Water Permeability Of Aquaporin Research Articles

Multiple pore lining residues modulate water permeability of GlpF.

The water permeability of aquaporins (AQPs) varies by more than an order of magnitude even though the pore structure, geometry, as well as the channel lining residues are highly conserved. However, channel gating by pH, divalent ions or phosphorylation was only shown for a minority of AQPs. Structural and in silico indications of water flux modulation by flexible side chains of channel lining residues have not been experimentally confirmed yet. Hence, the aquaporin “open state” is still considered to be a continuously open pore with water molecules permeating in a single‐file fashion. Using protein mutations outside the selectivity filter in the aqua(glycerol)facilitator GlpF of Escherichia coli we, to the best of our knowledge, for the first time, modulate the position of the highly conserved Arg in the selectivity filter. This in turn enhances or reduces the unitary water permeability of GlpF as shown in silico by molecular dynamics (MD) simulations and in vitro with purified and reconstituted GlpF. This finding suggests that AQP water permeability can indeed be regulated by lipid bilayer asymmetry and the transmembrane potential. Strikingly, our long‐term MD simulations reveal that not only the conserved Arg in the selectivity filter, but the position and dynamics of multiple other pore lining residues modulate water passage through GlpF. This finding is expected to trigger a wealth of future investigations on permeability and regulation of AQPs among others with the aim to tune water permeability for biotechnological applications.

Functional Analysis of Aquaporin Water Permeability Using an Escherichia coli-Based Cell-Free Protein Synthesis System

Aquaporins are essential water channel proteins found in all kingdoms of life. Although the water permeability of aquaporins has been well characterized, sample preparation for aquaporin water permeability assays remains challenging and time-consuming. Besides the difficulty in overexpressing membrane proteins in a cell-based expression system, the unique requirement for homogeneity in aquaporin proteoliposome sample preparations for water transport assays further increases the complexity. In this study, a complementary Cell-free Protein Synthesis (CFPS) method is described in detail, providing three different strategies for the preparation of aquaporin proteoliposome samples. Aquaporin can be produced either as a pellet fraction and then resolubilized, or co-translationally as a detergent-soluble fraction. Furthermore, aquaporin can be directly incorporated into liposomes, which was included in the CFPS reactions. Although proteoliposomes tend to fuse during the incubation of the CFPS reactions, an additional treatment of the fused samples with detergent, followed by a detergent removal step, can re-form homogenously sized proteoliposomes suitable for functional analysis. Using this method, we successfully characterized aquaporins from both prokaryotic and eukaryotic organisms. In particular, in the presence of liposomes, the developed CFPS expression system is a fast and convenient method for sample preparation for the functional analysis of aquaporins.

In vitro expression and functional characterization of NPA motifs in aquaporins of Nosema bombycis.

Nosema bombycis contains functional aquaporins (NbAQPs), which are key targets for exploring the mechanism of N. bombycis infection; however, the regulation of these NbAQPs remains unknown. The two highly conserved asparagine-proline-alanine sequences (NPA motifs) play important roles in AQP biogenesis. As part of this study, we constructed a series of NbAQP mutants (NbAQP_NPA1, NbAQP_NPA2, and NbAQP_NPA1,2) and expressed them in BmN cells. The results showed that mutations in either NPA motif or in both NPA motifs did not affect NbAQP expression in vitro. After expression in Xenopus laevis oocytes, those injected with wild-type NbAQP rapidly expanded, whereas oocytes injected with NbAQP_NPAs did not significantly change in size. The associated water permeability (pf) of NbAQP_NPAs was significantly reduced five-six times compared to that of wild-type NbAQP. These results indicated that NPA motifs are necessary for the water channel function of AQPs in N. bombycis. The present study shows for the first time that the NbAQP NPA motif has an impact on the water permeability of aquaporin in N. bombycis, thereby providing a platform for further research into the mechanisms underlying the regulation of NbAQP expression.

Effect of Lipid Layer on the Water Permeability of Aquaporin: A Molecular Dynamics Study

Aquaporin is a transmembrane protein, which facilitates selective water transport across cell membrane. High permeability of aquaporin led to many proposals to use aquaporin-embedded membranes for water purification. Recent experimental studies have shown that water permeability of aquaporin can be altered by the membrane composition. By far, there is no clear explanation how lipid composition changes the permeability of aquaporin. In this study, we simulated Aquaporin Z (AqpZ) with various conditions of lipid layers using molecular dynamics (MD) simulation. AqpZ was inserted into lipid layers having various compositions of lipids and cholesterol ratios. The osmotic permeability of aquaporin was calculated from the MD simulation and compared with experimental values. We further investigated the free energy and geometry change due to membrane composition. This study may shed light on the design optimization of aquaporin-embedded membrane.

