Reviews
MOLECULAR MEDICINE TODAY, FEBRUARY 2000 (VOL. 6)
Aquaporins in health and disease
Landon S. King, Masato Yasui and Peter Agre
The molecular basis of membrane water-permeability remained elusive until the recent discovery of the
aquaporin water-channel proteins. The fundamental importance of these proteins is suggested by their
conservation from bacteria through plants to mammals. Ten mammalian aquaporins have thus far been
identified, each with a distinct distribution. In the kidney, lung, eye and brain, multiple water-channel
homologs are expressed, providing a network for water transport in those locations. It is increasingly
clear that alterations in aquaporin expression or function can be rate-limiting for water transport across
certain membranes. Aquaporins are likely to prove central to the pathophysiology of a variety of clinical
conditions from diabetes insipidus to various forms of edema and, ultimately, they could be a target for
therapy in diseases of altered water homeostasis.
APPROPRIATE regulation of membrane water-permeability is a
fundamental requirement of all living organisms. In contrast to the
prevailing view of simple diffusion through a lipid bilayer, studies
performed over several decades have predicted the existence of a
water-specific channel protein in certain membranes1. The molecular
identity of such a channel protein remained elusive until the early
1990s. Investigation of the Rh protein of the erythrocyte membrane led
to the serendipitous identification of a novel 28 kDa protein2. As described below, this protein, now called Aquaporin-1 (AQP1), proved to
be the first molecular water channel3. Discovery of the aquaporin family of water-channel proteins has provided new insights into the molecular mechanisms of transcellular water movement. It is becoming
apparent that aquaporin biology will prove relevant to the pathophysiology and perhaps even therapy of a wide array of conditions.
The archetype: function and structure of aquaporin-1
Several features of AQP1 (initially called ‘CHIP 28’) suggested that it
could be the long-sought water-channel protein4. Hydropathy analysis
of the AQP1 cDNA predicted six membrane-spanning domains charLandon S. King MD*
Assistant Professor of Medicine
Masato Yasui
Postdoctoral Fellow
Peter Agre MD
Professor of Biological Chemistry and Medicine
Johns Hopkins University School of Medicine, 600 North Wolfe
Street, Blalock 910, Baltimore, MD 21287, USA.
Tel: 11 410 955 3467
Fax: 11 410 955 0036
*e-mail: [email protected]
60
acteristic of a channel protein. The size (28 kDa) and distribution
(abundant in erythrocytes and the renal proximal tubule) of AQP1 were
both similar to those of the physiologically defined water channel.
Finally, the number of copies of AQP1 in erythrocytes (~23105 per
cell) was similar to that predicted for the water channel. Expression of
AQP1 in Xenopus laevis oocytes allowed definitive identification.
When placed in hypotonic media, AQP1-expressing oocytes rapidly
swell and rupture, but control oocytes exhibit little change in volume3.
Further studies in oocytes as well as in proteoliposomes reconstituted
with purified AQP1 protein demonstrated that the channel was specifically permeable to water, and was not permeated by other small molecules including protons, ions, urea and glycerol5. Recent data suggest
that AQP1 is also permeable to CO2 (Ref. 6), but the magnitude of this
permeability is much lower than that for water; the functional relevance of this observation is unclear.
The structure of AQP1 is unique7. As noted, the AQP1 monomer consists of six transmembrane domains, with N- and C-termini that are
intracellular (Fig. 1). The first and second halves of the molecule have a
high degree of internal homology, but are oriented to opposite sides of
the lipid bilayer. A three amino acid motif of Asn-Pro-Ala (NPA) is found
in both the N- and C-terminal halves of the molecule, a feature common
to all members of the aquaporin family. A cysteine in position 189, adjacent to the carboxy-NPA motif, confers sensitivity to mercurial compounds, as described for the physiologically defined water channel8.
