Role of aquaporin water channels in kidney and lung

Several aquaporin-type water channels are expressed in mammalian kidney and lung: AQP1 in lung microvessels and kidney proximal tubule, thin descending limb of Henle, and vasa recta; AQP2 in apical membrane of collecting duct epithelium; AQP3 and AQP4 in basolateral membranes of airway and collecting duct epithelium; and AQP5 in alveolar epithelium. Novel quantitative fluorescence methods demonstrated very high water permeabilities of the alveolar epithelial and endothelial barriers, and moderately high water permeability across distal airways. In the kidney, water permeability is high in proximal tubule and thin descending limb of Henle, and regulated by vasopressin in collecting duct. The author's laboratory has studied the role of aquaporins in organ physiology using transgenic knockout mice lacking specific aquaporins. AQP1 null mice are mildly growth-retarded, manifest a severe urinary concentrating defect, and have reduced water permeability between airspace and capillary compartments. AQP4 null mice appear normal grossly except for a mild defect in maximum urinary concentrating ability. AQP2-deficient humans have hereditary non-X-linked nephrogenic diabetes insipidus (NDI). In transfected mammalian cells, many NDI-causing AQP2 mutants are retained in the endoplasmic reticulum. The author's laboratory has found that "chemical chaperones," that is, small compounds that promote protein folding in vitro, are able to correct defective AQP2 trafficking in cell culture models. The transgenic mouse and mammalian cell models are thus beginning to provide clues about the role of aquaporins in normal physiology and disease.     

Decreased aquaporin-2 expression and apical plasma membrane delivery in kidney collecting ducts of polyuric hypercalcemic rats

Hypercalcemia is frequently associated with a urinary concentrating defect and overt polyuria. The molecular mechanisms underlying this defect are poorly understood. Dysregulation of aquaporin-2 (AQP2), the predominant vasopressin-regulated water channel, is known to be associated with a range of congenital and acquired water balance disorders including nephrogenic diabetes insipidus and states of water retention. This study examines the effect of hypercalcemia on the expression of AQP2 in rat kidney. Rats were treated orally for 7 d with dihydrotachysterol, which produced significant hypercalcemia with a 15 +/- 2% increase in plasma calcium concentration. Immunoblotting and densitometry of membrane fractions revealed a significant decrease in AQP2 expression in kidney inner medulla of hypercalcemic rats to 45.7 +/- 6.8% (n = 11) of control levels (100 +/- 12%, n = 9). A similar reduction in AQP2 expression was seen in cortex (36.9 +/- 4.2% of control levels, n = 6). Urine production increased in parallel, from 11.3 +/- 1.4 to a maximum of 25.3 +/- 1.9 ml/d (P < 0.01), whereas urine osmolality decreased from 2007 +/- 186 mosmol/kg x H2O to 925 +/- 103 mosmol/kg x H2O (P < 0.01). Immunocytochemistry confirmed a decrease in total AQP2 labeling of collecting duct principal cells from kidneys of hypercalcemic rats, and reduced apical labeling. Immunoelectron microscopy demonstrated a significant reduction in AQP2 labeling of the apical plasma membrane, consistent with the development of polyuria. In summary, the results strongly suggest that AQP2 downregulation and reduced apical plasma membrane delivery of AQP2 play important roles in the development of polyuria in association with hypercalcemia.     

Comprehensive evaluation of long-term trends in occupational exposure: Part 1. Description of the database

The regulation of water excretion by the kidney is one of the few physiologic processes that are prominent in everyday life. This process predominantly occurs in renal collecting duct cells, where transcellular water reabsorption is induced after binding of the pituitary hormone arginine-vasopressin to its vasopressin type-2 receptor and the subsequent insertion of aquaporin-2 (AQP2) water channels in the apical membrane of these cells. Removal of the hormone triggers endocytosis of AQP2 and restores the water-impermeable state of the collecting duct cells. Nephrogenic diabetes insipidus is characterized by the inability of the kidney to concentrate urine in response to vasopressin; the vasopressin type-2 receptor and the AQP2 water channel have both been shown to be involved in this disease. This article focuses on mutations in the vasopressin V2 receptor and aquaporin-2 water channel identified in nephrogenic diabetes insipidus patients, and on the effects of these mutations on the transport and function of these proteins upon expression in cell systems.     

