The Arg233Lys AQP0 mutation disturbs aquaporin0-calmodulin interaction causing polymorphic congenital cataract

Calmodulin (CaM) directly interacts with the aquaporin 0 (AQP0) C-terminus in a calcium dependent manner to regulate the water permeability of AQP0. We previously identified a missense mutation (p.R233K) in the putative CaM binding domain of AQP0 C-terminus in a congenital cataract family. This study was aimed at exploring the potential pathogenesis of this mutation causative of cataract and mainly identifying how it influenced the binding of AQP0 to CaM. Wild type and R233K mutant AQP0 with EGFP-tag were transfected separately into Hela cells to determine the expression and subcellular localizations. The co-immunoprecipitation (CoIP) assay was used to detect the interaction between AQP0 and CaM. AQP0 C-terminus peptides were synthesized with and without R233K, and the binding abilities of these peptides to CaM were assessed using a fluorescence binding assay. Localizations of wild type and R233K mutant AQP0 were determined from EGFP fluorescence, and the chimeric proteins were both localized abundantly in the plasma membrane. Protein expression levels of the culture cells showed no significant difference between them. The results from CoIP assay implied that R233K mutant presented more weakly in association with CaM than wild type AQP0. The AQP0 C-terminal mutant peptide was found to have 2.5-fold lower binding affinity to CaM than wild type peptide. These results suggested that R233K mutation did not affect the expression, location and trafficking of the protein but did influence the interaction between AQP0 and CaM. The binding affinity of AQP0 C-terminus to CaM was significantly reduced. Due to lack of the modulation of the Ca2+-calmodulin complex, the water permeability of AQP0 was subsequently augmented, which might lead to the development of this cataract.     

Cooperativity and allostery in aquaporin 0 regulation by Ca2+

Aquaporin 0 (AQP0) is essential for eye lens homeostasis as is regulation of its water permeability by Ca²⁺, which occurs through interactions with calmodulin (CaM), but the underlying molecular mechanisms are not well understood. Here, we use molecular dynamics (MD) simulations on the microsecond timescale under an osmotic gradient to explicitly model water permeation through the AQP0 channel. To identify any structural features that are specific to water permeation through AQP0, we also performed simulations of aquaporin 1 (AQP1) and a pure mixed lipid bilayer under the same conditions. The relative single-channel water osmotic permeability coefficients (p_f) calculated from all of our simulations are in reasonable agreement with experiment. Our simulations allowed us to characterize the dynamics of the key structural elements that modulate the diffusion of water single-files through the AQP0 and AQP1 pores. We find that CaM binding influences the collective dynamics of the whole AQP0 tetramer, promoting the closing of both the extracellular and intracellular gates by inducing cooperativity between neighboring subunits.     

Regulation of AQP0 water permeability is enhanced by cooperativity

Aquaporin 0 (AQP0), essential for lens clarity, is a tetrameric protein composed of four identical monomers, each of which has its own water pore. The water permeability of AQP0 expressed in Xenopus laevis oocytes can be approximately doubled by changes in calcium concentration or pH. Although each monomer pore functions as a water channel, under certain conditions the pores act cooperatively. In other words, the tetramer is the functional unit. In this paper, we show that changes in external pH and calcium can induce an increase in water permeability that exhibits either a positive cooperativity switch-like increase in water permeability or an increase in water permeability in which each monomer acts independently and additively. Because the concentrations of calcium and hydrogen ions increase toward the center of the lens, a concentration signal could trigger a regulatory change in AQP0 water permeability. It thus seems plausible that the cooperative modes of water permeability regulation by AQP0 tetramers mediated by decreased pH and elevated calcium are the physiologically important ones in the living lens.     

Zinc modulation of water permeability reveals that aquaporin 0 functions as a cooperative tetramer

We previously showed that the water permeability of AQP0, the water channel of the lens, increases with acid pH and that His40 is required (Németh-Cahalan, K.L., and J.E. Hall. 2000. J. Biol. Chem. 275:6777-6782; Németh-Cahalan, K.L., K. Kalman, and J.E. Hall. 2004. J. Gen. Physiol. 123:573-580). We have now investigated the effect of zinc (and other transition metals) on the water permeability of AQP0 expressed in Xenopus oocytes and determined the amino acid residues that facilitate zinc modulation. Zinc (1 mM) increased AQP0 water permeability by a factor of two and prevented any additional increase induced by acid pH. Zinc had no effect on water permeability of AQP1, AQP4 or MIPfun (AQP0 from killifish), or on mutants of AQP1 and MIPfun with added external histidines. Nickel, but not copper, had the same effect on AQP0 water permeability as zinc. A fit of the concentration dependence of the zinc effect to the Hill equation gives a coefficient greater than three, suggesting that binding of more than one zinc ion is necessary to enhance water permeability. His40 and His122 are necessary for zinc modulation of AQP0 water permeability, implying structural constraints for zinc binding and functional modulation. The change in water permeability was highly sensitive to a coinjected zinc-insensitive mutant and a single insensitive monomer completely abolished zinc modulation. Our results suggest a model in which positive cooperativity among subunits of the AQP0 tetramer is required for zinc modulation, implying that the tetramer is the functional unit. The results also offer the possibility of a pharmacological approach to manipulate the water permeability and transparency of the lens.     