Mutation of residues surrounding the NPA motif in either half of the molecule reduces water permeability, strongly suggesting that these regions
contribute to formation of the aqueous pore9. From the above observations, an ‘hourglass’ model for the structure of the AQP1 monomer was
proposed. Loops B and E (Fig. 1) dip into the plane of the lipid bilayer,
and the N- and C-terminal halves of AQP1 fold together to form the aqueous pore.
Reconstitution of purified AQP1 from erythrocytes into proteoliposomes has allowed analysis of their crystal structure. Electron diffraction of cryopreserved specimens at tilts of up to 608 (3–6 Å resolution) demonstrates six bilayer-spanning domains and an intrasubunit
structure highly consistent with the proposed hourglass topology10, a
1357-4310/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S1357-4310(99)01636-6
Reviews
MOLECULAR MEDICINE TODAY, FEBRUARY 2000 (VOL. 6)
(a)
Outside
E
P
N A
Aquaporins: the growing family
Aquaporins have now been identified in all levels of life as far back as
prokaryotes. The first bacterial homolog, aqpZ, was identified in
Escherichia coli, and is upregulated by, and confers a survival advantage
in, hypotonic conditions13. Saccharomyces contains two aquaporin
genes, at least one of which forms a functional water channel14. As
might be predicted by their dependence on local environmental water,
plants express numerous aquaporins15. Arabidopsis thaliana contains at
least 23 aquaporin homologs in the expressed sequence tag library.
Two functional groups of mammalian aquaporins are now being
recognized16. The first, including AQP0, AQP1, AQP2, AQP4 and
AQP5, are permeable only to water, as classically defined. A second
group, including AQP3, AQP7 and AQP9, are highly permeable to
water, but are also permeated by glycerol and other small molecules.
Phylogenetically, this apparent dichotomy could have arisen from bacteria, which contain both a water channel gene (aqpZ in E. coli) and a
glycerol transporter (glpF in E. coli). The structural explanation and
physiological relevance of these differences is not known. Although
the sequence of AQP6 is similar to the water-selective group, the recent
surprising observations of gated anion conductance indicates the functional distinction is more complex than previously thought17. The sequence of AQP8 is intermediate between the water-selective and the
glycerol permeant groups, and functional definition is awaited18.
C
C
N A
P
B
Membrane
A
Inside
D
H2N
HOOC
C
(b)
A
Outside
E
N
A
P
P
B
A
N
C
Membrane
structure also confirmed by others11,12. Atomic resolution of the structure
and detailed analysis of the aqueous pore await further investigation.
Crystallographic studies also confirm the tetrameric arrangement of
AQP1 in membranes as previously described. The requirement for
tetramerization is not yet understood, but presumably results from instability of the asymmetric AQP1 monomer in a lipid environment.
D
Inside
H2N
Physiology and pathology of the aquaporins
Ten mammalian aquaporins have been identified to date, each with a
distinct tissue distribution. Multiple regulatory mechanisms are likely
to be identified that will explain the specificity of ontogeny, distribution and function of each aquaporin. With the exception of AQP2,
these regulatory mechanisms are poorly understood. Below, we describe aquaporin expression in several organs, with speculation about
their physiological roles.
Kidney
Of the 180 L of glomerular filtrate produced each day, 80–90% is reabsorbed in the proximal tubule and the descending thin limb of Henle’s
loop – segments known to have constitutively high water permeability.
The ascending thin and thick limbs of Henle’s loop, the distal convoluted
tubule and the connecting tubule are known to be largely impermeable
to water. The remaining 10–20% of the glomerular filtrate is reabsorbed
in a vasopressin-dependent fashion in the collecting duct. The water
permeability of the individual nephron segments correlates closely with
aquaporin expression within each segment19 (Fig. 2).
AQP1 is abundant in the proximal tubule and the descending thin
limb of the nephron, where it constitutes approximately 4% of the
brush-border protein20. AQP1 is present in reduced amounts on the
basolateral membrane, and there is no intracellular pool. The density
and unit water conductance of AQP1 are sufficient to account for the
known water permeability of the proximal tubule and the descending
thin limb21. The renal medulla vascular supply, the vasa recta, is critical to the generation of an axial osmotic gradient in the medulla.