Selective brain cooling reduces respiratory water loss during heat stress

It was shown recently that the aquaporin family of water channels exists in lacrimal gland cells. To determine the localization of aquaporin homologs and the pathophysiological modification of aquaporins by pilocarpine, a muscarinic agonist, we performed immunohistochemistry and immunoblotting in mouse lacrimal glands. By immunohistochemistry, aquaporin-4 (AQP4) and aquaporin-5 (AQP5) were found to be the lacrimal glands. AQP5 immunolabeling was detected in the apical membranes of the acinus and duct cells, while AQP4 was expressed in the basolateral membranes only. The tear secretion of mice systemically treated with pilocarpine was significantly (2.5-fold) higher than that of the saline-treated controls. The antibody to the AQP5 carboxy terminus showed high immunoreactivity on the apical membrane in the pilocarpine-treated lacrimal glands but not on that of saline-treated controls. However, the antibody to the extracellular domain of AQP5 showed similar immunolabeling in both groups of animals. In contrast, the immunoreactivity of AQP4 was not affected by pilocarpine stimulation. As shown by western blot analysis, the expression level of AQP5 on the apical membrane in the pilocarpine-stimulated lacrimal glands was not significantly different compared with the saline-treated controls. We conclude that AQP4 and AQP5 water channels are expressed on mouse lacrimal gland cells, with greater expression of AQP4 on the basolateral membrane and of AQP5 on the apical membrane. Furthermore, the AQP5 carboxy terminus region may undergo pathophysiological modification when tear secretion is increased by pilocarpine stimulation.     

Glycosylation is not essential for vasopressin-dependent routing of aquaporin-2 in transfected Madin-Darby canine kidney cells

Glycosylation has been shown to be important for proper routing and membrane insertion of a number of proteins. In the collecting duct, aquaporin-2 (AQP2) is inserted into the apical membrane after stimulation of vasopressin type-2 receptors and retrieved into an endosomal compartment after withdrawal of vasopressin. The extent of glycosylation of AQP2 in human kidney and urine and the effects of deglycoylation on routing of AQP2 in an AQP2-transfected Madin-Darby canine kidney cell line (clone WT10) were investigated. Semiquantitative immunoblotting of human kidney membranes and urine showed an AQP2 glycosylation of 35 to 45% for medulla, papilla, and urine, with low variation among individuals. The 1-desamino-8-D-arginine vasopressin-induced transcellular osmotic water permeability (Pf) of WT10 cells by a factor of 2.6 +/- 0.2 was reduced to 1.5 +/- 0.1 after pretreatment with the glycosylation inhibitor tunicamycin. However, when WT10 cells were incubated with 8-br-cAMP, the Pf increased by a factor 2.8 +/- 0.2 and by 2.9 +/- 0.2 after prior incubation with tunicamycin. Immunoblot analyses revealed that in WT10 cells, 34% of AQP2 is glycosylated, which was reduced to 2% after tunicamycin treatment. Surface biotinylation and subsequent semiquantitative immunoblotting revealed that stimulation by cAMP increased the level of AQP2 in the apical membrane of WT10 cells 1.5-fold. independent of the presence of tunicamycin. However, in tunicamycin-treated WT10 cells, all AQP2 in the apical membrane was unglycosylated, whereas in untreated cells 30% of AQP2 in the apical membrane was glycosylated. These results prove that glycosylation has no function in the routing of AQP2 in Madin-Darby canine kidney cells.     

Freeze-fracture analysis of plasma membranes of CHO cells stably expressing aquaporins 1-5

Several studies suggest that aquaporin water channels can be identified in membranes by freeze-fracture electron microscopy. For this report, Chinese Hamster ovary cells were stably transfected with cDNAs encoding aquaporins 1-5. Measurement of the osmotic water permeability of the cells confirmed that functional protein was expressed and delivered to the plasma membrane. By freeze-fracture electron microscopy, a 20% increase in intramembrane particle (IMP) density was found in plasma membranes of cells expressing AQP2, 3 and 5, and a 100% increase was measured in AQP1-expressing cells, when compared to mock-transfected cells. On membranes of cells expressing AQP4, large aggregates of IMPs were organized into orthogonal arrays, which occupied 10-20% of the membrane surface. IMP aggregates were never seen in AQP2-transfected cells. Hexagonally packed IMP clusters were detected in approximately 5% of the membranes from AQP3-expressing cells. Particle size-distribution analysis of rotary shadowed IMPs showed a significant shift from 13. 5 (control cells) to 8.5 nm or less in AQP-expressing cells; size distribution analysis of unidirectionally shadowed IMPs also showed a significant change when compared to control. Some IMPs in AQP expressing cells had features consistent with the idea that aquaporins are assembled as tetramers. The results demonstrate that in transfected CHO cells, AQP transfection modifies the general appearance and number of IMPs on the plasma membrane, and show that only AQP4 assembles into well-defined IMP arrays.     

