Purification and functional characterization of aquaporin-8

BACKGROUND INFORMATION: Aquaporins (AQPs) are a family of channels permeable to water and some small solutes. In mammals, 13 members (AQP0-AQP12) have been found. AQP8 is widely distributed in many tissues and organs. Previous studies in frog oocytes suggested that AQP8 was permeable to water, urea and ammonium, but no direct characterization had yet been reported. RESULTS: We expressed recombinant rAQP8, hAQP8 and mAQP8 (rat, human and mouse AQP8 respectively) in yeast, purified the proteins to homogeneity and reconstituted them into proteoliposomes. Although showing high sequence similarity, AQP8 proteins from the three species had to be purified with different detergents prior to reconstitution. In stopped-flow studies, all three AQP8 proteoliposomes showed water permeability, which was inhibited by mercuric chloride and rescued by 2-mercaptoethanol. rAQP8 and hAQP8 proteoliposomes did not transport glycerol or urea but were permeable to formamide, which was also inhibited by mercuric chloride. In the oocyte transport assay, hAQP8-injected oocytes showed significantly higher [14C]methylammonium uptake than water-injected oocytes. CONCLUSIONS: In the present study, we successfully purified rAQP8, hAQP8 and mAQP8 proteins and characterized their biochemical and biophysical properties. All three AQP8 proteins transport water. rAQP8 and hAQP8 are not permeable to urea or glycerol. Moreover, hAQP8 is permeable to ammonium analogues (formamide and methylammonium). Our results suggest that AQP8 may transport ammonium in vivo and physiologically contribute to the acid-base equilibrium.     

Characterization of Leishmania donovani aquaporins shows presence of subcellular aquaporins similar to tonoplast intrinsic proteins of plants

Leishmania donovani, a protozoan parasite, resides in the macrophages of the mammalian host. The aquaporin family of proteins form important components of the parasite-host interface. The parasite-host interface could be a potential target for chemotherapy. Analysis of L. major and L. infantum genomes showed the presence of five aquaporins (AQPs) annotated as AQP9 (230aa), AQP putative (294aa), AQP-like protein (279aa), AQP1 (314aa) and AQP-like protein (596aa). We report here the structural modeling, localization and functional characterization of the AQPs from L. donovani. LdAQP1, LdAQP9, LdAQP2860 and LdAQP2870 have the canonical NPA-NPA motifs, whereas LdAQP putative has a non-canonical NPM-NPA motif. In the carboxyl terminal to the second NPA box of all AQPs except AQP1, a valine/alanine residue was found instead of the arginine. In that respect these four AQPs are similar to tonoplast intrinsic proteins in plants, which are localized to intracellular organelles. Confocal microscopy of L. donovani expressing GFP-tagged AQPs showed an intracellular localization of LdAQP9 and LdAQP2870. Real-time PCR assays showed expression of all aquaporins except LdAQP2860, whose level was undetectable. Three-dimensional homology modeling of the AQPs showed that LdAQP1 structure bears greater topological similarity to the aquaglyceroporin than to aquaporin of E. coli. The pore of LdAQP1 was very different from the rest in shape and size. The cavity of LdAQP2860 was highly irregular and undefined in geometry. For functional characterization, four AQP proteins were heterologously expressed in yeast. In the fps1Δ yeast cells, which lacked the key aquaglyceroporin, LdAQP1 alone displayed an osmosensitive phenotype indicating glycerol transport activity. However, expression of LdAQP1 and LdAQP putative in a yeast gpd1Δ strain, deleted for glycerol production, conferred osmosensitive phenotype indicating water transport activity or aquaporin function. Our analysis for the first time shows the presence of subcellular aquaporins and provides structural and functional characterization of aquaporins in Leishmania donovani.     

Aquaporin-3 functions as a glycerol transporter in mammalian skin

The AQPs (aquaporins) are a family of homologous water transporting proteins expressed in many mammalian epithelial, endothelial and other cell types. Phenotype analysis of mice lacking individual AQPs has been informative in elucidating their role in mammalian physiology. For example, phenotype analysis has indicated an important role of AQPs in the renal urinary concentrating mechanism (AQP1-AQP4), brain water balance and neural signal transduction (AQP4), exocrine gland secretion (AQP5) and ocular fluid balance (AQP1, AQP5). In skin, the aquaglyceroporin AQP3 is expressed in the basal layer of epidermal keratinocytes. Mice deficient in AQP3 have dry skin with reduced SC (stratum corneum) hydration, decreased elasticity and impaired biosynthesis. Mechanistic analysis of the altered skin phenotype in AQP3 deficiency suggested that the glycerol rather than the water transporting function of AQP3 is important in skin physiology. The glycerol content of SC and epidermis of AQP3 deficient mice is reduced, whereas that of dermis and serum is normal. The dry, relatively inelastic skin in AQP3 null mice is probably related to the humectant properties of glycerol, and the impaired SC repair to impaired epidermal biosynthetic function. The key role of AQP3 in epidermal physiology might be exploited in the development of improved cosmetics and new therapies for skin diseases associated with altered skin water content.     

