The role of phosphorylation in calmodulin-mediated gating of human AQP0.

Aquaporin-0 (AQP0) is the main water channel in the mammalian lens and is involved in accommodation and maintaining lens transparency. AQP0 binds the Ca2+-sensing protein calmodulin (CaM) and this interaction is believed to gate its water permeability by closing the water-conducting pore. Here, we express recombinant and functional human AQP0 in Pichia pastoris and investigate how phosphorylation affects the interaction with CaM in vitro as well as the CaM-dependent water permeability of AQP0 in proteoliposomes. Using microscale thermophoresis and surface plasmon resonance technology we show that the introduction of the single phospho-mimicking mutations S229D and S235D in AQP0 reduces CaM binding. In contrast, CaM interacts with S231D with similar affinity as wild type, but in a different manner. Permeability studies of wild-type AQP0 showed that the water conductance was significantly reduced by CaM in a Ca2+-dependent manner, whereas AQP0 S229D, S231D and S235D were all locked in an open state, insensitive to CaM. We propose a model in which phosphorylation of AQP0 control CaM-mediated gating in two different ways (1) phosphorylation of S229 or S235 abolishes binding (the pore remains open) and (2) phosphorylation of S231 results in CaM binding without causing pore closure, the functional role of which remains to be elucidated. Our results suggest that site-dependent phosphorylation of AQP0 dynamically controls its CaM-mediated gating. Since the level of phosphorylation increases towards the lens inner cortex, AQP0 may become insensitive to CaM-dependent gating along this axis.

Effect of controlled hypotensive hemorrhage on plasma sodium levels in anesthetized pigs: An exploratory study.

Perioperative hyponatremia, due to non-osmotic release of the antidiuretic hormone arginine vasopressin, is a serious electrolyte disorder observed in connection with many types of surgery. Since blood loss during surgery contributes to the pathogenesis of hyponatremia, we explored the effect of bleeding on plasma sodium using a controlled hypotensive hemorrhage pig model. After 30-min baseline period, hemorrhage was induced by aspiration of blood during 30 min at mean arterial pressure <50 mmHg. Thereafter, the animals were resuscitated with retransfused blood and a near-isotonic balanced crystalloid solution and monitored for 180 min. Electrolyte and water balances, cardiovascular response, renal hemodynamics, and markers of volume regulation and osmoregulation were investigated. All pigs (n = 10) developed hyponatremia. All animals retained hypotonic fluid, and none could excrete net-free water. Urinary excretion of aquaporin 2, a surrogate marker of collecting duct responsiveness to antidiuretic hormone, was significantly reduced at the end of the study, whereas lysine vasopressin, i.e., the pig antidiuretic hormone remained high. In this animal model, hyponatremia developed due to net positive fluid balance and generation of electrolyte-free water by the kidneys. A decreased urinary aquaporin 2 excretion may indicate an escape from antidiuresis.

Structural and functional analysis of aquaporin-2 mutants involved in nephrogenic diabetes insipidus.

Aquaporins are water channels found in the cell membrane, where they allow the passage of water molecules in and out of the cells. In the kidney collecting duct, arginine vasopressin-dependent trafficking of aquaporin-2 (AQP2) fine-tunes reabsorption of water from pre-urine, allowing precise regulation of the final urine volume. Point mutations in the gene for AQP2 may disturb this process and lead to nephrogenic diabetes insipidus (NDI), whereby patients void large volumes of highly hypo-osmotic urine. In recessive NDI, mutants of AQP2 are retained in the endoplasmic reticulum due to misfolding. Here we describe the structural and functional characterization of three AQP2 mutations associated with recessive NDI: T125M and T126M, situated close to a glycosylation site and A147T in the transmembrane region. Using a proteoliposome assay, we show that all three mutants permit the transport of water. The crystal structures of T125M and T126M together with biophysical characterization of all three mutants support that they retain the native structure, but that there is a significant destabilization of A147T. Our work provides unique molecular insights into the mechanisms behind recessive NDI as well as deepens our understanding of how misfolded proteins are recognized by the ER quality control system.

Perioperative water and electrolyte balance and water homeostasis regulation in children with acute surgery.