Nielsen and colleagues recently demonstrated both mercury-
HOOC
Molecular Medicine Today
Figure 1. Hourglass model of aquaporin-1 (AQP1) topology. (a) The six transmembrane domains of the AQP1 monomer are represented, as well as the
extracellular loops A, C, and E, and intracellular loops B and D. The two NPA
motifs are located in loops B and E, which fold into the lipid bilayer. Cysteine
189 (C) is the residue that confers mercury-sensitivity to AQP1. (b) The NPA
motifs in loops B and E come together to form the aqueous channel.
inhibitable water permeability and the presence of AQP1 in endothelial
cells of the descending vasa recta, suggesting that AQP1 mediates
water transport in the vasa recta22.
Discovery of the Colton blood-group antigen on AQP1 allowed the
subsequent identification of rare Colton-null individuals who lack AQP1;
surprisingly, these individuals have no obvious clinical phenotype 23. In
contrast, targeted gene disruption of the Aqp1 gene in mice revealed a
marked urinary concentrating defect24. These studies add credence to
speculation that rare AQP1-null humans have compensated water transport defects that are subclinical under non-stressed conditions.
AQP2 is located in the principal cells of the collecting duct. Under
resting conditions, AQP2 is found primarily in intracellular vesicles
beneath the apical membrane. In response to arginine vasopressin
(AVP) binding to the V2 receptor at the basolateral membrane, Ser256
of the AQP2 C-terminus is phosphorylated25. Vesicle-associated pro61
Reviews
Proximal tubule
(AQP1 and AQP6)
MOLECULAR MEDICINE TODAY, FEBRUARY 2000 (VOL. 6)
Intercalated cell
where they probably provide an exit pathway
for fluid reabsorbed in the collecting duct.
Disruption of the Aqp4 gene in mice resulted
in a mild urinary concentrating defect37.
AQP3 transports glycerol as well as water; but
the functional significance of this observation
is unknown.
Respiratory tract
Fluid requirements in the respiratory tract are
complex. In the distal lung, removal of fluid in
the perinatal period is critical in the transition
from placental gas exchange to ex utero life.
Principal cell
Descending thin limb
Throughout life, appropriate handling of water
(AQP1)
in the vascular, interstitial and airspace comCollecting duct
partments of the lung is essential for normal
Aquaporins
(AQP2, AQP3,
gas exchange and lung defense. In the airways,
AQP1
AQP4
AQP4 and AQP6)
strict regulation of the airway surface liquid
AQP2
AQP6
AQP3
layer is required for effective mucociliary
Molecular Medicine Today
transport. In both the airways and nasopharynx, inspired air must be humidified to prevent
Figure 2. A single nephron showing aquaporin distribution. AQP1 is expressed in both the apical and basodrying of the distal airways, and water must be
lateral membrane of cells in the proximal tubule and descending thin limb, both highly water permeable.
extracted from the expired air stream to miniAQP6 is present in intracellular vesicles in proximal tubule epithelial cells, as well as in the acid secreting
mize breath-to-breath water loss.
intercalated cells of the collecting duct. AQP2 is the collecting duct water-channel that translocates from
Four water channels have been identified in
cytoplasmic vesicles to the apical membrane in the presence of vasopressin. AQP3 and AQP4 are present
in the basolateral membrane of principal cells of the collecting duct.
the respiratory tract and their distribution is
complex38,39 (Fig. 3). In the rat, AQP1 is abundant in the apical and basolateral membrane
of the microvasculature and visceral pleura.