An Arginine-Rich Loop is Critical for the Modulation of the Water Permeability of Aquaporin 0

Aquaporin 0 (AQP0), the major intrinsic protein of the eye lens, plays a vital role in maintaining lens clarity by facilitating water transport. AQP0 reduces its osmotic water permeability (Pf) in response to increases in the external calcium concentration, an effect that is mediated through an interaction with calmodulin (CaM). Calcium sensitivity of AQP0 Pf is further modified by serine phosphorylation. Despite recent structural characterization of the AQP0-CaM complex, the mechanism used by CaM to modulate AQP0 remains poorly understood. We employed a combination of Brownian and molecular dynamics simulations to identify the critical features of the AQP0-CaM interaction. Brownian dynamics (BD) simulations suggest that serine phosphorylation of AQP0 does not significantly reduce CaM-binding to the whole AQP0 protein, in contrast to the experimental observation that phosphorylation does significantly reduce binding to C-terminus AQP0 peptides. Comparative MD simulation studies show that AQP0 phosphorylation changes contacts between AQP0 and CaM, particularly at a small arginine-rich loop on the AQP0 cytosolic face. This charged loop allosterically couples CaM to the second constriction site residues of AQP0 through an interaction with R156. Additionally, we observe that R153 increases the size of the pore opening through an interaction with the Y149 hydroxyl group, which is necessary for maintaining high permeability states of AQP0. Experimental and simulation data support the notion that serine phosphorylation of AQP0 changes the calcium sensitivity of Pf by modifying CaM contacts with the cytosolic arginine-rich loop rather than by inhibiting CaM-binding.

The influence of natural mineral water on aquaporin water permeability and human natural killer cell activity

Aquaporins are the intrinsic membrane proteins functioning as water channel to transport water and/or mineral nutrients across the biological membrane systems. In this research, we aimed to clarify if the selected mineral water can affect aquaporin functions in vitro and the assumption of the mineral water can modify aquaporin expression and activate natural killer cell activity in human body. First, we expressed six human and eight plant aquaporin genes in oocytes and compared the effect of different kinds of natural mineral water on aquaporin activity. The oocyte assay data show that Hita tenryosui water could promote water permeability of almost all human and plant aquaporins in varying degrees, and freeze-dry and organic solvent extraction could reduce AQP2 activity but pH change and boiling could not. Second, each volunteer in two groups (10 in one group) received an oral Hita tenryosui or tap water load of 1000ml/day for total four weeks. We found that these two kinds of water did not directly affect the relative expression levels of AQP1 and AQP9 in the blood cells, but intriguingly, the natural killer cell activities of the volunteers drinking Hita tenryosui water were significantly improved, suggesting that Hita tenryosui water has obvious health function, which opens a new and interesting field of investigation related to the link between mineral water consumption and human health and the therapies for some chronic diseases.

Proinflammatory role of aquaporin‐4 in autoimmune neuroinflammation

Aquaporin-4 (AQP4) deficiency in mice reduces neuroinflammation in experimental autoimmune encephalomyelitis (EAE) produced by active immunization with myelin oligodendrocyte glycoprotein peptide (MOG). Potential mechanisms for the protective effect of AQP4 deficiency were investigated, including AQP4-dependent leukocyte and microglia cell function, immune cell entry in the central nervous system (CNS), intrinsic neuroinflammation, and humoral immune response. As we found with active-immunization EAE, neuroinflammation was greatly reduced in AQP4-knockout mice in adoptive-transfer EAE. AQP4 was absent in immune cells, including activated T lymphocytes. The CNS migration of fluorescently labeled, MOG-sensitized T lymphocytes was comparable in wild-type and AQP4-knockout mice. Microglia did not express AQP4. Serum anti-AQP4 antibodies were absent in EAE. Remarkably, intracerebral injection of LPS produced much greater neuroinflammation in wild-type than in AQP4-knockout mice, and cytokine (TNF-α and IL-6) secretion was reduced in astrocyte cultures from AQP4-knockout mice. Adenovirus-mediated expression of AQP4, or of an unrelated aquaporin, AQP1, increased cytokine secretion in astrocyte and nonastrocyte cell cultures, supporting the involvement of aquaporin water permeability in cytokine secretion. Our data suggest an intrinsic proinflammatory role of AQP4 involving AQP4-dependent astrocyte swelling and cytokine release. Reduction in AQP4 water transport may be protective in neuroinflammatory CNS diseases.