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.     

Single-channel permeability and glycerol affinity of human aquaglyceroporin AQP3

For its fundamental relevance, transport of water and glycerol across the erythrocyte membrane has long been investigated before and after the discovery of aquaporins (AQPs), the membrane proteins responsible for water and glycerol transport. AQP1 is abundantly expressed in the human erythrocyte for maintaining its hydrohomeostasis where AQP3 is also expressed (at a level ~30-folds lower than AQP1) facilitating glycerol transport. This research is focused on two of the remaining questions: How permeable is AQP3 to water? What is the glycerol-AQP3 affinity under near-physiological conditions? Through atomistic modelling and large-scale simulations, we found that AQP3 is two to three times more permeable to water than AQP1 and that the glycerol-AQP3 affinity is approximately 500/M. Using these computed values along with the data from the latest literature on AQP1 and on erythrocyte proteomics, we estimated the water and glycerol transport rates across the membrane of an entire erythrocyte. We used these rates to predict the time courses of erythrocyte swelling-shrinking in response to inward and outward osmotic gradients. Experimentally, we monitored the time course of human erythrocytes when subject to an osmotic or glycerol gradient with light scattering in a stopped-flow spectrometer. We observed close agreement between the experimentally measured and the computationally predicted time courses of erythrocytes, which corroborated our computational conclusions on the AQP3 water-permeability and the glycerol-AQP3 affinity.     

Stopped-flow spectrophotometric assay of glycerol permeation in Escherichia coli: applicability and limitations.

The passive permeation and facilitated diffusion of glycerol in various strains of Escherichia coli have been studied by stopped-flow spectrophotometry. Contrary to the prediction for glycerol entry by simple diffusion, the reciprocal relaxation time (1/tau, s-1) for the passive permeation of glycerol in cells grown in the presence of glucose was not constant but decreased as the glycerol concentration increased above 100 mM. This anomaly was not due to refractive index differences or to the presence of residual levels of the glycerol facilitator protein in non-induced cells. Although reciprocal relaxation times for glycerol-induced E. coli exhibited the expected elevation relative to non-induced cells, a similar anomalous decrease 1/tar (s-1) with increasing glycerol concentration was observed. In addition, at early times after suspension in dilute buffer, the 1/tau (s-1) values obtained for induced or non-induced E. coli swelling in glycerol were considerably greater than for organisms incubated in dilute buffer for longer times. We concluded that either this spectrophotometric technique was not monitoring solely the permeation of glycerol into E. coli, or concentrations of glycerol above 100 mM significantly perturbed the structure of the E. coli cell envelope.     

Molecular characterization, chromosomal and expression patterns of three aquaglyceroporins (AQP3, 7, 9) from pig

As one subgroup of aquaporin, aquaglyceroporin including AQP3, 7, 9, 10 facilitates glycerol transport as well as water transport. In this study, we cloned the full length coding sequences of porcine (Sus scrofa) AQP3, 7 and 9 and the genomic sequence of AQP3 including 6 exons and 5 introns. Additionally, as a first step toward understanding the regulatory mechanisms of AQP9 in pig, we cloned and analyzed the upstream genomic sequence of the ATG translation initiation codon and found two negative insulin response elements (TGTTTTC and TATTTTG.), glucocorticoid-responsive elements, several CCAAT enhancer binding protein (C/EBP) sites, hepatocyte nuclear factor (HNF) sites, and NF-kappaB sites in this region. Subsequently, semi-quantitative analysis showed that AQP3 selectively expressed in spleen, stomach, kidney and lung. AQP7 and AQP9 were ubiquitously detected in all tissues examined and highly expressed in adipose tissue and liver, respectively. Finally, both AQP3 and AQP7 were assigned to chromosome 10q while AQP9 was mapped to chromosome 1q. This is the first report of molecular characterization of aquaglyceroporin in pig, which provides basic observations useful for future assessing and characterizing the role of aquaglyceroporin.     

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.     