Select types of supporting cell in the inner ear express aquaporin-4 water channel protein

Aquaporins (AQPs) confer a high water permeability on cell membranes and play important parts in secretory and absorptive epithelia in kidney and other organs. Here we investigate whether AQPs are expressed in the sensory epithelia of the inner ear, where a precise volume regulation is crucial. By use of specific antibodies it was found that the inner ear contains AQP1 and 4 while being devoid of detectable levels of AQP2, 3 or 5. Immunofluorescence and postembedding immunogold labelling revealed a strictly non-epithelial distribution of AQP1, confirming previous data. In contrast, AQP4 protein and mRNA (visualized by in situ hybridization) were concentrated in select types of supporting cell, including Hensen's cells and inner sulcus cells. Immunogold particles signalling AQP4 were confined to the basolateral plasma membrane of Hensen's cells and to the basal plasma membrane of Claudius cells and inner sulcus cells. AQP4 was also found in supporting cells of the vestibular end organs, but was absent from transitional epithelial cells and dark cells. Strong labelling for AQP4 and AQP4-mRNA was associated with the central part of the cochlear and vestibular nerves. Hair cells were consistently unlabelled. Our findings indicate that AQP4 may facilitate osmotically driven water fluxes in the sensory epithelia of the inner ear and thus contribute to the volume and ion homeostasis at these sites.     

Upregulation of aquaporin 2 water channel expression in pregnant rats

Water retention is characteristic of pregnancy but the mechanism(s) of the altered water metabolism has yet to be elucidated. The collecting duct water channel, aquaporin 2 (AQP2), plays a pivotal role in the renal water regulation, and we hypothesized that AQP2 expression could be modified during pregnancy. Sprague-Dawley female rats were studied on days 7 (P7), 14 (P14), and 20 (P20) of pregnancy, and expression of AQP2 in papillae was examined. Nonpregnant (NP) littermates were used as controls. Plasma osmolalities were significantly lower in pregnant rats by day 7 of gestation (P7 283.8+/-1.82, P14 284.3+/-1.64, P < 0.001, P20 282. 4+/-1.32, P < 0.0001, vs. NP 291.8+/-1.06 mosmol/kgH2O). However, plasma vasopressin concentrations in pregnant rats were not significantly different than in nonpregnant rats (NP 1.03+/-0.14, P7 1.11+/-0.21, P14 1.15+/-0.21, P20 1.36+/-0.24 pg/ml, NS). The mRNA of AQP2 was increased early during pregnancy: AQP2/beta actin: P7 196+/-17.9, P14 200+/-6.8, and P20 208+/-15.5%, P < 0.005 vs. NP (100+/-11.1%). AQP2 protein was also increased during pregnancy: AQP2 protein: P7 269+/-10.0, P14 251+/-12.0, P < 0.0001, and P20 250+/-13.6%, P < 0.001 vs. NP (100+/-12.5%). The effect of V2 vasopressin receptor antagonist, OPC-31260, was then investigated. AQP2 mRNA was suppressed significantly by OPC-31260 administration to P14 rats (AQP2/beta actin: P14 with OPC-31260 39.6+/-1.7%, P < 0.001 vs. P14 with vehicle) and was decreased to the same level of expression as NP rats receiving OPC-31260. Similar findings were found with the analysis of AQP2 protein. The decreased plasma osmolality of P14 rats was not modified by OPC-31260. The results of the study indicate that upregulation of AQP2 contributes to the water retention in pregnancy through a V2 receptor-mediated effect. In addition to vasopressin, other factors may be involved in this upregulation.     

cis-Elements in the 5''-region of the Oct-1 gene. Possibilities of autoregulation