teins including vesicle-associated membrane protein-2 (VAMP-2) AQP5 is expressed in the apical membrane of both type I pneumocytes
have been colocalized with AQP2 and might facilitate targeting of the and secretory cells in airway submucosal glands. AQP3 and AQP4 are
AQP2-containing vesicles to the apical membrane26, where they could expressed in the basolateral membrane of different cells in airway and
interact with the targeting receptor syntaxin-4 (Ref. 27). AQP2 re- nasopharyngeal epithelium. This non-overlapping distribution of aquadistribution to the apical membrane has been closely correlated with a porins might provide a coordinated network for transcellular water
dramatic increase in membrane water permeability.
movement in the respiratory tract. The absence of known water chanIn contrast to genetically AQP1-deficient humans, AQP2 deficiency nels in the apical membrane of the airway epithelium and the basolatproduces a dramatic clinical phenotype. Nephrogenic diabetes insipidus eral membrane of type I pneumocytes is, however, provocative.
(NDI) is a disease whose etiology is renal resistance to AVP, and whose Clearly, undiscovered aquaporins might exist in those locations.
clinical hallmark is excretion of large volumes of dilute urine. Deen and Alternatively, transcellular water movement might not occur at every
colleagues have now described multiple patients with autosomal recessive point across the respiratory epithelium, but instead take place only at
NDI who have mutations in the AQP2 gene28, providing the first clear ex- select sites.
The phenomenon of perinatal lung water-clearance is well deample that aquaporins can be rate-limiting for water transport. Acquired
NDI is more common than the congenital form, and has a variety of scribed. In that context, the ontogeny of aquaporins in the lung is of
causes. Nielsen and colleagues recently demonstrated that lithium29, bi- considerable interest. AQP1 is expressed in fetal rat lung late in geslateral ureteral obstruction30 and chronic hypokalemia31 – known causes tation, increases dramatically at birth, and is sustained at high levels
of NDI – all produce marked reductions in AQP2 expression in animals, in adult animals39. Corticosteroids induce AQP1 expression in fetal
with a concomitant decrease in urinary concentrating ability. At the other (and adult) rat lung, consistent with known acceleration of fetal lung
end of the water imbalance spectrum, increased AQP2 expression has maturation by corticosteroids. AQP5 is expressed one to two days
been demonstrated in conditions of fluid retention including congestive after birth in rat lung, with high levels of expression in adult aniheart failure32,33, cirrhosis34 and pregnancy35. As we gain insight into mals40. In contrast to AQP1, AQP5 is not induced by corticosteroids.
mechanisms regulating its function, AQP2 could prove to be a therapeutic AQP4 exhibits transient high level expression in distal lung two days
target in numerous conditions of altered fluid balance.
after birth. Although these observations predict participation of water
Other aquaporins are also present in the nephron. AQP6 was re- channels in perinatal lung water-clearance, their precise roles in this
cently identified as the first intracellular water channel. AQP6 is ex- process and in the pathophysiology of the premature lung remain to be
pressed in vesicles in epithelial cells of the proximal tubule and in determined.
intercalated cells of the collecting duct36. The unique characteristics of
The distribution of aquaporins in the respiratory tract suggests inAQP6 predict functional differences from other water channels, in- volvement in a variety of conditions. In the distal lung, altered exprescluding regulation of acid–base balance17. AQP3 and AQP4 are ex- sion or function of AQP1 and AQP5 might play a role in the pathopressed in the basolateral membrane of the collecting duct epithelium, genesis of pulmonary edema and pleural effusions. A functional role for
62
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MOLECULAR MEDICINE TODAY, FEBRUARY 2000 (VOL. 6)
Figure 3. Aquaporin expression in the respiratory tract. (a) AQP1 is expressed in the
apical and basolateral membrane of endothelial cells. AQP5 is present in the apical
membrane of type I pneumocytes. (b) In tracheal epithelium, AQP3 is present in the
basal cells, and AQP4 is present in the basolateral membrane of the ciliated cells. No
aquaporins have been identified in the apical membrane or in the goblet cells. AQP1
is expressed in endothelial cells of the vascular plexus beneath the airway. (c) In secretory glands of the nasopharyngeal epithelium, AQP5 is present in the apical membrane of secretory cells, whereas AQP3 and AQP4 are expressed in the basolateral
membrane of the same cells.
