Aquaporin water channel in the salivary glands of the Formosan subterranean termite Coptotermes formosanus is predominant in workers and absent in soldiers

Termite society is unique because the worker caste fetches and carries free water, utilizing it as a solvent for nest construction and gallery building and to maintain wetness for their nestmates. Such water management in a social organization relies largely upon the function of the workers in the colony, as well as on the individuals controlling the location and movement of water inside their bodies. The movement of water via aquaporins (AQPs; water channels) into and out of cells is a key feature of the numerous physiological functions related to whole‐insect water balance. In the present study, the homologue of the water‐specific Drosophila AQP [Drosophila integral protein (DRIP)] is characterized in workers of the Formosan subterranean termite Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae), a highly active wood decomposer. Immunoblot analysis of DRIP‐type AQP using an antibody from the silkworm Bombyx mori reveals that the Coptotermes DRIP (formerly cloned as ‘CfAQP1’) with a molecular mass of approximately 25.7 kDa is expressed predominantly in the salivary (labial) gland of the workers. The Coptotermes DRIP is present at the basal plasma membrane of the parietal cells, as demonstrated by immunocytochemistry. By contrast, there is no DRIP detected within the salivary glands of soldier termites, and neither caste expresses DRIP in their labial gland reservoir (water sac), a tissue that is suggested to have a function as a water sink. The AQP present in the salivary glands is of physiological importance with respect to salivation, aiding in the secretion of cellulolytic enzymes for wood ingestion by the workers of the subterranean termite.     

Two water-specific aquaporins at the apical and basal plasma membranes of insect epithelia: molecular basis for water recycling through the cryptonephric rectal complex of lepidopteran larvae

Larval lepidopteran and coleopteran insects have evolved a specialised cryptonephric system in the hindgut in which water is constantly and rapidly taken up before defecation. In the silkworm, Bombyx mori, the movement of water through the epithelia within the cryptonephric rectal complex is likely facilitated by the two aquaporins, AQP-Bom1 and AQP-Bom3. Both are functionally water-specific and are predominantly expressed in the hindgut (colon and rectum). Phylogenetically, AQP-Bom1 and AQP-Bom3 belong to the DRIP (Drosophila integral protein) and PRIP (Pyrocoelia rufa integral protein) subfamilies, respectively, of the insect AQP clade. In immunoblot analyses using antipeptide antibodies for each Bombyx AQP, the predicted molecular mass for the respective AQPs were around 25 kDa, and further indicated that both tended to be oligomerised as a homotetramer (～110 kDa). AQP-Bom1 [DRIP] was exclusively expressed at the apical plasma membrane of colonic and rectal epithelial cells, whereas AQP-Bom3 [PRIP] was expressed at the basal plasma membrane of these cells. This polarised localisation of DRIP/PRIP was also observed in the outer cryptonephric Malpighian tubules (outer cMT) and in the six tubules just outside the cryptonephric rectal complex (rectal lead MT). In the rectal epithelia, water is transported from the rectal lumen to the perinephric space and then deposited into the lumen of the outer cMT; the water then goes through the tubular lumen to exit the complex and is finally transported across the rectal lead MT. We conclude that rectal water retrieval into the haemocoele occurs at the very limited region of the water-permeable sites in MT epithelia after passing the rectal and cMT epithelia and that the high osmotic permeability is due to the presence of two distinct water-specific AQPs (DRIP and PRIP) in the epithelial cells of lepidopteran hindgut.     

Prediction of Aquaporin Function by Integrating Evolutionary and Functional Analyses

Aquaporins (AQPs) are a family of channel proteins, which transport water and/or small solutes across cell membranes. AQPs are present in Bacteria, Eukarya, and Archaea. The classical AQP evolution paradigm explains the inconsistent phylogenetic trees by multiple transfer events and emphasizes that the assignment of orthologous AQPs is not possible, making it difficult to integrate functional information. Recently, a novel phylogenetic framework of eukaryotic AQP evolution showed congruence between eukaryotic AQPs and organismal trees identifying 32 orthologous clusters in plants and animals (Soto et al. Gene 503:165–176, 2012). In this article, we discuss in depth the methodological strength, the ability to predict functionality and the AQP community perception about the different paradigms of AQP evolution. Moreover, we show an updated review of AQPs transport functions in association with phylogenetic analyses. Finally, we discuss the possible effect of AQP data integration in the understanding of water and solute transport in eukaryotic cells.     