The anti-diuretic hormone vasopressin (AVP) regulates water excretion from the kidney by increasing the water permeability of the collecting duct. AVP binds to V2-receptors and induces the translocation of aquaporin-2 water channels (AQP-2) into the apical plasma membrane of principal cells. By this mechanism AVP controls water reabsorption in the kidney. The effects of AVP on the endolymphatic sac (ES) of the inner ear, which is thought to mediate reabsorption of endolymph, were investigated. Both the V2-receptor and the AQP-2 water channel were found to be expressed in the ES epithelium. In the ES AVP binds to receptors most probably of the V2-subtype. Application of AVP to organotypically cultured ES inhibits membrane turnover in ribosomal-rich cells of the ES epithelia, which is thought to mediate translocation of AQP-2 into the surface membrane. This suggests that AVP has contrasting effects in the inner ear and kidney, which may be physiologically useful for maintaining endolymphatic pressure during severe hypovolemia. Animal experiments show that AVP causes endolymphatic hydrops after systemic application to guinea-pigs, which suggests a causal role for the increased AVP levels found in humans suffering from Ménière's disease.     

Epithelial galectin-3 during human nephrogenesis and childhood cystic diseases

Galectin-3 is a beta-galactoside-binding protein with putative roles in development, oncogenesis, and inflammation. Its expression in human nephrogenesis has not been previously reported. This study examines galectin-3 expression in early human embryos by Western blot and immunohistochemistry. This 33-kD protein was detected in the apical domain of distal tubules of the mesonephros and also in the mesonephric duct. In the metanephros, the adult kidney precursor, galectin-3 was detected in the apical domains of ureteric bud branches, and there was intense expression in fetal medullary and papillary collecting ducts in both the cytoplasm and plasma membranes. Low levels of galectin-3 were detected in the cytoplasm of a subset of cells in adult collecting ducts; these were alpha-intercalated cells because they expressed basal band 3 protein. In human multicystic dysplastic kidneys, all diseased epithelia had an embryonic apical expression pattern of galectin-3 and, in addition, all cystic epithelia in autosomal recessive polycystic kidneys expressed this molecule. It is concluded that galectin-3 is expressed by cells of the mesonephric duct/ureteric bud lineage, and it is speculated that the different subcellular locations may be implicated in both the regulation of normal growth and differentiation of this lineage, as well as in the pathogenesis of cystic epithelia.     

Three-dimensional organization of a human water channel

Aquaporins (AQP) are members of the major intrinsic protein (MIP) superfamily of integral membrane proteins and facilitate water transport in various eukaryotes and prokaryotes. The archetypal aquaporin AQP1 is a partly glycosylated water-selective channel that is widely expressed in the plasma membranes of several water-permeable epithelial and endothelial cells. Here we report the three-dimensional structure of deglycosylated, human erythrocyte AQP1, determined at 7 A resolution in the membrane plane by electron crystallography of frozen-hydrated two-dimensional crystals. The structure has an inplane, intramolecular 2-fold axis of symmetry located in the hydrophobic core of the bilayer. The AQP1 monomer is composed of six membrane-spanning, tilted alpha-helices. These helices form a barrel that encloses a vestibular region leading to the water-selective channel, which is outlined by densities attributed to the functionally important NPA boxes and their bridges to the surrounding helices. The intramolecular symmetry within the AQP1 molecule represents a new motif for the topology and design of membrane protein channels, and is a simple and elegant solution to the problem of bidirectional transport across the bilayer.     

Bilateral deafness due to congenital CMV and not rubella reinfection

Two cases of autosomal recessive nephrogenic diabetes insipidus (NDI) were evaluated. Both cases were found to be compound heterozygote for missense mutations in the aquaporin-2 (AQP2) gene. To determine the structural-functional relationship, the mutated AQP2 proteins, T125M, G175R, A190T, and P262L, were expressed in Xenopus oocytes and examined by measurement of water permeability, immunoblot, and immunocytochemistry. Our results suggest that T125M and G175R are nonfunctional water channels, whereas the translocation to the plasma membrane is impaired in A190T and P262L.     

The apical Cl/HCO3 exchanger of beta intercalated cells

There are two types of intercalated cells of the renal collecting duct; one secretes H+ and the other secretes HCO3-. The H(+)-secreting form has an apical vacuolar H(+)-ATPase and a basolateral Cl/HCO3 exchanger that cross-reacts with antibodies to band 3, the product of the AE1 gene. The HCO3(-)-secreting form has a basolateral vacuolar H(+)-ATPase and an apical Cl/HCO3 exchanger, whose identity has not been established previously. Apical membrane vesicles of beta intercalated cells purified from rabbit kidney cortex contain both an electroneutral Cl/HCO3 exchange activity and polypeptides that react with antibodies to band 3 on Western blots. Furthermore, both primary cultures of HCO3(-)-secreting intercalated cells and an immortalized cell line derived from these cells express AE1 and have an apical Cl/HCO3 exchanger. Apical membranes purified from these cells contain a 100-kDa polypeptide that cross-reacts with antibody to the cytoplasmic domain of band 3. These data suggest that the apical Cl/HCO3 exchanger of HCO3(-)-secreting intercalated cells is band 3.     