Influence of the Cytoplasmic Domains of Aquaporin-4 on Water Conduction and Array Formation

Phosphorylation of Ser180 in cytoplasmic loop D has been shown to reduce the water permeability of aquaporin (AQP) 4, the predominant water channel in the brain. However, when the structure of the S180D mutant (AQP4M23S180D), which was generated to mimic phosphorylated Ser180, was determined to 2.8 Å resolution using electron diffraction patterns, it showed no significant differences from the structure of the wild-type channel. High-resolution density maps usually do not resolve protein regions that are only partially ordered, but these can sometimes be seen in lower-resolution density maps calculated from electron micrographs. We therefore used images of two-dimensional crystals and determined the structure of AQP4M23S180D at 10 Å resolution. The features of the 10-Å density map are consistent with those of the previously determined atomic model; in particular, there were no indications of any obstruction near the cytoplasmic pore entrance. In addition, water conductance measurements, both in vitro and in vivo, show the same water permeability for wild-type and mutant AQP4M23, suggesting that the S180D mutation neither reduces water conduction through a conformational change nor reduces water conduction by interacting with a protein that would obstruct the cytoplasmic channel entrance. Finally, the 10-Å map shows a cytoplasmic density in between four adjacent tetramers that most likely represents the association of four N termini. This finding supports the critical role of the N terminus of AQP4 in the stabilization of orthogonal arrays, as well as their interference through lipid modification of cysteine residues in the longer N-terminal isoform.

Oocyte hydration in the Japanese eel (Anguilla japonica) during meiosis resumption and ovulation

In this study, to elucidate the mechanisms of oocyte hydration in the Japanese eel (Anguilla japonica), we examined the in vivo and in vitro morphological changes and hydration process occurring during oocyte maturation and ovulation. We also investigated the effects of the presence of ovarian follicles, aquaporin water permeability (HgCl(2)), and yolk proteolysis (bafilomycin A1) inhibitors, gap junction uncouplers (carbenoxolone and 1-octanol), a 3beta-hydroxysteroid dehydrogenase inhibitor (trilostane), and a P450 antagonist (aminoglutethimide) on oocyte hydration during in vitro oocyte maturation and ovulation. The oocytes underwent more than threefold increase in volume during maturation and ovulation, which was artificially induced by injecting salmon pituitary extracts and 17,20beta-dihydroxy-4-pregnen-3-one (DHP). Wet and dry weight measurements indicated that water accumulation during oocyte maturation is the major factor contributing to the follicular diameter increase, suggesting that follicular diameter measurements can be used as a hydration index. In the in vitro experiments, human chorionic gonadotropin (HCG) and DHP caused an increase in the diameter of follicle-enclosed oocytes but not defolliculated oocytes. Addition of HgCl(2) and bafilomycin A1 to the incubation media inhibited the HCG- and DHP-induced increase in the follicular diameter in a dose-dependent manner. Neither carbenoxolone nor 1-octanol influenced the HCG-induced increase in the follicular diameter. Trilostane and aminoglutethimide slightly but significantly inhibited HCG-induced oocyte hydration. Consequently, we concluded that ovarian follicles are essential for HCG- and DHP-induced oocyte hydration. Furthermore, aquaporin facilitates water uptake by acting as a water channel, and yolk proteolysis is essential for water influx into oocytes via osmotic mechanisms.

Water transport in aquaporins: molecular dynamics simulations

Aquaporins and aquaglyceroporins are membrane channel proteins that selectively transport water and small molecules such as glycerol across biological membranes. Molecular dynamics simulations have made substantial contributions toward understanding the permeation mechanisms of aquaporins in atomic detail. Osmotic pressure is the driving force of the transport by aquaporins. The osmotic water permeability of aquaporins can be estimated from equilibrium molecular dynamics simulations by using linear response theory. The relationship between osmotic permeability and channel structure was investigated by the recently proposed pf-matrix method. In addition to the water transport, other functions of aquaporins and aquaglyceroporins, i.e., glycerol permeation, proton blockage, and gating, have also been extensively studied by molecular dynamics simulations.