AQP1 in the distal lung is supported by recent investigation in Aqp1
knockout mice41. When compared with control animals, Aqp1-null
mice demonstrated a tenfold reduction in osmotic water permeability,
and a twofold decrease in hydrostatic permeability. Additionally, perfusion
of isolated distal airways has demonstrated water-channel-mediated
transport across the distal airway epithelium42. Water channel expression
in the epithelium, subepithelial vasculature and subepithelial glands of
the airways and nasopharynx (Fig. 3) predicts participation in airway humidification, as well as generation and regulation of the airway surface
liquid38. Alterations in the airway surface liquid layer are central to the
pulmonary manifestations of cystic fibrosis, and might play a role
in some forms of asthma. As with the kidney, it is increasingly
likely that aquaporins will participate in multiple aspects of lung water
homeostasis.
(a) Alveolar airspace
Type II
pneumocyte
H2O
Type I
pneumocyte
Basement
membrane
Capillary
(b) Respiratory airspace
Goblet cell
Ciliated
cell
Brain
The physical constraints of the bony cranium demand tight regulation of
intracranial fluid. Cerebrospinal fluid is made by the choroid plexus, a
specialized structure located in the walls of the lateral, third and fourth
ventricles consisting of a vascular core covered by a secretory epithelium. Immunolocalization studies demonstrate that AQP1 is abundant
in apical membrane microvilli of the choroid epithelium; it is expressed
there from early gestation in the fetal rat. Colocalization of AQP1 with a
Na–K ATPase in the apical membrane strongly suggests a role for the
water channel in cerebrospinal fluid production1.
AQP4 was cloned from both lung43 and brain44 cDNA libraries, although the brain appears to be its predominant distribution. AQP4 is expressed in the foot processes of astroglial cells, specifically in the perivascular membrane, suggesting that it plays a role in the regulation of
extravascular brain water45. Additionally, AQP4 is abundant in the lamellae adjacent to magnocellular cells in the supraoptic and paraventricular
nuclei, the site of arginine vasopressin production. From there, AVP is
then transported along axons to the posterior pituitary, where it can subsequently be released. These AQP4-containing lamellar structures might
participate in the sensation and/or the transduction of osmotic signals to
the magnocellular cells. Recent studies have demonstrated a marked reduction in AQP4-mediated water permeability by phorbol diesters46.
Phosphorylation-mediated gating or trafficking of water channels would
allow rapid regulation of intracerebral membrane water permeability.
Basal
cells
Basement
membrane
H2O
Fibroblasts
Capillary
(c) Airway gland
Eye
Five aquaporins have been identified in non-overlapping domains in the
eye47 (Fig. 4). MIP (AQP0) comprises approximately half of the lensfiber-cell protein, and has recently been shown to function as a low capacity water channel. Two different mutations of the Mip gene in mice
lead to congenital cataracts48. AQP1 in the corneal endothelium and in
the lens anterior epithelium, as well as AQP5 in the corneal epithelium,
might participate in reducing the water content of those tissues, an important feature for maintaining transparency of the cornea and the lens.
Basement
membrane
H2O
Aquaporins
AQP1
AQP3
AQP4
AQP5
Molecular Medicine Today
63
Reviews
MOLECULAR MEDICINE TODAY, FEBRUARY 2000 (VOL. 6)
Glossary
The outstanding questions
Intercalated cells – Renal collecting duct epithelial cells that participate in acid secretion (in contrast to collecting duct principal cells,
which reabsorb water).
structural features of the aquaporin molecules dictate
• What
the specificity of their permeability?
aquaporins always open and permeable when inserted
• Are
in the membrane, or can they be gated?
is aquaporin expression regulated and, in organs with
• How
more than one aquaporin, is this regulation coordinated?
aquaporin expression or function be altered to affect
• Can
pathophysiologic processes?
role do aquaporins play in epithelia or endothelia that
• What
are thought to have high paracellular permeability?