Water transporting properties of hepatocyte basolateral and canalicular plasma membrane domains

Previous work from our laboratory supports an important role for aquaporins (AQPs), a family of water channel proteins, in bile secretion by hepatocytes. To further define the pathways and molecular mechanisms for water movement across hepatocytes, we directly assessed osmotic water permeability (Pf) and activation energy (Ea) in highly purified, rat hepatocytes basolateral membrane vesicles (BLMV) and canalicular membrane (CMV) vesicles by measuring scattered light intensity using stopped-flow spectrophotometry. The time course of scattered light for BLMV and CMV fit well to a single-exponential function. In BLMV, Pf was 108 +/- 4 mum.s-1 (25 degrees C) with an Ea of 7.7 kcal/mol; in CMV, Pf was 86 +/- 5 mum.s-1 (25 degrees C) with an Ea of 8.0 kcal/mol. The AQP blocker, dimethyl sulfoxide, significantly inhibited the Pf of both basolateral (81 +/- 4 mum.s-1; -25%) and canalicular (59 +/- 4 mum.s-1; -30%) membrane vesicles. When CMV were isolated from hepatocytes treated with dibutyryl cAMP, a double-exponential fit was needed, implying two functionally different vesicle populations; one population had Pf and Ea values similar to those of CMV from untreated hepatocytes, but the other population had a very high Pf (655 +/- 135 mum.s-1, 25 degrees C) and very low Ea (2.8 kcal/mol). Dimethyl sulfoxide completely inhibited the high Pf value in this second vesicle population. In contrast, Pf and Ea of BLMV were unaltered by cAMP treatment of hepatocytes. Our results are consistent with the presence of both lipid- and AQP-mediated pathways for basolateral and canalicular water movement across the hepatocyte plasma membrane barrier. Our data also suggest that the hepatocyte canalicular membrane domain is rate-limiting for transcellular water transport and that this domain becomes more permeable to water when hepatocytes are exposed to a choleretic agonist, presumably by insertion of AQP molecules. These data suggest a molecular mechanism for the efficient coupling of osmotically active solutes and water transport during canalicular bile formation.     

Involvement of Aquaporin-5 Water Channel in Osmoregulation in Parotid Secretory Granules

Aquaporins (AQPs) are a family of channel proteins that allow water or very small solutes to pass, functioning in tissues where the rapid and regulated transport of fluid is necessary, such as the kidney, lung, and salivary glands. Aquaporin-5 (AQP5) has been demonstrated to localize on the luminal surface of the acinar cells of the salivary glands. In this paper, we investigated the expression and function of AQP5 in the secretory granules of the rat parotid gland. AQP5 was detected in the secretory granule membranes by immunoblot analysis. The immunoelectron microscopy experiments confirmed that AQP5 was to be found in the secretory granule membrane. Anti-AQP5 antibody evoked lysis of the secretory granules but anti-aquaporin-1 antibody did not and AQP1 was not detected in the secretory granule membranes by immunoblot analysis. When chloride ions were removed from the solution prepared for suspending secretory granules, the granule lysis induced by anti-AQP5 antibody was inhibited. Furthermore, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, an anion channel blocker, blocked the anti-AQP5 antibody-induced secretory granule lysis. These results suggest that AQP5 is, expressed in the parotid gland secretory granule membrane and is involved in osmoregulation in the secretory granules.     

Roles of Aquaporin-3 Water Channels in Volume-Regulatory Water Flow in a Human Epithelial Cell Line

Membrane water transport is an essential event not only in the osmotic cell volume change but also in the subsequent cell volume regulation. Here we investigated the route of water transport involved in the regulatory volume decrease (RVD) that occurs after osmotic swelling in human epithelial Intestine 407 cells. The diffusion water permeability coefficient (Pd) measured by NMR under isotonic conditions was much smaller than the osmotic water permeability coefficient (Pf) measured under an osmotic gradient. Temperature dependence of Pf showed the Arrhenius activation energy (Ea) of a low value (1.6 kcal/mol). These results indicate an involvement of a facilitated diffusion mechanism in osmotic water transport. A mercurial water channel blocker (HgCl₂) diminished the Pf value. A non-mercurial sulfhydryl reagent (MMTS) was also effective. These blockers of water channels suppressed the RVD. RT-PCR and immunocytochemistry demonstrated predominant expression of AQP3 water channel in this cell line. Downregulation of AQP3 expression induced by treatment with antisense oligodeoxynucleotides was found to suppress the RVD response. Thus, it is concluded that AQP3 water channels serve as an essential pathway for volume-regulatory water transport in, human epithelial cells.     

