-3002,1995.
Frigeri,
A., M. Gropper,
C. W. Turck,
and A. S. Verkman.
Immunolocalization
of the mercurial-insensitive
water channel
and glycerol
intrinsic
protein
in epithelial
cell plasma
membranes. Proc. NatZ. Acad. Sci. USA 92: 4328-4331,1995.
Fushimi,
K., S. Uchida,
Y. Hara,
Y. Hirata,
F. Marumo,
and
S. Sasaki.
Cloning
and expression
of apical membrane
water
channel
of rat kidney
collecting
tubule.
Nature
Lond.
361:
549-552,1993.

REVIEW

C27

41. Handler,
J. S. Antidiuretic
hormone
moves membranes.
Am. J.
Physiol.
255 (Renal Fluid Electrolyte
Physiol.
24): F375-F382,
1988.
42. Harris,
H. W., B. Botelho,
M. L. Zeidel,
and K. Strange.
Cytoplasmic
dilution
induces antidiuretic
hormone
water channel retrieval
in toad urinary
bladder. Am. J. Physiol.
263 (Renal
Fluid Electrolyte
Physiol.
32): F163-F170,
1992.
43. Harris,
H. W., J. S. Handler,
and R. Blumenthal.
Apical
membrane
vesicles
of ADH-stimulated
toad bladder
are highly
water
permeable.
Am. J. Physiol.
258 (Renal FZuid EZectroZyte
Physiol.
27): F237-F243,
1990.
44. Harris,
H. W., K. Strange,
and M. Zeidel.
Current
understanding of the cellular
biology
and molecular
structure
of the
antidiuretic
hormone-stimulated
water
transport
pathway.
J.
CZin. Invest. 91: 1-8, 1991.
45. Hasegawa,
H., S. C. Lian,
W. E. Finkbeiner,
and A. S.
Verkman.
Extrarenal
tissue
distribution
of CHIP28
water
channels
by in situ hybridization
and antibody
staining.
Am. J.
Physiol.
266 (Cell Physiol.
35): C893-C903,
1994.
46. Hasegawa,
H., T. Ma, W. Skach,
M. Matthay,
and A. S.
Verkman.
Molecular
cloning
of a mercurial-insensitive
water
channel
expressed
in selected
water
transporting
tissues.
J.
BioZ. Chem. 269: 5497-5500,1994.
47. Hasegawa,
H., W. Skach,
0. Baker,
M. C. Calayag,
V.
Lingappa,
and A. S. Verkman.
A multi-functional
aqueous
channel
formed
by CFTR. Science Wash. DC 258: 1477-1479,
1992.
48. Hasegawa,
H., R. Zhang,A.
Dohrman,
and A. S. Verkman.
Tissue-specific
expression
of mRNA
encoding
the rat kidney
water channel CHIP28k
by in situ hybridization.
Am. J. PhysioZ.
264 (Cell PhysioZ. 33): C237-C245,
1993.
49. Hayashi,
M., S. Sasaki,
H. Tsuganezawa,
T. Monkawa,
W.
Kitajima,
K. Konishi,