Proteoliposomes – Vesicles made in vitro by mixing different
combinations of lipid and proteins.
Targeting receptor – A membrane protein that interacts with
vesicle-associated proteins to direct trafficking of vesicles to the
plasma membrane.
Vesicle-associated proteins – One of a small group of integral
membrane-proteins found in intracellular vesicles, which facilitate
specificity of docking and fusion with the plasma membrane.
Expression of AQP1 in the anterior ciliary epithelium and canals of
Schlemm suggests a role in secretion and uptake of aqueous humor.
AQP1 is also present in the iris, where high water permeability is
thought to facilitate the rapid shape changes that occur with pupillary
constriction. The presence of AQP4 in the end-feet of Müller cells in the
retina suggests that it plays a role in the light-dependent hydration of the
space around photoreceptors49. Finally, AQP3 in the bulbar conjunctiva
might play a role in the hydration of the protective covering of the eye.
Erythrocytes
Erythrocytes were the source for the initial identification and purification of AQP1 (Ref. 50). Although water channels are thought to
facilitate erythrocyte survival during the transit through the hypertonic
renal medulla, AQP1-deficient humans are not anemic and have evi-
AQP5
Lacrimal gland
Corneal epithelium
Salivary and lacrimal glands
AQP5 was cloned from a salivary gland library, and is similar to the
other aquaporins in water transport capacity52. AQP5 is abundantly expressed in the apical membrane of secretory cells in the salivary and
lacrimal glands, but is not present in the basolateral membrane or in
duct cells38. AQP5 has a protein kinase A consensus in a cytoplasmic
loop, similar to that of AQP2. Phosphorylation of the protein is an appealing, though as yet unproven, explanation for the rapid onset of salivation and lacrimation in response to the appropriate stimuli.
Adenoviral-mediated transfer of the AQP5 gene is being evaluated as
a potential therapy for damaged salivary glands53.
Curiously, the distribution of AQP5 coincides almost exactly with
the organ involvement of Sjögren’s disease, an immunologically mediated process causing dry eyes, dry mouth and desiccation of the tracheobronchial secretions. The antigen(s) driving immune destruction of
the involved organs is unknown, but it is compelling to consider that
either primary or secondary dysfunction of AQP5 could be involved.
Concluding remarks
AQP1
Lens epithelium
Corneal endothelium
Nonpigmented epithelium
(ciliary and iris)
Trabecular meshwork
MIP (AQP0)
Lens fiber cells
AQP3
Conjunctiva
dence of only low-grade hemolysis. A combination of washout of the
medullary interstitial gradient and low-level expression of AQP3 in
erythrocytes51 could partially explain the surprisingly normal erythrocyte survival in AQP1-null humans.
AQP4
Retinal glia
(Müller cells)
Discovery of the aquaporin family of membrane proteins has provided
new insights into the molecular mechanisms of membrane water-permeability. The conservation of aquaporins from prokaryotes to mammals is consistent with the central role that water plays in all forms of
life. Historically, the focus of discussions on membrane permeability
has been solute transport. However, it is increasingly clear that, under
some circumstances, membrane water permeability might be regulated
independently from solute transport, and that aquaporin expression or
function can be rate-limiting for water movement. Further investigation
of the structure, regulation and function of aquaporins should greatly
enhance our ability to both understand and manipulate membrane
water-permeability.
Molecular Medicine Today
Figure 4. Aquaporin expression in the eye. Five aquaporins have been identified in the eye, with non-overlapping distribution in different parts of the eye as
shown above.
Acknowledgments. This work was supported by the National Institutes of Health (L.K. and
P.A.), the Cystic Fibrosis Foundation (L.K. and P.A.) and the Human Frontier Science
Program (M.Y.).
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