Function and immuno-localization of aquaporins in the Antarctic midge Belgica antarctica

Aquaporin (AQP) water channel proteins play key roles in water movement across cell membranes. Extending previous reports of cryoprotective functions in insects, this study examines roles of AQPs in response to dehydration, rehydration, and freezing, and their distribution in specific tissues of the Antarctic midge, Belgica antarctica (Diptera, Chironomidae). When AQPs were blocked using mercuric chloride, tissue dehydration tolerance increased in response to hypertonic challenge, and susceptibility to overhydration decreased in a hypotonic solution. Blocking AQPs decreased the ability of tissues from the midgut and Malpighian tubules to tolerate freezing, but only minimal changes were noted in cellular viability of the fat body. Immuno-localization revealed that a DRIP-like protein (a Drosophila aquaporin), AQP2- and AQP3 (aquaglyceroporin)-like proteins were present in most larval tissues. DRIP- and AQP2-like proteins were also present in the gut of adult midges, but AQP4-like protein was not detectable in any tissues we examined. Western blotting indicated that larval AQP2-like protein levels were increased in response to dehydration, rehydration and freezing, whereas, in adults DRIP-, AQP2-, and AQP3-like proteins were elevated by dehydration. These results imply a vital role for aquaporin/aquaglyceroporins in water relations and freezing tolerance in B. antarctica.     

Lung edema clearance: 20 years of progress: invited review: role of aquaporin water channels in fluid transport in lung and airways.

Water transport across epithelial and endothelial barriers in bronchopulmonary tissues occurs during airway hydration, alveolar fluid transport, and submucosal gland secretion. Many of the tissues involved in these processes are highly water permeable and express aquaporin (AQP) water channels. AQP1 is expressed in microvascular endothelia throughout the lung and airways, AQP3 in epithelia in large airways, AQP4 in epithelia throughout the airways, and AQP5 in type I alveolar epithelial cells and submucosal gland acinar cells. The expression of some of these AQPs increases near the time of birth and is regulated by growth factors, inflammation, and osmotic stress. Transgenic mouse models of AQP deletion have provided information about their physiological role. In lung, AQP1 and AQP5 provide the principal route for osmotically driven water transport; however, alveolar fluid clearance in the neonatal and adult lung is not affected by AQP deletion nor is lung CO(2) transport or fluid accumulation in experimental models of lung injury. In the airways, AQP3 and AQP4 facilitate water transport; however, airway hydration, regulation of the airway surface liquid layer, and isosmolar fluid absorption are not impaired by AQP deletion. In contrast to these negative findings, AQP5 deletion in submucosal glands in upper airways reduced fluid secretion and increased protein content by greater than twofold. Thus, although AQPs play a major physiological role outside of the airways and lung, AQPs appear to be important mainly in airway submucosal gland function. The substantially slower rates of fluid transport in airways, pleura, and lung compared with renal and some secretory epithelia may account for the apparent lack of functional significance of AQPs at these sites. However, the possibility remains that AQPs may play a role in lung physiology under conditions of stress and/or injury not yet tested or in functions unrelated to transepithelial fluid transport.     

Invertebrate aquaporins: a review

Aquaporins (AQPs) or water channels render the lipid bilayer of cell membranes permeable to water. The numerous AQP subtypes present in any given species, the transport properties of each subtype and the variety of methods of their regulation allows different cell types to be transiently or permanently permeable to water or other solutes that AQPs are capable of transporting (e.g. urea or glycerol). AQPs have been well characterized in all vertebrate classes, other than reptilia. Here we review the current state of knowledge of invertebrate AQPs set in the context of the much more thoroughly studied vertebrate AQPs. By phylogenetic analysis of the total AQP complement of several completed insect genomes, we propose a classification system of insect AQPs including three sub-families (DRIP, BIB and PRIP) that have one representative from all the complete insect genomes. The physiological role of AQPs in invertebrates (insects, ticks and nematodes) is discussed, including their function in common invertebrate phenomena such as high-volume liquid diets, cryoprotection and anhydrobiosis.     