Biophysical Assessment of Human Aquaporin-7 as a Water and Glycerol Channel in 3T3-L1 Adipocytes

The plasma membrane aquaporin-7 (AQP7) has been shown to be expressed in adipose tissue and its role in glycerol release/uptake in adipocytes has been postulated and correlated with obesity onset. However, some studies have contradicted this view. Based on this situation, we have re-assessed the precise localization of AQP7 in adipose tissue and analyzed its function as a water and/or glycerol channel in adipose cells. Fractionation of mice adipose tissue revealed that AQP7 is located in both adipose and stromal vascular fractions. Moreover, AQP7 was the only aquaglyceroporin expressed in adipose tissue and in 3T3-L1 adipocytes. By overexpressing the human AQP7 in 3T3-L1 adipocytes it was possible to ascertain its role as a water and glycerol channel in a gain-of-function scenario. AQP7 expression had no effect in equilibrium cell volume but AQP7 loss of function correlated with higher triglyceride content. Furthermore it is also reported for the first time a negative correlation between water permeability and the cell non-osmotic volume supporting the observation that AQP7 depleted cells are more prone to lipid accumulation. Additionally, the strong positive correlation between the rates of water and glycerol transport highlights the role of AQP7 as both a water and a glycerol channel and reflects its expression levels in cells. In all, our results clearly document a direct involvement of AQP7 in water and glycerol transport, as well as in triglyceride content in adipocytes.     

Role of NHERF1, cystic fibrosis transmembrane conductance regulator, and cAMP in the regulation of aquaporin 9

Water and solute transport across the plasma membrane of cells is a crucial biological function that is mediated mainly by aquaporins and aquaglyceroporins. The regulation of these membrane proteins is still incompletely understood. Using the male reproductive tract as a model system in which water and glycerol transport are critical for the establishment of fertility, we now report a novel pathway for the regulation of aquaporin 9 (AQP9) permeability. AQP9 is the major aquaglyceroporin of the epididymis, liver, and peripheral leukocytes, and its COOH-terminal portion contains a putative PDZ binding motif (SVIM). Here we show that NHERF1, cystic fibrosis transmembrane conductance regulator (CFTR), and AQP9 co-localize in the apical membrane of principal cells of the epididymis and the vas deferens, and that both NHERF1 and CFTR co-immunoprecipitate with AQP9. Overlay assays revealed that AQP9 binds to both the PDZ1 and PDZ2 domains of NHERF1, with an apparently higher affinity for PDZ1 versus PDZ2. Pull-down assays showed that the AQP9 COOH-terminal SVIM motif is essential for interaction with NHERF1. Functional assays on isolated tubules perfused in vitro showed a high permeability of the apical membrane to glycerol, which is inhibited by the AQP9 inhibitor, phloretin, and is markedly activated by cAMP. The CFTR inhibitors DPC, GlyH-101 and CFTRinh-172 all significantly reduced the cAMP-activated glycerol-induced cell swelling. We propose that CFTR is an important regulator of AQP9 and that the interaction between AQP9, NHERF1, and CFTR may facilitate the activation of AQP9 by cAMP.     

Requirement for asparagine in the aquaporin NPA sequence signature motifs for cation exclusion

Two highly conserved NPA motifs are a hallmark of the aquaporin (AQP) family. The NPA triplets form N-terminal helix capping structures with the Asn side chains located in the centre of the water or solute-conducting channel, and are considered to play an important role in AQP selectivity. Although another AQP selectivity filter site, the aromatic/Arg (ar/R) constriction, has been well characterized by mutational analysis, experimental data concerning the NPA region--in particular, the Asn position--is missing. Here, we report on the cloning and mutational analysis of a novel aquaglyceroporin carrying one SPA motif instead of the NPA motif from Burkholderia cenocepacia, an epidemic pathogen of cystic fibrosis patients. Of 1357 AQP sequences deposited in RefSeq, we identified only 15 with an Asn exchange. Using direct and phenotypic permeability assays, we found that Asn and Ser are freely interchangeable at both NPA sites without affecting protein expression or water, glycerol and methylamine permeability. However, other mutations in the NPA region led to reduced permeability (S186C and S186D), to nonfunctional channels (N64D), or even to lack of protein expression (S186A and S186T). Using electrophysiology, we found that an analogous mammalian AQP1 N76S mutant excluded protons and potassium ions, but leaked sodium ions, providing an argument for the overwhelming prevalence of Asn over other amino acids. We conclude that, at the first position in the NPA motifs, only Asn provides efficient helix cap stabilization and cation exclusion, whereas other small residues compromise structural stability or cation exclusion but not necessarily water and solute permeability.     