Protein kinase A anchoring proteins are required for vasopressin-mediated translocation of aquaporin-2 into cell membranes of renal principal cells

The antidiuretic hormone arginine-vasopressin (AVP) regulates water reabsorption in renal collecting duct principal cells by inducing a cAMP-dependent translocation of water channels (aquaporin-2, AQP-2) from intracellular vesicles into the apical cell membranes. In subcellular fractions from primary cultured rat inner medullary collecting duct (IMCD) cells, enriched for intracellular AQP-2-bearing vesicles, catalytic protein kinase A (PKA) subunits and several protein kinase A anchoring proteins (AKAPs) were detected. In nonstimulated IMCD cells the majority of AQP-2 staining was detected intracellularly but became mainly localized within the cell membrane after stimulation with AVP or forskolin. Quantitative analysis revealed that preincubation of the cells with the synthetic peptide S-Ht31, which prevents the binding between AKAPs and regulatory subunits of PKA, strongly inhibited AQP-2 translocation in response to forskolin. Preincubation of the cells with the PKA inhibitor H89 prior to forskolin stimulation abolished AQP-2 translocation. In contrast to H89, S-Ht31 did not affect the catalytic activity of PKA. These data demonstrate that not only the activity of PKA, but also its tethering to subcellular compartments, are prerequisites for cAMP-dependent AQP-2 translocation.     

Membrane dynamics of the water transport protein aquaporin-1 in intact human red cells

Aquaporin-1 (AQP1) is the prototype integral membrane protein water channel. Although the three-dimensional structure and water transport function of the molecule have been described, the physical interactions between AQP1 and other membrane components have not been characterized. Using fluorescein isothiocyanate-anti-Co3 (FITC-anti-Co3), a reagent specific for an extracellular epitope on AQP1, the fluorescence photobleaching recovery (FPR) and fluorescence imaged microdeformation (FIMD) techniques were performed on intact human red cells. By FPR, the fractional mobility of fluorescently labeled AQP1 (F-alphaAQP1) in the undeformed red cell membrane is 66 +/- 10% and the average lateral diffusion coefficient is (3.1 +/- 0.5) x 10(-11) cm2/s. F-alphaAQP1 fractional mobility is not significantly affected by antibody-induced immobilization of the major integral proteins band 3 or glycophorin A, indicating that AQP1 does not exist as a complex with these proteins. FIMD uses pipette aspiration of individual red cells to create a constant but reversible skeletal density gradient. F-alphaAQP1 distribution, like that of lipid-anchored proteins, is not at equilibrium after microdeformation. Over time, approximately 50% of the aspirated F-alphaAQP1 molecules migrate toward the membrane portion that had been maximally dilated, the aspirated cap. Based on the kinetics of migration, the F-alphaAQP1 lateral diffusion coefficient in the membrane projection is estimated to be 6 x 10(-10) cm2/s. These results suggest that AQP1 lateral mobility is regulated in the unperturbed membrane by passive steric hindrance imposed by the spectrin-based membrane skeleton and/or by skeleton-linked membrane components, and that release of these constraints by dilatation of the skeleton allows AQP1 to diffuse much more rapidly in the plane of the membrane.     

Secondary structures comparison of aquaporin-1 and bacteriorhodopsin: a Fourier transform infrared spectroscopy study of two-dimensional membrane crystals

Aquaporins are integral membrane proteins found in diverse animal and plant tissues that mediate the permeability of plasma membranes to water molecules. Projection maps of two-dimensional crystals of aquaporin-1 (AQP1) reconstituted in lipid membranes suggested the presence of six to eight transmembrane helices in the protein. However, data from other sequence and spectroscopic analyses indicate that this protein may adopt a porin-like beta-barrel fold. In this paper, we use Fourier transform infrared spectroscopy to characterize the secondary structure of highly purified native and proteolyzed AQP1 reconstituted in membrane crystalline arrays and compare it to bacteriorhodopsin. For this analysis the fractional secondary structure contents have been determined by using several different algorithms. In addition, a neural network-based evaluation of the Fourier transform infrared spectra in terms of numbers of secondary structure segments and their interconnections [sij] has been performed. The following conclusions were reached: 1) AQP1 is a highly helical protein (42-48% alpha-helix) with little or no beta-sheet content. 2) The alpha-helices have a transmembrane orientation, but are more tilted (21 degrees or 27 degrees, depending on the considered refractive index) than the bacteriorhodopsin helices. 3) The helices in AQP1 undergo limited hydrogen/deuterium exchange and thus are not readily accessible to solvent. Our data support the AQP1 structural model derived from sequence prediction and epitope insertion experiments: AQP1 is a protein with at least six closely associated alpha-helices that span the lipid membrane.     