Voltage-regulated water flux through aquaporin channels in silico

Aquaporins (AQPs) facilitate the passive flux of water across biological membranes in response to an osmotic pressure. A number of AQPs, for instance in plants and yeast, have been proposed to be regulated by phosphorylation, cation concentration, pH change, or membrane-mediated mechanical stress. Here we report an extensive set of molecular dynamics simulations of AQP1 and AQP4 subject to large membrane potentials in the range of ±1.5 V, suggesting that AQPs may in addition be regulated by an electrostatic potential. As the regulatory mechanism we identified the relative population of two different states of the conserved arginine in the aromatic/arginine constriction region. A positive membrane potential was found to stabilize the arginine in an up-state, which allows rapid water flux, whereas a negative potential favors a down-state, which reduces the single-channel water permeability.     

Light inactivation of water transport and protein-protein interactions of aquaporin-Killer Red chimeras

Aquaporins (AQPs) have a broad range of cellular and organ functions; however, nontoxic inhibitors of AQP water transport are not available. Here, we applied chromophore-assisted light inactivation (CALI) to inhibit the water permeability of AQP1, and of two AQP4 isoforms (M1 and M23), one of which (M23) forms aggregates at the cell plasma membrane. Chimeras containing Killer Red (KR) and AQPs were generated with linkers of different lengths. Osmotic water permeability of cells expressing KR/AQP chimeras was measured from osmotic swelling-induced dilution of cytoplasmic chloride, which was detected using a genetically encoded chloride-sensing fluorescent protein. KR-AQP1 red fluorescence was bleached rapidly (~10% per second) by wide-field epifluorescence microscopy. After KR bleaching, KR-AQP1 water permeability was reduced by up to 80% for the chimera with the shortest linker. Remarkably, CALI-induced reduction in AQP4-KR water permeability was approximately twice as efficient for the aggregate-forming M23 isoform; this suggests intermolecular CALI, which was confirmed by native gel electrophoresis on cells coexpressing M23-AQP4-KR and myc-tagged M23-AQP4. CALI also disrupted the interaction of AQP4 with a neuromyelitis optica autoantibody directed against an extracellular epitope on AQP4. CALI thus permits rapid, spatially targeted and irreversible reduction in AQP water permeability and interactions in live cells. Our data also support the utility of CALI to study protein-protein interactions as well as other membrane transporters and receptors.     

Expression and function of aquaporins in human skin: Is aquaporin-3 just a glycerol transporter?

The aquaporins (AQPs) are a family of transmembrane proteins forming water channels. In mammals, water transport through AQPs is important in kidney and other tissues involved in water transport. Some AQPs (aquaglyceroporins) also exhibit glycerol and urea permeability. Skin is the limiting tissue of the body and within skin, the stratum corneum (SC) of the epidermis is the limiting barrier to water loss by evaporation. The aquaglyceroporin AQP3 is abundantly expressed in keratinocytes of mammalian skin epidermis. Mice lacking AQP3 have dry skin and reduced SC hydration. Interestingly, however, results suggested that impaired glycerol, rather than water transport was responsible for this phenotype. In the present work, we examined the overall expression of AQPs in cells from human skin and we reviewed data on the functional role of AQPs in skin, particularly in the epidermis. By RT-PCR on primary cell cultures, we found that up to 6 different AQPs (AQP1, 3, 5, 7, 9 and 10) may be selectively expressed in various cells from human skin. AQP1, 5 are strictly water channels. But in keratinocytes, the major cell type of the epidermis, only the aquaglyceroporins AQP3, 10 were found. To understand the role of aquaglyceroporins in skin, we examined the relevance to human skin of the conclusion, from studies on mice, that skin AQP3 is only important for glycerol transport. In particular, we find a correlation between the absence of AQP3 and intercellular edema in the epidermis in two different experimental models: eczema and hyperplastic epidermis. In conclusion, we suggest that in addition to glycerol, AQP3 may be important for water transport and hydration in human skin epidermis.     