On the Nature of the Heat Stabilizing Effect of Inorganic Ions on Escherichia coli

The growth lag of Escherichia coli at 45°C was reduced by the addition of sodium, potassium, magnesium and calcium ions to the growth medium. A method to quantitatively determine the lag-reducing effect of these ions was developed. The results obtained showed that equivalent amounts of the ions produced the same reduction of the growth lag. According to the results of plasmolysis experiments cells of E. coli suspended in peptone broth were permeable to all four ions. The course of plasmolysis and subsequent deplasmolysis was registered as changes in the cells' ability to scatter light. The heat stability of catalase from E. coli was increased by addition of the four ions. This was observed in experiments with intact cells and with a crude cell-free preparation of catalase. The results of our experiments are most easily explained by assuming a stabilizing effect of the ions tested on the intracellular bacterial proteins.     

Copper inhibits the water and glycerol permeability of aquaporin-3

Aquaporin-3 (AQP3) is an aquaglyceroporin expressed in erythrocytes and several other tissues. Erythrocytes are, together with kidney and liver, the main targets for copper toxicity. Here we report that both water and glycerol permeability of human AQP3 is inhibited by copper. Inhibition is fast, dose-dependent, and reversible. If copper is dissolved in carbonic acid-bicarbonate buffer, the natural buffer system in our body, doses in the range of those observed in Wilson disease and in copper poisoning caused significant inhibition. AQP7, another aquaglyceroporin, was insensitive to copper. Three extracellular amino acid residues, Trp128, Ser152, and His241, were identified as responsible for the effect of copper on AQP3. We have previously shown that Ser152 is involved in regulation of AQP3 by pH. The fact that Ser152 mediates regulation of AQP3 by copper may explain the phenomenon of exquisite sensitivity of human erythrocytes to copper at acidic pH. When AQP3 was co-expressed with another AQP, only glycerol but not water permeability was inhibited by copper. Our results provide a better understanding of processes that occur in severe copper metabolism defects such as Wilson disease and in copper poisoning.     

Intra-helical salt-bridge and helix destabilizing residues within the same helical turn: Role of functionally important loop E half-helix in channel regulation of major intrinsic proteins

The superfamily of major intrinsic proteins (MIPs) includes aquaporin (AQP) and aquaglyceroporin (AQGP) and it is involved in the transport of water and neutral solutes across the membrane. Diverse MIP sequences adopt a unique hour-glass fold with six transmembrane helices (TM1 to TM6) and two half-helices (LB and LE). Loop E contains one of the two conserved NPA motifs and contributes two residues to the aromatic/arginine selectivity filter. Function and regulation of majority of MIP channels are not yet characterized. We have analyzed the loop E region of 1468 MIP sequences and their structural models from six different organism groups. They can be phylogenetically clustered into AQGPs, AQPs, plant MIPs and other MIPs. The LE half-helix in all AQGPs contains an intra-helical salt-bridge and helix-breaking residues Gly/Pro within the same helical turn. All non-AQGPs lack this salt-bridge but have the helix destabilizing Gly and/or Pro in the same positions. However, the segment connecting LE half-helix and TM6 is longer by 10–15 residues in AQGPs compared to all non-AQGPs. We speculate that this longer loop in AQGPs and the LE half-helix of non-AQGPs will be relatively more flexible and this could be functionally important. Molecular dynamics simulations on glycerol-specific GlpF, water-transporting AQP1, its mutant and a fungal AQP channel confirm these predictions. Thus two distinct regions of loop E, one in AQGPs and the other in non-AQGPs, seem to be capable of modulating the transport. These regions can also act in conjunction with other extracellular residues/segments to regulate MIP channel transport.     