K. Fushimi,
F. Marumo,
and T.
Saruta.
Expression
and distribution
of aquaporin
of collecting
duct are regulated
by vasopressin
V-2 receptor
in rat kidney.
J.
CZin. Invest. 94: 1778-1783,1994.
50. Hernandez,
J. A., and J. Fischbarg.
Kinetic
analysis
of
water transport
through
a single-file
pore. J. Gen. PhysioZ. 99:
645-662,1992.
51. Hernandez,
J. A., and J. Fischbarg.
The independence
principle
in the processes
of water
transport.
Biophys.
J. 67:
1464-1472,1994.
52. Hill, A. E. Osmotic
flow in membrane
pores of molecular
size. J.
Membr.
BioZ. 137: 197-203,1994.
53. Ishibashi,
K., S. Sasaki,
K. Fushimi,
S. Uchida,
M. Kuwahara,
H. Saito,
T. Furukawa,
K. Nakajima,
Y. Yamaguchi,
T. Gojobori,
and F. Marumo.
Molecular
cloning
and expression of a member
of the aquaporin
family with permeability
to
glycerol
and urea in addition
to water expressed
at the basolatera1 membrane
of kidney
collecting
duct cells. Proc. NatZ. Acad.
Sci. USA 91: 6269-6273,1994.
54. Jiang,
C., W. E. Finkbeiner,
J. H. Widdicombe,
P. B.
McCray,
Jr., and S. S. Miller.
Altered
fluid transport
across
airway
epithelium
in cystic fibrosis.
Science
Wash. DC 262:
424-427,1993.
55. Johnson,
K. D., H. Hofte,
and M. J. Chrispeels.
An intrinsic
tonoplast
protein
of protein storage vacuoles in seeds is structurally related
to a bacterial
solute transporter
(GlpF).
Plant Cell
2: 525-532,199O.
56. Jung,
J. S., R. V. Bhat, G. M. Preston,
W. B. Guggino,
J. M.
Baraban,
and
P. Agre.
Molecular
characterization
of an
aquaporin
cDNA from brain: candidate
osmoreceptor
and regulation of water balance.
Proc. NatZ. Acad. Sci. USA 91: 1305213056,1994.
57. Jung,
J. S., G. M. Preston,
B. L. Smith,
W. B. Guggino,
and
P. Agre.
Molecular
structure
of the water
channel
through
aquaporin
CHIP.
The hourglass
model. J. BioZ. Chem.
269:
14648-14654,1994.
58. Kao, H. P., and A. S. Verkman.
Tracking
of single fluorescent
particles
in three dimensions:
use of cylindrical
optics to encode
particle
position.
Biophys.
J. 67: 1291-1300,
1994.
59. Katsura,
T., J.-M.
Verbavatz,
J. Farinas,
T. Ma, D. A.
Ausiello,
A. S. Verkman,
and D. Brown.
Constitutive
and
regulated
membrane
expression
of aquaporin-CHIP
and aqua-