BACKGROUND: Hospital-acquired hyponatremia remains a feared event in patients receiving hypotonic fluid therapy. Our objectives were to assess post-operative plasma-sodium concentration and to provide a physiological explanation for plasma-sodium levels over time in children with acute appendicitis. METHODS: Thirteen normonatremic (plasma-sodium ≥135 mmol/L) children (8 males), median age 12.3 (IQR 11.5-13.5) years participated in this prospective observational study (ACTRN12621000587808). Urine was collected and analyzed. Blood tests, including renin, aldosterone, arginine-vasopressin, and circulating nitric oxide substrates were determined on admission, at induction of anesthesia, and at the end of surgery. RESULTS: On admission, participants were assumed to be mildly dehydrated and were prescribed 50 mL/kg of Ringer's acetate intravenously followed by half-isotonic saline as maintenance fluid therapy. Blood tests, urinary indices, plasma levels of aldosterone, arginine-vasopressin, and net water-electrolyte balance indicated that participants were dehydrated on admission. Although nearly 50% of participants still had arginine-vasopressin levels that would have been expected to produce maximum antidiuresis at the end of surgery, electrolyte-free water clearance indicated that almost all participants were able to excrete net free water. No participant became hyponatremic. CONCLUSIONS: The use of moderately hypotonic fluid therapy after correction of extracellular fluid deficit is not necessarily associated with post-operative hyponatremia. IMPACT: Our observations show that in acutely ill normonatremic children not only the composition but also the amount of volume infused influence on the risk of hyponatremia. Our observations also suggest that perioperative administration of hypotonic fluid therapy is followed by a tendency towards hyponatremia if extracellular fluid depletion is left untreated. After correcting extracellular deficit almost all patients were able to excrete net free water. This occurred despite nearly 50% of the cohort having high circulating plasma levels of arginine-vasopressin at the end of surgery, suggesting a phenomenon of renal escape from arginine-vasopressin-induced antidiuresis.

High-resolution structure of a fish aquaporin reveals a novel extracellular fold.

Aquaporins are protein channels embedded in the lipid bilayer in cells from all organisms on earth that are crucial for water homeostasis. In fish, aquaporins are believed to be important for osmoregulation; however, the molecular mechanism behind this is poorly understood. Here, we present the first structural and functional characterization of a fish aquaporin; cpAQP1aa from the fresh water fish climbing perch (<i>Anabas testudineus</i>), a species that is of high osmoregulatory interest because of its ability to spend time in seawater and on land. These studies show that cpAQP1aa is a water-specific aquaporin with a unique fold on the extracellular side that results in a constriction region. Functional analysis combined with molecular dynamic simulations suggests that phosphorylation at two sites causes structural perturbations in this region that may have implications for channel gating from the extracellular side.

Insight into the Mammalian Aquaporin Interactome.

Aquaporins (AQPs) are a family of transmembrane water channels expressed in all living organisms. AQPs facilitate osmotically driven water flux across biological membranes and, in some cases, the movement of small molecules (such as glycerol, urea, CO2, NH3, H2O2). Protein-protein interactions play essential roles in protein regulation and function. This review provides a comprehensive overview of the current knowledge of the AQP interactomes and addresses the molecular basis and functional significance of these protein-protein interactions in health and diseases. Targeting AQP interactomes may offer new therapeutic avenues as targeting individual AQPs remains challenging despite intense efforts.

Assessing water permeability of aquaporins in a proteoliposome-based stopped-flow setup.

Aquaporins (AQPs) are water channels embedded in the cell membrane that are critical in maintaining water homeostasis. We describe a protocol for determining the water permeation capacity of AQPs reconstituted into proteoliposomes. Using a stopped-flow setup, AQP embedded in proteoliposomes are exposed to an osmogenic gradient that triggers water flux. The consequent effects on proteoliposome size can be tracked using the fluorescence of an internalized fluorophore. This enables controlled characterization of water flux by AQPs. For complete details on the use and execution of this protocol, please refer to Kitchen et al. (2020).

High-yield overproduction and purification of human aquaporins from Pichia pastoris.