Phosphorylation of aquaporin-2 regulates its water permeability

Vasopressin-regulated water reabsorption through the water channel aquaporin-2 (AQP2) in renal collecting ducts maintains body water homeostasis. Vasopressin activates PKA, which phosphorylates AQP2, and this phosphorylation event is required to increase the water permeability and water reabsorption of the collecting duct cells. It has been established that the phosphorylation of AQP2 induces its apical membrane insertion, rendering the cell water-permeable. However, whether this phosphorylation regulates the water permeability of this channel still remains unclear. To clarify the role of AQP2 phosphorylation in water permeability, we expressed recombinant human AQP2 in Escherichia coli, purified it, and reconstituted it into proteoliposomes. AQP2 proteins not reconstituted into liposomes were removed by fractionating on density step gradients. AQP2-reconstituted liposomes were then extruded through polycarbonate filters to obtain unilamellar vesicles. PKA phosphorylation significantly increased the osmotic water permeability of AQP2-reconstituted liposomes. We then examined the roles of AQP2 phosphorylation at Ser-256 and Ser-261 in the regulation of water permeability using phosphorylation mutants reconstituted into proteoliposomes. The water permeability of the non-phosphorylation-mimicking mutant S256A-AQP2 and non-phosphorylated WT-AQP2 was similar, and that of the phosphorylation-mimicking mutant S256D-AQP2 and phosphorylated WT-AQP2 was similar. The water permeability of S261A-AQP2 and S261D-AQP2 was similar to that of non-phosphorylated WT-AQP2. This study shows that PKA phosphorylation of AQP2 at Ser-256 enhances its water permeability.     

Permeability of Bacteria to Inorganic Cations. A Comparison between a Psychrophilic Achromobacter Strain and Escherichia coli

The permeability of a psychrophilic Acbromobacter strain to the chlorides of Na, K, Mg and Ca was investigated with light-scattering technique. Comparisons, were made with cells of Escherichia coli B. Cells of both strains suspended in “water were plasmolyzed by 0.1 or 0.2 M solutions of MgCl₂ or CaCl₂ without subsequent deplasmolysis. NaCl or KCl also plasmolyzed the cells, but deplasmolysis followed.” When suspended in growth medium E. coli became completely de-plasmolyzed., whereas the psychrophile still excluded MgCl₂ and CaCl₂ to a great extent. The plasmolysis and deplasmolysis were reversible. Electron micrographs of the psychrophile exposed to CaCl₂ confirmed the presence of plasrnolysis.     

The effect of plasmolysis and deplasmolysis on the permeability of plant membranes

The overall washing out of ions, especially⁸⁶Rb⁺ (as the tracer for K⁺), from hypocotyl segments of pumpkin (Cucurbita pepo L.) into distilled water or a CaCl₂ solution was studied, during plasmolysis with a saccharose solution and during deplasmolysis. Compartimental analysis was used to evaluate the⁸⁶Rb⁺ washing out kinetics. During plasmolysis, the washing out of⁸⁶Rb⁺ increases, due to two processes whose half-times are lower than those during washing out into the CaCl₂ solution. During deplasmolysis, the permeability of plasmalemma and tonoplast is substantially descreased, leading to washing out of most⁸⁶Rb⁺ from the cells. Plasmolysis differs from a mere decrease in the turgor pressure in the fact that after exchange for a hypotonic solution the membranes are irreversibly damaged. The aim of this work was to monitor the changes in the cell membrane permeability due to a change in the water potential of the cells, especially during plasmolysis and deplasmolysis.     

Mercury chloride decreases the water permeability of aquaporin-4-reconstituted proteoliposomes.

BACKGROUND INFORMATION: Mercurials inhibit AQPs (aquaporins), and site-directed mutagenesis has identified Cys(189) as a site of the mercurial inhibition of AQP1. On the other hand, AQP4 has been considered to be a mercury-insensitive water channel because it does not have the reactive cysteine residue corresponding to Cys(189) of AQP1. Indeed, the osmotic water permeability (P(f)) of AQP4 expressed in various types of cells, including Xenopus oocytes, is not inhibited by HgCl2. To examine the direct effects of mercurials on AQP4 in a proteoliposome reconstitution system, His-tagged rAQP4 [corrected] (rat AQP4) M23 was expressed in Saccharomyces cerevisiae, purified with an Ni2+-nitrilotriacetate affinity column, and reconstituted into liposomes with the dilution method. RESULTS: The water permeability of AQP4 proteoliposomes with or without HgCl2 was measured with a stopped-flow apparatus. Surprisingly, the P(f) of AQP4 proteoliposomes was significantly decreased by 5 microM HgCl2 within 30 s, and this effect was completely reversed by 2-mercaptoethanol. The dose- and time-dependent inhibitory effects of Hg2+ suggest that the sensitivity to mercury of AQP4 is different from that of AQP1. Site-directed mutagenesis of six cysteine residues of AQP4 demonstrated that Cys(178), which is located at loop D facing the intracellular side, is a target responding to Hg2+. We confirmed that AQP4 is reconstituted into liposome in a bidirectional orientation. CONCLUSIONS: Our results suggest that mercury inhibits the P(f) of AQP4 by mechanisms different from those for AQP1 and that AQP4 may be gated by modification of a cysteine residue in cytoplasmic loop D.