C28

60.

61.
62.

63.

64.

65.

66.

67.

68.

69.

70.

71.
72.

73.

74.

75.

76.

77.
78.

79.

INVITED
porin-2
water
channels
in stably
transfected
LLC-PKi
cells.
Proc. Natl. Acad. Sci. USA. 92: 7212-7216,1995.
Keen, T. L., C. F. Inglehearn,
R. J. Patel,
E. D. Green,
D. C.
Peluso,
and S. S. Bhattacharya.
Localization
of the aquaporin 1 (AQPl)
gene within
a YAC containing
the polymorphic
markers
D7S632 and D7S526.
Genomics
25: 599-600,1995.
Knepper,
M. A. The aquaporin
family
of molecular
water
channels.
Proc. Natl. Acad. Sci. USA 91: 6255-6258,1994.
Kuwahara,
M., C. A. Berry,
and A. S. Verkman.
Rapid
development
of vasopressin-induced
hydroosmosis
in kidney
collecting
tubules
measured
by a new fluorescence
technique.
Biophys.
J. 54: 595-602,1988.
Kuwahara,
M., L.-B. Shi, F. Marumo,
and A. S. Verkman.
Transcellular
water
flow modulates
water
channel
exocytosis
and endocytosis
in kidney
collecting
tubule. J. CZin. Invest.
88:
423-429,199l.
Kuwahara,
M., and A. S. Verkman.
Direct
fluorescence
measurement
of diffusional
water permeability
in the vasopressin-sensitive
kidney
collecting
tubule. Biophys.
J. 54: 587-593,
1988.
Kuwahara,
M., and A. S. Verkman.
Pre-steady-state
analysis
of the regulation
of water permeability
in the kidney
collecting
tubule. J. Membr.
Biol. 110: 57-65,
1989.
Lee, J. W., C. D. Weaver,
N. H. Shomer,
C. F. Louis,
and
D. M. Roberts.
Nodulin
26 ion channel
activity:
effect of
phosphorylation
and mutagenesis
of Ser 262 (Abstract).
Biophys.
J. 6:A147,
1995.
Lencer,
W., D. Brown,
D. A. Ausiello,
and A. S. Verkman.
Endocytosis
of water channels
in rat kidney:
cell specificity
and
correlation
with in vivo antidiuretic
states. Am. J. Physiol.
259
(CeZZ Physiol.
28): C920-C932,
1990.
Lencer,
W. I., A. S. Verkman,
D. A. Ausiello,
A. Arnaout,
and D. Brown.
Endocytic
vesicles
from renal papilla
which
retrieve
the vasopressin-sensitive
water channel do not contain
an H+ ATPase. J. CeZZ BioZ. 111: 379-389,199O.
Lencer,
W. I., P. Weyer,
A. S. Verkman,
D. A. Ausiello,
and
D. Brown.
FITC-dextran
as a probe for endosome
function
and
localization
in kidney.
Am. J. Physiol.
258 (CeZZ Physiol.
27):
c309-c317,1990.
Levitt,
D. G., and H. J. Mlekoday.
Reflection
coefficient
and
permeability
of urea and ethylene
glycol in the human
red cell
membrane.
J. Gen. Physiol.
81: 239-254,1983.
Li, X., H. Yu, and S. S. Koide.
The water
channel
gene in
human uterus. Biochem.
Mol. Biol. Int. 32: 371-377,1994.
Ma, T., A. Frigeri,
H. Hasegawa,
and A. S. Verkman.
Cloning
of a water channel
homolog
expressed
in brain meningeal cells and kidney collecting
duct that functions
as a stilbenesensitive
glycerol
transporter.
J. BioZ. Chem.
269: 2184521849,1994.
Ma, T., A. Frigeri,
W. Skach,
and A. S. Verkman.
Cloning
of a
cDNA from rat kidney
with homology
to CHIP28
and WCH-CD
water
channels.
Biochim.
Biophys.
Res. Commun.
197: 654659,1993.
Ma, T., A. Frigeri,
S.-T. Tsai, J.-M.
Verbavatz,
and A. S.
Verkman.
Localization
and functional
analysis
of CHIP28k
water channels
in stably transfected
CHO cells. J. BioZ. Chem.
268: 22756-22764,1993.
Ma, T., H. Hasegawa,
W. Skach,
A. Frigeri,
and A. S.
Verkman.
Expression,
functional
analysis
and in situ hybridization
of a cloned rat kidney
collecting
duct water
channel.
Am. J. Physiol.
266 (Cell Physiol.
35): C189-C197,
1994.
Ma, T., B. Yang, and A. S. Verkman.
Molecular
cloning
of a
human water channel homolog
expressed
exclusively
in kidney:
evidence
for a gene cluster
of MIP family
members
at chromosome locus 12q13 (Abstract).
J. Am. Sot. Nephrol.
6: 325,1995.
Macey,
R. I. Transport
of water
and urea in red blood cells.
Am. J. Physiol.
246 (Cell Physiol.
15): C195C203,
1984.
Maeda,
Y., B. L. Smith,
P. Agre,
and M. A. Knepper.
Quantification
of aquaporin-CHIP
water
channel
protein
in
microdissected
renal tubules
by fluorescence-based
ELISA.
9.
CZin. Invest. 95: 422-428,
1995.
Maurel,
C., J. Reizer,
I. Schroeder,
and M. J. Chrispeels.
The vacuolar
membrane
protein
y-TIP
creates water
specific
channels
inxenopus
oocytes. EMBO
J. 12: 2241-2247,1993.