Renal expression and functions of aquaporin 1 and aquaporin 4 in cattle

Abstract

Aquaporin (AQP) 1 and AQP 4 are members of the aquaporin water channel family that play an important role in reabsorption of water from the renal tubular fluid to concentrate urine. Studies of renal AQPs have been performed in human, rodents, sheep, dogs and horses. We studied nephron segment-specific expression of AQP 1 and AQP 4 using immunohistochemical staining on paraffin sections of bovine kidneys. AQP 1 was moderately expressed in endothelium of the cortical capillary network, vasa recta, and glomerular capillaries. AQP 4 was moderately expressed only in cytoplasm of epithelial cells in proximal tubules. We concluded that AQP 1 and AQP 4 in the bovine kidney showed some differences from other species in renal trans-epithelial water transport.     

Concerted action of two cation filters in the aquaporin water channel

Aquaporin (AQP) facilitated water transport is common to virtually all cell membranes and is marked by almost perfect specificity and high flux rates. Simultaneously, protons and cations are strictly excluded to maintain ionic transmembrane gradients. Yet, the AQP cation filters have not been identified experimentally. We report that three point mutations turned the water-specific AQP1 into a proton/alkali cation channel with reduced water permeability and the permeability sequence: H⁺ ≫K⁺ >Rb⁺ >Na⁺ >Cs⁺ >Li⁺. Contrary to theoretical models, we found that electrostatic repulsion at the central asn-pro-ala (NPA) region does not suffice to exclude protons. Full proton exclusion is reached only in conjunction with the aromatic/arginine (ar/R) constriction at the pore mouth. In contrast, alkali cations are blocked by the NPA region but leak through the ar/R constriction. Expression of alkali-leaking AQPs depolarized membrane potentials and compromised cell survival. Our results hint at the alkali-tight but solute-unselective NPA region as a feature of primordial channels and the proton-tight and solute-selective ar/R constriction variants as later adaptations within the AQP superfamily.     

Aquaporins in complex tissues. I.Developmental patterns in respiratory and glandular tissues of rat

Developmentalexpression of aquaporin water transport proteins is not well understoodin respiratory tract or secretory glands; here we define aquaporinprotein ontogeny in rat. Expression of aquaporin-3 (AQP3), AQP4, andAQP5 proteins occurs within 2 wk after birth, whereas AQP1 firstappears before birth. In most tissues, aquaporin protein expressionincreases progressively, although transient high-level expression isnoted in distal lung (AQP4 at postnatal day+2) and trachea (AQP5 at postnatalday +21 and AQP3 at postnatal day+42). In mature animals, AQP5 is abundant in distallung and salivary glands, AQP3 and AQP4 are present in trachea, andAQP1 is present in all of these tissues except salivary glands.Surprisingly, all four aquaporin proteins are highly abundant innasopharynx. Unlike AQP1, corticosteroids did not induce expression ofAQP3, AQP4, or AQP5 in lung. Our results seemingly implicate aquaporinsin proximal airway humidification, glandular secretion, and perinatalclearance of fluid from distal airways. However, the studies underscorea need for detailed immunohistochemical characterizations anddefinitive functional studies.

     

Distribution and quantitative changes in amounts of aquaporin 1, 5 and 9 in the pig uterus during the estrous cycle and early pregnancy

Background  

Aquaporins (AQPs) are a family of membrane channel proteins that facilitate bulk water transport. To date, 11 isoforms of AQPs have been reported to be expressed in the female and male reproductive systems. The purpose of our study was to determine the localization and quantitative changes in the expression of AQP1, 5 and 9 within the pig uterus during different stages of the estrous cycle and early pregnancy.     