The in vivo balance between B cell clonal expansion and elimination is regulated by CD95 both on B cells and in their micro-environment

In congenital nephrogenic diabetes insipidus, the renal collecting ducts are resistant to the antidiuretic action of arginine vasopressin or to its antidiuretic analog 1-deamino[8-D-arginine] vasopressin (dDAVP). This is a rare, but now well described entity secondary to either mutations in the AVPR2 gene that codes for the vasopressin antidiuretic (V2) receptor or to mutations in the AQP2 gene that codes for the vasopressin-dependent water channel. A majority (> 90%) of congenital nephrogenic diabetes insipidus patients have AVPR2 mutations: Of 115 families with congenital nephrogenic diabetes insipidus, 105 families had AVPR2 mutations, and 10 had AQP2 mutations. When studied in vitro, most AVPR2 mutations lead to receptors that are trapped intracellularly and are unable to reach the plasma membrane. A minority of the mutant receptors reach the cell surface but are unable to bind vasopressin or to trigger an intracellular adenosine 3:5-cyclic phosphate signal properly. Most of the reported mutations are secondary to a complete loss of function of the receptor, and only a few mutations have been associated with a mild phenotype. These advances provide diagnostic tools for physicians caring for these patients because, when the disease causing mutation has been identified, carrier and perinatal testing could be done by mutation analysis.     

Direct immunogold labeling of aquaporin-4 in square arrays of astrocyte and ependymocyte plasma membranes in rat brain and spinal cord

Aquaporin (AQP) water channels are abundant in the brain and spinal cord, where AQP1 and AQP4 are believed to play major roles in water metabolism and osmoregulation. Immunocytochemical analysis of the brain recently revealed that AQP4 has a highly polarized distribution, with marked expression in astrocyte end-feet that surround capillaries and form the glia limitans; however, the structural organization of AQP4 has remained unknown. In freeze-fracture replicas, astrocyte end-feet contain abundant square arrays of intramembrane particles that parallel the distribution of AQP4. To determine whether astrocyte and ependymocyte square arrays contain AQP4, we employed immunogold labeling of SDS-washed freeze-fracture replicas and stereoscopic confirmation of tissue binding. Antibodies to AQP4 directly labeled approximately 33% of square arrays in astrocyte and ependymocyte plasma membranes in rat brain and spinal cord. Overall, 84% of labels were present beneath square arrays; 11% were beneath particle clusters that resembled square arrays that had been altered during fixation or cleaving; and 5% were beneath the much larger areas of glial plasma membrane that were devoid of square arrays. Based on this evidence that AQP4 is concentrated in glial square arrays, freeze-fracture methods may now provide biophysical insights regarding neuropathological states in which abnormal fluid shifts are accompanied by alterations in the aggregation state or the molecular architecture of square arrays.     

The three-dimensional structure of aquaporin-1

The entry and exit of water from cells is a fundamental process of life. Recognition of the high water permeability of red blood cells led to the proposal that specialized water pores exist in the plasma membrane. Expression in Xenopus oocytes and functional studies of an erythrocyte integral membrane protein of relative molecular mass 28,000, identified it as the mercury-sensitive water channel, aquaporin-1 (AQP1). Many related proteins, all belonging to the major intrinsic protein (MIP) family, are found throughout nature. AQP1 is a homotetramer containing four independent aqueous channels. When reconstituted into lipid bilayers, the protein forms two-dimensional lattices with a unit cell containing two tetramers in opposite orientation. Here we present the three-dimensional structure of AQP1 determined at 6A resolution by cryo-electron microscopy. Each AQP1 monomer has six tilted, bilayer-spanning alpha-helices which form a right-handed bundle surrounding a central density. These results, together with functional studies, provide a model that identifies the aqueous pore in the AQP1 molecule and indicates the organization of the tetrameric complex in the membrane.