REVIEW
80. Maurel,
C., J. Reizer,
I. Schroeder,
M. J. Chrispeels,
and
M. H. Saier.
Functional
characteristics
of Escherichia
coli
glycerol
facilitator
GlpF in Xenopus
oocytes. J. BioZ. Chem. 269:
11869-11872,1994.
81. Meyer,
M. M., and A. S. Verkman.
Evidence
for water
channels
in proximal
tubule
cell membranes.
J. Membr.
BioZ.
96: 107-119,1987.
82. Mitra,A.,A.
N. van Hoek,
M. C. Wiener,A.
S. Verkman,
and
M. Yaeger.
The CHIP28
water
channel
visualized
in ice by
electron
cryo-crystallography.
Nature
Struct.
BioZ. 2: 726-729,
1995.
83. Mitra,
A. K., M. Yaeger,
A. N. van Hoek,
M. C. Wiener,
and
A. S. Verkman.
Projection
structure
of the CHIP28
water
channel
at 12 angstrom
resolution.
Biochemistry
33: 1273512740,1994.
84. Moon,
C., G. M. Preston,
C. A. Griffin,
E. W. Jabs, and P.
Agre.
The human
aquaporin-CHIP
gene: structure,
organization and chromosomal
localization.
J. BioZ. Chem. 268: 1577215778,1993.
85. Muallem,
S., R. Zhang,
P. A. Loessberg,
and R. A. Star.
Simultaneous
recording
of cell volume changes intracellular
pH
or Ca2+ concentration
in single osteosarcoma
cells UMR- 106-01.
J. Biol. Chem. 267: 17658-17664,1992.
85a. Nielsen,
S., C. L. Chou, D. Marples,
E. I. Christensen,
B. K.
Kishore,
and M. A. Knepper.
Vasopressin
increases
water
permeability
of kidney
collecting
duct by inducting
translocation of aquaporin-CD
water
channels
to plasma
membrane.
Proc. Natl. Acad. Sci. USA 92: 1013-1017,1995.
86. Nielsen,
S., S. R. Digiovani,
E. I. Christensen,
M. A.
Knepper,
and H. W. Harris.
Cellular
and subcellular
immunolocalization
of vasopressin-regulated
water channel
in rat kidney. Proc. Natl. Acad. Sci. USA 90: 11663-11667,1993.
87. Nielsen,
S., B. L. Smith,
E. I. Christensen,
and P. Agre.
Distribution
of the aquaporin
CHIP in secretory
and resorptive
epithelia
and capillary
endothelia.
Proc. Natl. Acad. Sci. USA
90: 7275-7279,1993.
88. Nielsen,
S., B. L. Smith,
E. I. Christensen,
M. A. Knepper,
and P. Agre.
CHIP28
water channels
are localized
in constitutively water-permeable
segments
of the nephron.
J. Cell BioZ.
120: 371-383,1993.
89. Pao, G. M., L.-F.
Wu, K. D. Johnson,
H. Hofte,
M. J.
Chrispeels,
G. Sweet,
N. N. Sandal,
and M. H. Saier.
Evolution
of the MIP family
of integral
membrane
transport
of
proteins.
Mol. Microbial.
5: 33-37,
1991.
90. Parisi,
M., M. Pisam,
G. Calamita,
R. Gobin,
R. Toriano,
and J. Bouquet.
Water
pathways
across a reconstituted
epithelial
barrier
formed
by Caco-2
cells: effects of medium
hypertonicity.
J. Membr.
BioZ. 143: 237-245,
1995.
91. Pearce,
D., and A. S. Verkman.
NaCl reflection
coefficients
in
proximal
tubule
apical
and basolateral
membrane
vesicles:
measurement
by induced osmosis and solvent drag. Biophys.
J.
55: 1251-1259,1989.
92. Pisano,
M. M., and A. B. Chepelinsky.
Genomic
cloning,
complete
nucleotide
sequence,
and structure
of the human
gene
encoding the major intrinsic
protein
(MIP) of the lens. Genomics
11: 981-990,199l.
93. Preston,
G. M., and P. Agre.
Isolation
of the cDNA
for
erythrocyte
integral
membrane
protein
of 28 kilodaltons:
member of an ancient channel
family. Proc. NatZ. Acad. Sci. USA 88:
llllO-11114,199l.
94. Preston,
G. M., T. P. Carroll,
W. B. Guggino,
and P. Agre.
Appearance
of water
channels
in Xenopus
oocytes expressing
red cell CHIP28
protein.
Science Wash. DC 256: 385-387,
1992.
95. Preston,
G. M., J. S. Jung,
W. B. Guggino,
and P. Agre. The
mercury-sensitive
residue
at cysteine
189 in the CHIP28
water
channel.
J. Biol. Chem. 268: 17-20,
1993.
96. Preston,
G. M., J. S. Jung,
W. B. Guggino,
and P. Agre.
Membrane
topology
of aquaporin
CHIP-analysis
of function
epitope-scanning
mutants
by vectorial
proteolysis.
J. BioZ.
Chem. 269: 1668-1673,1994.
97. Preston,
G. M., B. L. Smith,
M. L. Zeidel,
J. J. Moulds,
and
P. Agre.
Mutations
in aquaporin-1
in phenotypically
normal
human
without
functional
CHIP water channels.
Science Wash.
DC 265: 1585-1587,1994.