Expression and function of aquaporins in human skin: Is aquaporin-3 just a glycerol transporter?

The aquaporins (AQPs) are a family of transmembrane proteins forming water channels. In mammals, water transport through AQPs is important in kidney and other tissues involved in water transport. Some AQPs (aquaglyceroporins) also exhibit glycerol and urea permeability. Skin is the limiting tissue of the body and within skin, the stratum corneum (SC) of the epidermis is the limiting barrier to water loss by evaporation. The aquaglyceroporin AQP3 is abundantly expressed in keratinocytes of mammalian skin epidermis. Mice lacking AQP3 have dry skin and reduced SC hydration. Interestingly, however, results suggested that impaired glycerol, rather than water transport was responsible for this phenotype. In the present work, we examined the overall expression of AQPs in cells from human skin and we reviewed data on the functional role of AQPs in skin, particularly in the epidermis. By RT-PCR on primary cell cultures, we found that up to 6 different AQPs (AQP1, 3, 5, 7, 9 and 10) may be selectively expressed in various cells from human skin. AQP1, 5 are strictly water channels. But in keratinocytes, the major cell type of the epidermis, only the aquaglyceroporins AQP3, 10 were found. To understand the role of aquaglyceroporins in skin, we examined the relevance to human skin of the conclusion, from studies on mice, that skin AQP3 is only important for glycerol transport. In particular, we find a correlation between the absence of AQP3 and intercellular edema in the epidermis in two different experimental models: eczema and hyperplastic epidermis. In conclusion, we suggest that in addition to glycerol, AQP3 may be important for water transport and hydration in human skin epidermis.     

Aquaporin-11 containing a divergent NPA motif has normal water channel activity

Recently, two novel mammalian aquaporins (AQPs), AQPs 11 and 12, have been identified and classified as members of a new AQP subfamily, the “subcellular AQPs”. In members of this subfamily one of the two asparagine-proline-alanine (NPA) motifs, which play a crucial role in selective water conduction, are not completely conserved. Mouse AQP11 (mAQP11) was expressed in Sf9 cells and purified using the detergent Fos-choline 10. The protein was reconstituted into liposomes, which were used for water conduction studies with a stopped-flow device. Single water permeability (pf) of AQP11 was measured to be 1.72 ± 0.03 × 10^− 13 cm³/s, suggesting that other members of the subfamily with incompletely conserved NPA motifs may also function as water channels.     

Aquaporin-11 containing a divergent NPA motif has normal water channel activity

Recently, two novel mammalian aquaporins (AQPs), AQPs 11 and 12, have been identified and classified as members of a new AQP subfamily, the "subcellular AQPs". In members of this subfamily one of the two asparagine-proline-alanine (NPA) motifs, which play a crucial role in selective water conduction, are not completely conserved. Mouse AQP11 (mAQP11) was expressed in Sf9 cells and purified using the detergent Fos-choline 10. The protein was reconstituted into liposomes, which were used for water conduction studies with a stopped-flow device. Single water permeability (pf) of AQP11 was measured to be 1.72+/-0.03x10(-13) cm(3)/s, suggesting that other members of the subfamily with incompletely conserved NPA motifs may also function as water channels.     

The structural basis of water permeation and proton exclusion in aquaporins

Aquaporins (AQPs) represent a ubiquitous class of integral membrane proteins that play critical roles in cellular osmoregulations in microbes, plants and mammals. AQPs primarily function as water-conducting channels, whereas members of a sub-class of AQPs, termed aquaglyceroporins, are permeable to small neutral solutes such as glycerol. While AQPs facilitate transmembrane permeation of water and/or small neutral solutes, they preclude the conduction of protons. Consequently, openings of AQP channels allow rapid water diffusion down an osmotic gradient without dissipating electrochemical potentials. Molecular structures of AQPs portray unique features that define the two central functions of AQP channels: effective water permeation and strict proton exclusion. This review describes AQP structures known to date and discusses the mechanisms underlying water permeation, proton exclusion and water permeability regulation.     

The structural basis of water permeation and proton exclusion in aquaporins (Review)

Aquaporins (AQPs) represent a ubiquitous class of integral membrane proteins that play critical roles in cellular osmoregulations in microbes, plants and mammals. AQPs primarily function as water-conducting channels, whereas members of a sub-class of AQPs, termed aquaglyceroporins, are permeable to small neutral solutes such as glycerol. While AQPs facilitate transmembrane permeation of water and/or small neutral solutes, they preclude the conduction of protons. Consequently, openings of AQP channels allow rapid water diffusion down an osmotic gradient without dissipating electrochemical potentials. Molecular structures of AQPs portray unique features that define the two central functions of AQP channels: effective water permeation and strict proton exclusion. This review describes AQP structures known to date and discusses the mechanisms underlying water permeation, proton exclusion and water permeability regulation.     