1,3‐propanediol binds deep inside the channel to inhibit water permeation through aquaporins

Aquaporins and aquaglyceroporins (AQPs) are membrane channel proteins responsible for transport of water and for transport of glycerol in addition to water across the cell membrane, respectively. They are expressed throughout the human body and also in other forms of life. Inhibitors of human AQPs have been sought for therapeutic treatment for various medical conditions including hypertension, refractory edema, neurotoxic brain edema, and so forth. Conducting all‐atom molecular dynamics simulations, we computed the binding affinity of acetazolamide to human AQP4 that agrees closely with in vitro experiments. Using this validated computational method, we found that 1,3‐propanediol (PDO) binds deep inside the AQP4 channel to inhibit that particular aquaporin efficaciously. Furthermore, we used the same method to compute the affinities of PDO binding to four other AQPs and one aquaglyceroporin whose atomic coordinates are available from the protein data bank (PDB). For bovine AQP1, human AQP2, AQP4, AQP5, and Plasmodium falciparum PfAQP whose structures were resolved with high resolution, we obtained definitive predictions on the PDO dissociation constant. For human AQP1 whose PDB coordinates are less accurate, we estimated the dissociation constant with a rather large error bar. Taking into account the fact that PDO is generally recognized as safe by the US FDA, we predict that PDO can be an effective diuretic which directly modulates water flow through the protein channels. It should be free from the serious side effects associated with other diuretics that change the hydro‐homeostasis indirectly by altering the osmotic gradients.     

Transport of water and glycerol in aquaporin 3 is gated by H(+).

Aquaporins (AQPs) were expressed in Xenopus laevis oocytes in order to study the effects of external pH and solute structure on permeabilities. For AQP3 the osmotic water permeability, L(p), was abolished at acid pH values with a pK of 6.4 and a Hill coefficient of 3. The L(p) values of AQP0, AQP1, AQP2, AQP4, and AQP5 were independent of pH. For AQP3 the glycerol permeability P(Gl), obtained from [(14)C]glycerol uptake, was abolished at acid pH values with a pK of 6.1 and a Hill coefficient of 6. Consequently, AQP3 acts as a glycerol and water channel at physiological pH, but predominantly as a glycerol channel at pH values around 6.1. The pH effects were reversible. The interactions between fluxes of water and straight chain polyols were inferred from reflection coefficients (sigma). For AQP3, water and glycerol interacted by competing for titratable site(s): sigma(Gl) was 0.15 at neutral pH but doubled at pH 6.4. The sigma values were smaller for polyols in which the -OH groups were free to form hydrogen bonds. The activation energy for the transport processes was around 5 kcal mol(-1). We suggest that water and polyols permeate AQP3 by forming successive hydrogen bonds with titratable sites.     

Reciprocity in the Developmental Regulation of Aquaporins 1, 3 and 5 during Pregnancy and Lactation in the Rat

Milk secretion involves significant flux of water, driven largely by synthesis of lactose within the Golgi apparatus. It has not been determined whether this flux is simply a passive consequence of the osmotic potential between cytosol and Golgi, or whether it involves regulated flow. Aquaporins (AQPs) are membrane water channels that regulate water flux. AQP1, AQP3 and AQP5 have previously been detected in mammary tissue, but evidence of developmental regulation (altered expression according to the developmental and physiological state of the mammary gland) is lacking and their cellular/subcellular location is not well understood. In this paper we present evidence of developmental regulation of all three of these AQPs. Further, there was evidence of reciprocity since expression of the rather abundant AQP3 and less abundant AQP1 increased significantly from pregnancy into lactation, whereas expression of the least abundant AQP5 decreased. It would be tempting to suggest that AQP3 and AQP1 are involved in the secretion of water into milk. Paradoxically, however, it was AQP5 that demonstrated most evidence of expression located at the apical (secretory) membrane. The possibility is discussed that AQP5 is synthesized during pregnancy as a stable protein that functions to regulate water secretion during lactation. AQP3 was identified primarily at the basal and lateral membranes of the secretory cells, suggesting a possible involvement in regulated uptake of water and glycerol. AQP1 was identified primarily at the capillary and secretory cell cytoplasmic level and may again be more concerned with uptake and hence milk synthesis, rather than secretion. The fact that expression was developmentally regulated supports, but does not prove, a regulatory involvement of AQPs in water flux through the milk secretory cell.