INVITED
98. Priver,
N. A., E. C. Rabon,
and M. L. Zeidel.
Apical
membrane of the gastric parietal
cell: water, proton,
and nonelectrolyte permeabilities.
Biochemistry
32: 2459-2468,
1993.
99. Raina,
S., G. M. Preston,
W. B. Guggino,
and P. Agre.
Molecular
cloning
and characterization
of an aquaporin
cDNA
from salivary,
lacrimal,
and respiratory
tissues.
J. BioZ. @hem.
270: 1908-1912,1995.
100. Rao, Y., L. Y. Jan, and Y. N. Jan. Similarity
of the product
the
DrosophiZa
neurogenic
gene big brain to transmembrane
channel proteins.
Nature
Lond. 345: 163-167,
1990.
101. Reizer,
J., A. Reizer,
and M. H. Saier.
The MIP family
of
integral
membrane
channel
proteins:
sequence
comparisons,
evolutional
relationships,
reconstructed
pathway
of evolution,
and proposed
functional
differentiation
of two repeated
halves
of the protein.
Crit. Rev. Biochem.
Mol. Biol. 28: 235-257,
1993.
102. Roberts,
S. K., M. Yano, Y. Ueno,
L. Pham,
G. Alpini,
P.
Agre,
and N. F. LaRusso.
Cholangiocytes
express the aquaporin CHIP and transport
water via a channel-mediated
mechanism. Proc. NatZ. Acad. Sci. USA 91: 13009-13013,1994.
103. Sabolic,
I., T. Katsura,
J.-M. Verbavatz,
and D. Brown.
The
AQP2 water channel:
effect of vasopressin
treatment,
microtubule disruption,
and distribution
in neonatal
rats. J. Membr.
Biol. 143: 165-175,
1995.
104. Sabolic,
I., G. Valenti,
J.-M.
Verbavatz,
A. N. van Hoek,
A. S. Verkman,
D.A. Ausiello,
and D. Brown.
Localization
of
the CHIP28
water
channel
in rat kidney.
Am. J. Physiol.
263
(CeZZ Physiol.
32): Cl225-C1233,
1992.
105. Sabolic,
I., F. Wuarin,
L.-B.
Shi, A. S. Verkman,
D. A.
Ausiello,
S. Gluck,
and D. Brown.
Apical
endosomes
from
collecting
duct principal
cells lack subunits
of the proton
pumpingATPase.
J. CeZZ Biol. 119: 111-122,1992.
106. Sasaki,
S., K. Fushimi,
H. Saito,
F. Saito,
S. Uchida,
K.
Ishibashi,
M. Kuwahara,
T. Ikeuchi,
K. Inui, K. Nakajima,
T. X. Watanabe,
and F. Marumo.
Cloning,
characterization,
and chromosomal
mapping
of human
aquaporin
of collecting
duct. J. Clin. Invest. 93: 1250-1256,
1994.
107. Shahrohk,
Z., S. Bicknese,
S. B. Shohet,
and A. S. Verkman. Single photon radioluminescence.
II. Experimental
detection and biological
applications.
Biophys.
J. 63: 1267-1279,
1992.
108. Shi, L.-B., D. Brown,
and A. S. Verkman.
Water,
urea and
proton
transport
properties
of endosomes
containing
the vasopressin-sensitive
water
channel
from
toad bladder.
J. Gen.
Physiol.
95: 941-960,
1990.
109. Shi, L.-B.,
K. Fushimi,
and A. S. Verkman.
Solvent
drag
measurement
of transcellular
and basolateral
membrane
NaCl
reflection
coefficient
in mammalian
proximal
tubule.
J. Gen.
Physiol.
98: 379-398,
1991.
110. Shi, L. B., W. R. Skach,
T. Ma, and A. S. Verkman.
Distinct
biogenesis
mechanisms
for water channels
MIWC
and CHIP28
at the endoplasmic
reticulum.
Biochemistry.
34: 8250-8256,
1995.
111. Shi, L.-B.,
W. R. Skach,
and A. S. Verkman.
Functional
independence
of monomeric
CHIP28
water
channels
revealed
by expression
of wild type-mutant
heterodimers.
J. Biol. Chem.
269: 10417-10422,1994.
112. Shi, L.-B., and A. S. Verkman.
Very high water permeability
in vasopressin-dependent
endocytic
vesicles
in toad urinary
bladder.
J. Gen. Physiol.
94: 1101-1115,
1989.
113. Shi, L.-B., and A. S. Verkman.
Selected
cysteine
point mutations confer mercurial
sensitivity
to the mercurial-insensitive
water channel MIWUAQP-4.
Biochemistry.
In press.
114. Skach,
W. R., L. B. Shi, M. C. Calayag,
A. Frigeri,
V. R.
Lingappa,
and A. S. Verkman.
Topology
and biogenesis
of the
CHIP28
water
channel
at the endoplasmic
reticulum.
J. Cell
BioZ. 125: 803-816,
1994.
115. Skach,
W. R., and A. S. Verkman.
Topological
maturation
of
aquaporin
CHIP
at the endoplasmic
reticulum
(Abstract).
Biophys.
J. 68: A344,1995.
116. Smith,
B. L., and P. Agre.
Erythrocyte
Mr 28,000 transmembrane
protein
exists
as a multisubunit
oligomer
similar
to
channel proteins.
J. BioZ. Chem. 266: 6407-6415,199l.
117. Smith,
B. L., G. M. Preston,
F. A. Spring,
D. J. Anstee,
and
P. Agre.
Human
red cell aquaporin
CHIP
I. Molecular
charac-