Aquaporin water channels and lung physiology

Fluid transport across epithelial and endothelial barriers occurs in the neonatal and adult lungs. Biophysical measurements in the intact lung and cell isolates have indicated that osmotic water permeability is exceptionally high across alveolar epithelia and endothelia and moderately high across airway epithelia. This review is focused on the role of membrane water-transporting proteins, the aquaporins (AQPs), in high lung water permeability and lung physiology. The lung expresses several AQPs: AQP1 in microvascular endothelia, AQP3 in large airways, AQP4 in large- and small-airway epithelia, and AQP5 in type I alveolar epithelial cells. Lung phenotype analysis of transgenic mice lacking each of these AQPs has been informative. Osmotically driven water permeability between the air space and capillary compartments is reduced approximately 10-fold by deletion of AQP1 or AQP5 and reduced even more by deletion of AQP1 and AQP4 or AQP1 and AQP5 together. AQP1 deletion greatly reduces osmotically driven water transport across alveolar capillaries but has only a minor effect on hydrostatic lung filtration, which primarily involves paracellular water movement. However, despite the major role of AQPs in lung osmotic water permeabilities, AQP deletion has little or no effect on physiologically important lung functions, such as alveolar fluid clearance in adult and neonatal lung, and edema accumulation after lung injury. Although AQPs play a major role in renal and central nervous system physiology, the data to date on AQP knockout mice do not support an important role of high lung water permeabilities or AQPs in lung physiology. However, there remain unresolved questions about possible non-water-transporting roles of AQPs and about the role of AQPs in airway physiology, pleural fluid dynamics, and edema after lung infection.     

Membrane water permeability and aquaporin expression increase during growth of maize suspension cultured cells

Aquaporins (AQPs) are water channels that allow cells to rapidly alter their membrane water permeability. A convenient model for studying AQP expression and activity regulation is Black Mexican Sweet (BMS) maize cultured cells. In an attempt to correlate membrane osmotic water permeability coefficient (P_f) with AQP gene expression, we first examined the expression pattern of 33 AQP genes using macro-array hybridization. We detected the expression of 18 different isoforms representing the four AQP subfamilies, i.e. eight plasma membrane (PIP), five tonoplast (TIP), three small basic (SIP) and two NOD26-like (NIP) AQPs. While the expression of most of these genes was constant throughout all growth phases, mRNA levels of ZmPIP1;3 , ZmPIP2;1 , ZmPIP2;2, ZmPIP2;4 and ZmPIP2;6 increased significantly during the logarithmic growth phase and the beginning of the stationary phase. The use of specific anti-ZmPIP antisera showed that the protein expression pattern correlated well with mRNA levels. Cell pressure probe and protoplast swelling measurements were then performed to determine the P_f. Interestingly, we found that the P_f were significantly increased at the end of the logarithmic growth phase and during the steady-state phase compared to the lag phase, demonstrating a positive correlation between AQP abundance in the plasma membrane and the cell P_f.     

Excretion and conservation of glycerol, and expression of aquaporins and glyceroporins, during cold acclimation in Cope's gray tree frog Hyla chrysoscelis

Cope's gray tree frog Hyla chrysoscelis accumulates glycerol during cold acclimation. We hypothesized that, during this process, gray tree frogs adjust renal filtration and/or reabsorption rates to retain accumulated glycerol. During cold acclimation, plasma concentrations of glycerol rose >200-fold, to 51 mmol/l. Although fractional water reabsorption decreased, glomerular filtration rate (GFR) and, consequently, urine flow were <5% of warm levels, and fractional glycerol reabsorption increased. In contrast, dehydrated frogs increased fractional water reabsorption, decreased GFR, and did not accumulate glycerol. We hypothesized that expression of proteins from the aquaporin (AQP)/glyceroporin (GLP) family was associated with changing patterns of water and glycerol movement. We cloned the cDNA for three such proteins, quantified mRNA expression in nine tissues using real-time quantitative PCR, and functionally characterized them using a Xenopus oocyte expression system. HC-1, an AQP1-like water channel conferring low glycerol permeability, is expressed ubiquitously in warm- and cold-acclimated tissues. HC-2, a water channel most similar to AQP2, is primarily expressed in organs of osmoregulation. HC-3, which is most similar to AQP3, is functionally characterized as a GLP, with low permeability to water but high permeability to glycerol. Aspects of expression levels and functional characteristics varied between cold and warm conditions for each of the three AQPs, suggesting a complex pattern of involvement in osmoregulation related to thermal acclimation.     