Aquaporins (AQPs) are membrane-bound water channels that play crucial roles in maintaining the water homeostasis of the human body. Here, we present a protocol for high-yield recombinant expression of human AQPs in the methylotropic yeast Pichia pastoris and subsequent AQP purification. The protocol typically yields 1-5 mg AQP per g of yeast cell at >95% purity and is compatible with any membrane protein cloned into Pichia pastoris, although expression levels may vary. For complete details on the use and execution of this protocol, please refer to Kitchen et al. (2020) and Frick et al. (2014).

Characterization of human aquaporin protein-protein interactions using microscale thermophoresis (MST).

Aquaporin water channels (AQPs) are membrane proteins that maintain cellular water homeostasis. The interactions between human AQPs and other proteins play crucial roles in AQP regulation by both gating and trafficking. Here, we describe a protocol for characterizing the interaction between a human AQP and a soluble interaction partner using microscale thermophoresis (MST). MST has the advantage of low sample consumption and high detergent compatibility enabling AQP protein-protein interaction investigation with a high level of control of components and environment. For complete details on the use and execution of this protocol, please refer to Kitchen et al. (2020) and Roche et al. (2017).

Molecular mechanisms governing aquaporin relocalisation.

The aquaporins (AQPs) form a family of integral membrane proteins that facilitate the movement of water across biological membrane by osmosis, as well as facilitating the diffusion of small polar solutes. AQPs have been recognised as drug targets for a variety of disorders associated with disrupted water or solute transport, including brain oedema following stroke or trauma, epilepsy, cancer cell migration and tumour angiogenesis, metabolic disorders, and inflammation. Despite this, drug discovery for AQPs has made little progress due to a lack of reproducible high-throughput assays and difficulties with the druggability of AQP proteins. However, recent studies have suggested that targetting the trafficking of AQP proteins to the plasma membrane is a viable alternative drug target to direct inhibition of the water-conducting pore. Here we review the literature on the trafficking of mammalian AQPs with a view to highlighting potential new drug targets for a variety of conditions associated with disrupted water and solute homeostasis.

Emerging roles for dynamic aquaporin-4 subcellular relocalization in CNS water homeostasis.

Aquaporin channels facilitate bidirectional water flow in all cells and tissues. AQP4 is highly expressed in astrocytes. In the CNS, it is enriched in astrocyte endfeet, at synapses, and at the glia limitans, where it mediates water exchange across the blood-spinal cord and blood-brain barriers (BSCB/BBB), and controls cell volume, extracellular space volume, and astrocyte migration. Perivascular enrichment of AQP4 at the BSCB/BBB suggests a role in glymphatic function. Recently, we have demonstrated that AQP4 localization is also dynamically regulated at the subcellular level, affecting membrane water permeability. Ageing, cerebrovascular disease, traumatic CNS injury, and sleep disruption are established and emerging risk factors in developing neurodegeneration, and in animal models of each, impairment of glymphatic function is associated with changes in perivascular AQP4 localization. CNS oedema is caused by passive water influx through AQP4 in response to osmotic imbalances. We have demonstrated that reducing dynamic relocalization of AQP4 to the BSCB/BBB reduces CNS oedema and accelerates functional recovery in rodent models. Given the difficulties in developing pore-blocking AQP4 inhibitors, targeting AQP4 subcellular localization opens up new treatment avenues for CNS oedema, neurovascular and neurodegenerative diseases, and provides a framework to address fundamental questions about water homeostasis in health and disease.

Ezrin Is a Novel Protein Partner of Aquaporin-5 in Human Salivary Glands and Shows Altered Expression and Cellular Localization in Sjögren's Syndrome.

Sjögren's syndrome (SS) is an exocrinopathy characterized by the hypofunction of salivary glands (SGs). Aquaporin-5 (AQP5); a water channel involved in saliva formation; is aberrantly distributed in SS SG acini and contributes to glandular dysfunction. We aimed to investigate the role of ezrin in AQP5 mislocalization in SS SGs. The AQP5-ezrin interaction was assessed by immunoprecipitation and proteome analysis and by proximity ligation assay in immortalized human SG cells. We demonstrated, for the first time, an interaction between ezrin and AQP5. A model of the complex was derived by computer modeling and in silico docking; suggesting that AQP5 interacts with the ezrin FERM-domain via its C-terminus. The interaction was also investigated in human minor salivary gland (hMSG) acini from SS patients (SICCA-SS); showing that AQP5-ezrin complexes were absent or mislocalized to the basolateral side of SG acini rather than the apical region compared to controls (SICCA-NS). Furthermore, in SICCA-SS hMSG acinar cells, ezrin immunoreactivity was decreased at the acinar apical region and higher at basal or lateral regions, accounting for altered AQP5-ezrin co-localization. Our data reveal that AQP5-ezrin interactions in human SGs could be involved in the regulation of AQP5 trafficking and may contribute to AQP5-altered localization in SS patients.