REVIEW

118.

c29
terization
of ABH and Colton
blood
Invest. 94: 1043-1049,1994.
Stamer,
W. D., R. W. Snyder,
B. L.
Regan.
Localization
of aquaporin
implications
in the pathogenesis
of
ders of ocular fluid balance.
Invest.

group

antigens.

J. Clin.

Smith,
P.Agre,
and J. W.
CHIP
in the human
eye:
glaucoma
and other disorOpthamol.
Visual Sci. 35:

3867-3872,1994.
119.

120.

Tsai, S.-T., R. Zhang,

and A. S. Verkman.
High channelmediated
water permeability
in rabbit
erythrocytes:
characterization
in native
cells and expression
in Xenopus
oocytes.
Biochemistry
30: 2087-2092,
1991.
Uchida,
S., S. Sasaki,
K. Fushimi,
and F. Marumo.
Isolation
of human
aquaporin-CD
gene. J. BioZ. Chem.
269: 23451-

23455,1994.
121.

Valenti,
Verkman,
related
parietal

G., J.-M. Verbavatz,

I. Sabolic,
D. A. Ausiello,
A. S.
and
D. Brown.
A basolateral
CHIP28/MIP26protein
(BLIP)
in kidney
principal
cells and gastric
cells. Am. J. Physiol.
267 (Cell Physiol.
36): C812-

122.

Van Hoek,
A. N., M. L. Horn, L. H. Luthjens,
M. D. de Jong,
J. A. Dempster,
and C. H. van OS. Functional
unit of 30 kDa
for proximal
tubule
water
channels
as revealed
by radiation
inactivation.
J. Biol. Chem. 226: 16633-16635,
1991.
Van Hoek,
A. N., L. H. Luthjens,
M. L. Horn,
C. H. van OS,
and J. A. Demster.
A 30 kDa functional
size for the erythrocyte
water
channel
determined
in situ by radiation
inactivation.
Biochem.
Biophys.
Res. Commun.
184: 1331-1335,1992.
Van Hoek,
A. N., and A. S. Verkman.
Functional
reconstitution of the isolated
erythrocyte
water channel
CHIP28.
J. Biol.
Chem.267:
18267-18269,1992.
Van Hoek,
A. N., M. C. Wiener,
S. Bicknese,
L. Miercke,
J.
Biwersi,
and A. S. Verkman.
Secondary
structure
analysis
of
purified
CHIP28
water channels
by CD and FTIR spectroscopy.
Biochemistry
32: 11847-11856,
1993.
Van Hoek,
A. N., M. C. Wiener,
J.-M. Verbavatz,
D. Brown,
R. R. Townsend,
P. H. Lipniunas,
and A. S. Verkman.
Purification
and structure-function
analysis
of PNGase
F- and
endo-p-galactosidase-treated
CHIP28
water channels.
Biochemistry 34: 2212-2219,1995.
Van Lieburg,
A. F., M. A. Verdijk,
V. V. Knoers,
A. J. Van
Essen,
W. Proesmans,
R. Mallmann,
L. A. Monnens,
B. A.
van
Oost,
C. H. van OS, and P. M. Deen.
Patients
with
autosomal
nephrogenic
diabetes
insipidus
homozygous
for mutations
in the aquaporin
2 water
channel
gene. Am. J. Hum.
Genet. 55: 648-652,1994.
Van OS, C. H., P. M. T. Deen,
and J. A. Dempster.
Aquaporins-water
selective
channels
in biological
membranesmolecular
structure
and tissue distribution.
Biochim.
Biophys.
ActaRev.Biomembr.
1197:291-309,1994.
Verbavatz,
J.-M.,
D. Brown,
I. Sabolic,
G. Valenti,
D. A.
Ausiello,
A. N. van
Hoek,
T. Ma,
and A. S. Verkman.
Tetrameric
assembly
of CHIP28
water
channels
in liposomes
and cell membranes.
A freeze-fracture
study. J. Cell Biol. 123:

C82OJ994.

123.

124.

125.

126.

127.

128.

129.

605-618,1993.
130.

131.

Verbavatz,
J.-M.,
A. N. van Hoek,
T. Ma, I. Sabolic,
G.
Valenti,
M. Ellisman,
D.A. Ausiello,
A. S. Verkman,
and D.
Brown.
A 28 kD sarcolemmal
antigen
in kidney
principal
cell
basolateral
membranes:
relationship
to orthogonal
arrays
and
MIP26.
J. CeZZSci. 107:1083-1094,1994.
Verkman,
A. S. Mechanisms
and regulation
of water permeability in renal epithelia.
Am. J. Physiol.
257 (Cell Physiol.
26):

C837-C850,1989.
132. Verkman,
A. S. Water channels
in cell membranes.
Annu. Rev.
Physiol.
54: 97-108,1992.
133. Verkman,
A. S. Water ChanneZs.
Austin,
TX: Landes,
1993.
134. Verkman,
A. S. Molecular
biophysics
of kidney water channels.

135.

In: Molecular
Biology
of the Kidney
in Health
and Disease,
edited by D. Schlondorff
and J. V. Bonventre.
New York: Dekker,
1995, p. 459-468.
Verkman,
A. S., J. A. Dix, and J. L. Seifter.
Water and urea
transport
in renal
microvillus
membrane
vesicles.
Am. J.
Physiol.
248 (Renal Fluid Electrolyte
Physiol.
17): F650-F655,

1985.

c30
136.

137.

138.

139.

140.

141.

142.

143.

144.

145.

146.