Effect of isomerization of amino acid residues on the structure of aquaporin

The effect of isomerization of amino acid residues on the structure and water permeability of aquaporin has been investigated. It was established that the equilibrium configuration of the model D isomer is energetically less favorable than that of its natural L isomer. It is shown that the model D isomer of aquaporin is waterproof.

Quaternary Ammonium Compounds as Water Channel Blockers

Excessive water uptake through Aquaporins (AQP) can be life-threatening and reversible AQP inhibitors are needed. Here, we determined the specificity, potency, and binding site of tetraethylammonium (TEA) to block Aquaporin water permeability. Using oocytes, externally applied TEA blocked AQP1/AQP2/AQP4 with IC50 values of 1.4, 6.2, and 9.8 microM, respectively. Related tetraammonium compounds yielded some (propyl) or no (methyl, butyl, or pentyl) inhibition. TEA inhibition was lost upon a Tyr to Phe amino acid switch in the external water pore of AQP1/AQP2/AQP4, whereas the water permeability of AQP3 and AQP5, which lack a corresponding Tyr, was not blocked by TEA. Consistent with experimental data, multi-nanosecond molecular dynamics simulations showed one stable binding site for TEA, but not tetramethyl (TMA), in AQP1, resulting in a nearly 50% water permeability inhibition, which was reduced in AQP1-Y186F due to effects on the TEA inhibitory binding region. Moreover, in the simulation TEA interacted with charged residues in the C (Asp128) and E (Asp185) loop, and the A(Tyr37-Asn42-Thr44) loop of the neighboring monomer, but not directly with Tyr186. The loss of TEA inhibition in oocytes expressing properly folded AQP1-N42A or -T44A is in line with the computationally predicted binding mode. Our data reveal that the molecular interaction of TEA with AQP1 differs and is about 1000-fold more effective on AQPs than on potassium channels. Moreover, the observed experimental and simulated similarities open the way for rational design and virtual screening for AQP-specific inhibitors, with quaternary ammonium compounds in general, and TEA in particular as a lead compound.

Regulation of Aquaporin Water Permeability in the Lens

To examine Ca(2+)- and pH-mediated regulation of water permeability of endogenously expressed aquaporin (AQP)0 in lens fiber cells and AQP1 in lens epithelial cells. Large, right-side-out membrane vesicles were formed from freshly isolated groups of lens fiber cells. Osmotic shrinking or swelling of these vesicles was used to determine the water permeability of endogenously expressed AQP0. The results were compared with those in similar studies of freshly isolated lens epithelial cells, which endogenously expressed AQP1, and of oocytes, which exogenously expressed AQP0. In the lens or in oocytes, decreasing external pH from 7.5 to 6.5 caused a two- to fourfold increase in the water permeability of mammalian AQP0. Several lines of evidence suggest that this effect is mediated by the binding of H(+) to a histidine in the first extracellular loop (His40). Lens AQP1 lacks His40 and also lacks pH sensitivity. Increasing Ca(2+) caused a two- to fourfold increase in the water permeability of endogenous AQP0. The Ca(2+) effect on mouse AQP0 was a 2.5-fold increase in the lens, whereas in oocytes, it was a 4-fold decrease. In either environment, the effect was mediated through calmodulin, most likely through its binding to the proximal domain of the C terminus. Lens AQP1 does not have a similar domain and does not have calcium sensitivity. In either the lens or oocytes, Ca(2+) and H(+) appear to affect the same mechanism, probably either the open probability of the water channel, or open-channel permeability. The difference between calcium's effects in lens versus oocytes was remarkable and is not understood. However, in the lens, Ca(2+) and H(+) are both increased in inner fiber cells, and so in the physiologically relevant environment, both may act to increase the water permeability of AQP0.

Molecular Basis of pH and Ca2+ Regulation of Aquaporin Water Permeability

Aquaporins facilitate the diffusion of water across cell membranes. We previously showed that acid pH or low Ca2+ increase the water permeability of bovine AQP0 expressed in Xenopus oocytes. We now show that external histidines in loops A and C mediate the pH dependence. Furthermore, the position of histidines in different members of the aquaporin family can “tune” the pH sensitivity toward alkaline or acid pH ranges. In bovine AQP0, replacement of His40 in loop A by Cys, while keeping His122 in loop C, shifted the pH sensitivity from acid to alkaline. In the killifish AQP0 homologue, MIPfun, with His at position 39 in loop A, alkaline rather than acid pH increased water permeability. Moving His39 to His40 in MIPfun, to mimic bovine AQP0 loop A, shifted the pH sensitivity back to the acid range. pH regulation was also found in two other members of the aquaporin family. Alkaline pH increased the water permeability of AQP4 that contains His at position 129 in loop C. Acid and alkaline pH sensitivity was induced in AQP1 by adding histidines 48 (in loop A) and 130 (in loop C). We conclude that external histidines in loops A and C that span the outer vestibule contribute to pH sensitivity. In addition, we show that when AQP0 (bovine or killifish) and a crippled calmodulin mutant were coexpressed, Ca2+ sensitivity was lost but pH sensitivity was maintained. These results demonstrate that Ca2+ and pH modulation are separable and arise from processes on opposite sides of the membrane.