Unraveling Human AQP5-PIP Molecular Interaction and Effect on AQP5 Salivary Glands Localization in SS Patients.

Saliva secretion requires effective translocation of aquaporin 5 (AQP5) water channel to the salivary glands (SGs) acinar apical membrane. Patients with Sjögren's syndrome (SS) display abnormal AQP5 localization within acinar cells from SGs that correlate with sicca manifestation and glands hypofunction. Several proteins such as Prolactin-inducible protein (PIP) may regulate AQP5 trafficking as observed in lacrimal glands from mice. However, the role of the AQP5-PIP complex remains poorly understood. In the present study, we show that PIP interacts with AQP5 in vitro and in mice as well as in human SGs and that PIP misexpression correlates with an altered AQP5 distribution at the acinar apical membrane in PIP knockout mice and SS hMSG. Furthermore, our data show that the protein-protein interaction involves the AQP5 C-terminus and the N-terminal of PIP (one molecule of PIP per AQP5 tetramer). In conclusion, our findings highlight for the first time the role of PIP as a protein controlling AQP5 localization in human salivary glands but extend beyond due to the PIP-AQP5 interaction described in lung and breast cancers.

Calcein Fluorescence Quenching to Measure Plasma Membrane Water Flux in Live Mammalian Cells.

Aquaporins (AQPs) are membrane channel proteins that facilitate the movement of water down osmotic gradients across biological membranes. This protocol allows measurements of AQP-mediated water transport across the plasma membrane of live mammalian cells. Calcein is a fluorescent dye that is quenched in a concentration-dependent manner. Therefore, on short timescales, its concentration-dependent fluorescence can be used as a probe of cell volume, and therefore a probe of water transport into or out of cells. For complete details on the use and execution of this protocol, please refer to Kitchen et al. (2020) and Kitchen and Conner (2015). For the underlying methodology development, please refer to Fenton et al. (2010) and Solenov et al. (2004).

A microfluidic strategy for the detection of membrane protein interactions.

Membrane proteins perform a vast range of vital biological functions and are the gatekeepers for exchange of information and matter between the intracellular and extracellular environment. However, membrane protein interactions can be challenging to characterise in a quantitative manner due to the low solubility and large size of the membrane protein complex with associated lipid or detergent molecules. Here, we show that measurements of the changes in charge and diffusivity on the micron scale allow for non-disruptive studies of membrane protein interactions in solution. The approach presented here uses measurements of key physical properties of membrane proteins and their ligands to characterise the binding equilibrium parameters. We demonstrate this approach for human aquaporins (AQPs), key membrane proteins in the regulation of water homeostasis in cells. We perform quantitative measurements to characterise the interactions between two full-length AQP isoforms and the regulatory protein, calmodulin (CaM), and show that CaM selectively binds AQP0. Through direct measurements of the diffusivity and mobility in an external electric field, the diffusion coefficients and electrophoretic mobilities are determined for the individual components and the resulting AQP0-CaM complex. Furthermore, we obtain directly the binding equilibrium parameters and effective charge of each component. These results open up a route towards the use of microfluidics as a general platform in protein science and open up new possibilities for the characterisation of membrane protein interactions in solution.

Targeting Aquaporin-4 Subcellular Localization to Treat Central Nervous System Edema.