INVITED
Verkman,
A. S., W. Lencer,
D. Brown,
and D. A. Ausiello.
Endosomes
from kidney
collecting
tubule contain the vasopressin-sensitive
water channel.
Nature
Lond. 333: 268-269,
1988.
Verkman,
A. S., A. N. van Hoek,
and R. Zhang.
Identification
and molecular
cloning
of water
transporting
proteins.
In: Isotonic Dansport
in Leaky
Epithelia,
edited by H. H. Ussing,
J.
Fischbarg,
0. Sten-Knudsen,
E. H. Larsen,
N. J. Willumson,
and J. H. Thaysen.
Copenhagen:
Munksgaard,
1993, p. 388395 (Alfred Benzon
Symp. 34).
Verkman,
A. S., P. Weyer,
D. Brown,
and D. A. Ausiello.
Functional
water channels are present in clathrin
coated vesicles
from bovine
kidney
but not from brain.
J. Biol.
Chem. 264:
20608-20613,1989.
Verkman,
A. S., and K. Wong.
Proton
NMR measurement
of
diffusional
water
permeability
in suspended
renal
proximal
tubule. Biophys.
J. 51: 717-723,1987.
Verkman,
A. S., R. Zhang,
Y.-X. Wang,
and L.-B. Shi. The
vasopressin
sensitive
water channel
in toad bladder:
functional
localization
in endosomes
and mRNA
expression
in Xenopus
oocytes.
In: Vasopressin,
edited
by S. Jard and R. Jamison.
Paris: Libbey Eurotext,
1991, vol. 208, p. 85-93.
Walz, T., B. L. Smith,
P. Agre,
and A. Engel.
The threedimensional
structure
of human
erythrocyte
aquaporin
CHIP
EMBO
J. 13: 2985-2993,1994.
Walz, T., B. L. Smith,
M. L. Zeidel,
A. Engel,
and P. Agre.
Biologically
active 2-dimensional
crystals
of aquaporin
CHIP. J.
Biol. Chem. 269: 1383-1386,1994.
Wang, Y.-X., L.-B. Shi, and A. S. Verkman.
Functional
water
channels
and proton
pumps
are in separate
populations
of
endocytic
vesicles from toad bladder
granular
cells. Biochemistry 30: 2888-2894,199l.
Wistow,
G. J., M. M. Pisano,
and A. B. Chepelinski.
Tandem
sequence
repeats
in transmembrane
channel
proteins.
fiends
Biochem.
Sci. 16: 170-171,199l.
Worman,
H. J., T.A. Brasitus,
P. K. Dudeja,
H.A. Fozzard,
and M. Field.
Relationship
between
lipid fluidity
and water
permeability
of bovine
tracheal
epithelial
cell apical
membranes. Biochemistry
25: 1549-1555,1986.
Yang,
B., T. Ma, and A. S. Verkman.
cDNA cloning,
gene
organization
and chromosomal
localization
of a human
mercu-

REVIEW

147.

148.

149.

150.

151.

152.

153.

154.

155.

156.

rial-insensitive
water channel:
evidence
for distinct
transcriptional units. J. BioZ. Chem. 270: 22907-22913,
1995.
Ye, R., L.-B. Shi, W. Lencer,
and A. S. Verkman.
Functional
colocalization
of water channels
and proton pumps on endocytic
vesicles from proximal
tubule. J. Gen. Physiol.
93: 885-902,1989.
Ye, R., and A. S. Verkman.
Osmotic
and diffusional
water
permeability
measured
simultaneously
in cells and liposomes.
Biochemistry
28: 824-829,1989.
Zampighi,
G. A., J. E. Hall, G. R. Ehring,
and S. A. Simon.
The structural
organization
and protein
composition
of lens
fiber junctions.
J. Cell BioZ. 108: 2255-2275,
1989.
Zeidel,
M. L., S. V. Ambudkar,
B. L. Smith,
and P. Agre.
Reconstitution
of functional
water channels in liposomes
containing purified
red cell CHIP28
protein.
Biochemistry
31: 74367440,1992.
Zeidel,
M. L., T. G. Hammond,
J. B. Wade, J. Tucker,
and
H. W. Harris.
Fate of antidiuretic
hormone
water
channel
proteins
after retrieval
from apical membrane.
Am. J. Physiol.
265 (CeZZ Physiol.
34): C822-C833,
1993.
Zhang,
R., S. Alper,
B. Thorens,
and A. S. Verkman.
Evidence
from oocyte expression
that the erythrocyte
water
channel
is distinct
from band 3 and the glucose transporter.
J.
CZin. Invest. 88: 1553-1558,199l.
Zhang,
R., K. Logee,
and A. S. Verkman.
Expression
of
mRNA coding for kidney
and red cell water channels
in Xenopus
oocytes. J. BioZ. Chem. 265: 15375-15378,199O.
Zhang,
R., W. Skach,
H. Hasegawa,
A. N. van Hoek,
and
A. S. Verkman.
Cloning,
functional
analysis
and cell localization of a kidney
proximal
tubule water transporter
homologous
to CHIP28.
J. Cell BioZ. 120: 359-369,
1993.
Zhang,
R., A. N. van Hoek,
J. Biwersi,
and A. S. Verkman.
A point mutation
at cysteine
189 blocks the water permeability
of rat kidney
water channel
CHIP28k.
Biochemistry
32: 29382941,1993.
Zhang,
R., and A. S. Verkman.
Water and urea transport
in
Xenopus
oocytes: expression
of mRNA from toad urinary
bladder. Am. J. Phwiol.
260 (Cell Phvsiol.
29): C26-C34,
1991.