Phosphorylation determines the calmodulin-mediated Ca2+ response and water permeability of AQP0

In Xenopus oocytes, the water permeability of AQP0 (P(f)) increases with removal of external calcium, an effect that is mediated by cytoplasmic calmodulin (CaM) bound to the C terminus of AQP0. To investigate the effects of serine phosphorylation on CaM-mediated Ca(2+) regulation of P(f), we tested the effects of kinase activation, CaM inhibition, and a series of mutations in the C terminus CaM binding site. Calcium regulation of AQP0 P(f) manifests four distinct phenotypes: Group 1, with high P(f) upon removal of external Ca(2+) (wild-type, S229N, R233A, S235A, S235K, K238A, and R241E); Group 2, with high P(f) in elevated (5 mm) external Ca(2+) (S235D and R241A); Group 3, with high P(f) and no Ca(2+) regulation (S229D, S231N, S231D, S235N, and S235N/I236S); and Group 4, with low P(f) and no Ca(2+) regulation (protein kinase A and protein kinase C activators, S229D/S235D and S235N/I236S). Within each group, we tested whether CaM binding mediates the phenotype, as shown previously for wild-type AQP0. In the presence of calmidazolium, a CaM inhibitor, S235D showed high P(f) and no Ca(2+) regulation, suggesting that S235D still binds CaM. Contrarily, S229D showed a decrease in recruitment of CaM, suggesting that S229D is unable to bind CaM. Taken together, our results suggest a model in which CaM acts as an inhibitor of AQP0 P(f). CaM binding is associated with a low P(f) state, and a lack of CaM binding is associated with a high P(f) state. Pathological conditions of inappropriate phosphorylation or calcium/CaM regulation could induce P(f) changes contributing to the development of a cataract.     

Junction-forming aquaporins

Aquaporins (AQPs) are a family of ubiquitous membrane channels that conduct water and solutes across membranes. This review focuses on AQP0 and AQP4, which in addition to forming water channels also appear to play a role in cell adhesion. We discuss the recently determined structures of the membrane junctions mediated by these two AQPs, the mechanisms that regulate junction formation, and evidence that supports a role for AQP0 and AQP4 in cell adhesion.     

Knockdown of aquaporin 3 is involved in intestinal barrier integrity impairment

AQP3 is a water/glycerol transporter expressed at the basolateral membrane of colonic epithelial cells. Although AQPs are expressed in the gastrointestinal tract, their effect on intestinal barrier has not been clear. Here, we showed that knockdown of AQP3 caused a dramatic, dose-dependent increase in E. coli C25 translocation, with the reduction of TEER and increasing LY permeability. Western blots revealed that expression of Claudin-1 and Occludin were significantly decreased in the AQP3 knockdown group, demonstrating that this treatment enhances paracellular permeability via an opening of the tight junction complex. These data not only describe the correlation between transcellular and paracellular pathways in human intestines, but also show that targeted knockdown of AQP3 might impair the intestinal barrier integrity.     

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.     

Effect of hyperosmotic stimulation on aquaporins gene expression in chick kidney

Birds can produce hyperosmotic urine, but their renal morphology differs from that of mammals. Recent studies in mammals, suggested that various aquaporins (AQPs) are present in the kidney and play crucial roles in urine production. To elucidate the role of AQPs in the avian kidney, we first examined for the presence of AQP1, 2, 3, 4, 7 and 9 mRNAs in the chick (Gallus gallus) kidney by RT-PCR analysis. Next, we quantified variations of AQPs mRNAs levels in chick kidney after hyperosmotic stimulation (water-deprivation or salt-loading) by real-time RT-PCR analysis. Our study showed that in addition to AQP1, 2, 3, 4 and 7, chick kidney also expressed AQP9 and that hyperosmotic stimulation induced changes in AQPs expression. In particular, water-deprivation increased AQP2 and AQP3 mRNAs levels, whereas salt-loading induced a significant increase in AQP1, AQP2 and AQP9 mRNAs levels. AQP4 and AQP7 mRNA levels were not affected by any hyperosmotic stimulation. Taken together, these results indicated that the presence of AQPs in chick kidney is similar to that in mammals, that the chick kidney has an additional AQP9 and that AQP1, 2, 3 and 9 may play a crucial but different role in water permeability in this organ.