Swelling of the brain or spinal cord (CNS edema) affects millions of people every year. All potential pharmacological interventions have failed in clinical trials, meaning that symptom management is the only treatment option. The water channel protein aquaporin-4 (AQP4) is expressed in astrocytes and mediates water flux across the blood-brain and blood-spinal cord barriers. Here we show that AQP4 cell-surface abundance increases in response to hypoxia-induced cell swelling in a calmodulin-dependent manner. Calmodulin directly binds the AQP4 carboxyl terminus, causing a specific conformational change and driving AQP4 cell-surface localization. Inhibition of calmodulin in a rat spinal cord injury model with the licensed drug trifluoperazine inhibited AQP4 localization to the blood-spinal cord barrier, ablated CNS edema, and led to accelerated functional recovery compared with untreated animals. We propose that targeting the mechanism of calmodulin-mediated cell-surface localization of AQP4 is a viable strategy for development of CNS edema therapies.

Phosphorylation of human AQP2 and its role in trafficking.

Human Aquaporin 2 (AQP2) is a membrane-bound water channel found in the kidney collecting duct whose regulation by trafficking plays a key role in regulating urine volume. AQP2 trafficking is tightly controlled by the pituitary hormone arginine vasopressin (AVP), which stimulates translocation of AQP2 residing in storage vesicles to the apical membrane. The AVP-dependent translocation of AQP2 to and from the apical membrane is controlled by multiple phosphorylation sites in the AQP2 C-terminus, the phosphorylation of which alters its affinity to proteins within the cellular membrane protein trafficking machinery. The aim of this chapter is to provide a summary of what is currently known about AVP-mediated AQP2 trafficking, dissecting the roles of individual phosphorylation sites, kinases and phosphatases and interacting proteins. From this, the picture of an immensely complex process emerges, of which many structural and molecular details remains to be elucidated.

Structural Insights into AQP2 Targeting to Multivesicular Bodies.

Vasopressin-dependent trafficking of AQP2 in the renal collecting duct is crucial for the regulation of water homeostasis. This process involves the targeting of AQP2 to the apical membrane during dehydration as well as its removal when hydration levels have been restored. The latter involves AQP2 endocytosis and sorting into multivesicular bodies (MVB), from where it may be recycled, degraded in lysosomes, or released into urine via exosomes. The lysosomal trafficking regulator-interacting protein 5 (LIP5) plays a crucial role in this by coordinating the actions of the endosomal sorting complex required for transport III (ESCRT-III) and vacuolar protein sorting 4 (Vps4) ATPase, resulting in the insertion of AQP2 into MVB inner vesicles. While the interaction between LIP5 and the ESCRT-III complex and Vps4 is well characterized, very little is known about how LIP5 interacts with AQP2 or any other membrane protein cargo. Here, we use a combination of fluorescence spectroscopy and computer modeling to provide a structural model of how LIP5 interacts with human AQP2. We demonstrate that, the AQP2 tetramer binds up to two LIP5 molecules and that the interaction is similar to that seen in the complex between LIP5 and the ESCRT-III component, charged multivesicular body protein 1B (CHMP1B). These studies give the very first structural insights into how LIP5 enables membrane protein insertion into MVB inner vesicles and significantly increase our understanding of the AQP2 trafficking mechanism.

On-chip crystallization for serial crystallography experiments and on-chip ligand-binding studies.

Efficient and reliable sample delivery has remained one of the bottlenecks for serial crystallography experiments. Compared with other methods, fixed-target sample delivery offers the advantage of significantly reduced sample consumption and shorter data collection times owing to higher hit rates. Here, a new method of on-chip crystallization is reported which allows the efficient and reproducible growth of large numbers of protein crystals directly on micro-patterned silicon chips for in-situ serial crystallography experiments. Crystals are grown by sitting-drop vapor diffusion and previously established crystallization conditions can be directly applied. By reducing the number of crystal-handling steps, the method is particularly well suited for sensitive crystal systems. Excessive mother liquor can be efficiently removed from the crystals by blotting, and no sealing of the fixed-target sample holders is required to prevent the crystals from dehydrating. As a consequence, 'naked' crystals are obtained on the chip, resulting in very low background scattering levels and making the crystals highly accessible for external manipulation such as the application of ligand solutions. Serial diffraction experiments carried out at cryogenic temperatures at a synchrotron and at room temperature at an X-ray free-electron laser yielded high-quality X-ray structures of the human membrane protein aquaporin 2 and two new ligand-bound structures of thermolysin and the human kinase DRAK2. The results highlight the applicability of the method for future high-throughput on-chip screening of pharmaceutical compounds.