Positive and negative regulation of water channel aquaporins in human small intestine by cholera toxin

Analysis of osmotic water permeability of aquaporin (AQP) 1, AQP3 and AQP4, which are expressed in human small intestine, in the presence or absence of cholera toxin (CT) was performed using a Xenopus oocyte expression system. When treated with CT, water permeability of AQP4 was facilitated while that of AQP3 was suppressed. AQP1 did not show any significant change of water permeability when treated with CT. An adenylyl cyclase accelerator forskolin showed similar effects as CT did, suggesting that changes of the water permeability of AQP4 and AQP3 were due to an increase of intracellular cAMP concentration. A possibility that these AQPs are responsible molecules for causing acute secretory diarrhoea as in cholera is considered.

PH and Calcium Regulate the Water Permeability of Aquaporin 0

Aquaporins increase the water permeability in many cell types across many species. We investigated the effects of external pH and Ca(2+) on water permeability of Xenopus oocytes injected with aquaporin cRNA by measuring the rate of swelling in hypotonic solutions. Lowering pH to 6.5 increased the water permeability of aquaporin (AQP0) 3.4 +/- 0.4-fold. Diethylpyrocarbonate pretreatment increased water permeability 4.2 +/- 0.5-fold and abolished pH sensitivity, suggesting that the pH regulation is mediated by an external histidine. Lowering Ca(2+) increased water permeability 4.1 +/- 0. 4-fold. The effects of Ca(2+) and pH each required the presence of histidine 40, indicating a critical role of this amino acid in facilitating the modulation of water permeability. Clamping intracellular Ca(2+) at high or low values abolished sensitivity to external Ca(2+), suggesting that Ca(2+) acts at an internal site. Three different calmodulin inhibitors each increased AQP0 water permeability, suggesting that Ca(2+) may act through calmodulin. None of the above altered the water permeability induced by AQP1 or AQP4. Because the greatest change in AQP0 water permeability is in the normal pH range found in the lens (7.2-6.5), this paper provides evidence for regulation of an aquaporin by pH under physiological conditions.

Transmembrane helix 5 is critical for the high water permeability of aquaporin.

Aquaporin-2 (AQP2), a vasopressin-regulated water channel, plays a major role in urinary concentration. AQP2 and the major intrinsic protein (MIP) of lens fiber are highly homologous (58% amino acid identity) and share a topology of six transmembrane helices connected by five loops (loops A-E). Despite the similarities of these proteins, however, the water channel activity of AQP2 is much higher than that of MIP. To determine the site responsible for this gain of activity in AQP2, several parts of MIP were replaced with the corresponding parts of AQP2. When expressed in Xenopus oocytes, the osmotic water permeability (P(f)) of MIP and AQP2 was 48 and 245 x 10(-)(4) cm/s, respectively. Substitutions in loops B-D failed to increase P(f), whereas substitution of loop E significantly increased P(f) 1.5-fold. A similar increase in P(f) was observed with the substitution of the front half of loop E. P(f) measurements taken in a yeast vesicle expression system also confirmed that loop E had a complementary effect, whereas loops B-D did not. However, P(f) values of the loop E chimeras were only approximately 30% of that of AQP2. Simultaneous exchanges of loop E and a distal half of transmembrane helix 5 just proximal to loop E increased P(f) to the level of that of AQP2. Replacement of helix 5 alone stimulated P(f) 2.7-fold. Conversely, P(f) was decreased by 73% when helix 5 of AQP2 was replaced with that of MIP. Moreover, P(f) was stimulated 2.6- and 3.3-fold after helix 5 of AQP1 and AQP4 was spliced into MIP, respectively. Our findings suggested that the distal half of helix 5 is necessary for maximum water channel activity in AQP. We speculate that this portion contributes to the formation of the aqueous pore and the determination of the flux rate.