Formation Of Water Channels Research Articles

Recognition of Hydrophobicity Class of Polymeric Insulators Employing Residual Morphological Neural Network and Granulometry-Based Image Analysis

Hydrophobicity is an important property of polymeric insulators that allow formation of discrete water droplets on the insulator surface. However, with aging or under extreme humidification, the insulator surface loses its hydrophobic property, resulting in continuous water channel formation, which may lead to dry band arcing and subsequently flashover, thereby affecting the long-term performance of the insulator. Therefore, accurate recognition of hydrophobic class (HC) is important as it provides information regarding the surface condition of the insulator. Considering the above-said facts, this article investigates the feasibility of employing residual morphological neural network (Res-Morph-NN) to accurately recognize different HCs of polymeric insulators. In addition, for better understanding of the distribution of water droplets on the insulator surface, a novel approach based on multiscale mathematical morphology, i.e., granulometry, is used in this article. Our investigations revealed significant alterations in the granulometric patterns with the change in HC of polymeric insulators. The efficacy of the proposed framework employing Res-Morph-NN has been validated on HC images obtained from the experiment as well as on large set of images obtained from an online database, which returned satisfactory results. The proposed method can be efficiently used to discriminate different hydrophobicity classes, which can be implemented for monitoring insulator surface condition in real time.

Lipid II Binding and Transmembrane Properties of Various Antimicrobial Lanthipeptides.

There has been an alarming rise in antibacterial resistant infections in recent years due to the widespread use of antibiotics, and there is a dire need for the development of new antibiotics utilizing novel modes of action. Lantibiotics are promising candidates to engage in the fight against resistant strains of bacteria due to their unique modes of action, including interference with cell wall synthesis by binding to lipid II and creating pores in bacterial membranes. In this study, we use atomic-scale molecular dynamics computational studies to compare both the lipid II binding ability and the membrane interactions of five lanthipeptides that are commonly used in antimicrobial research: nisin, Mutacin 1140 (MU1140), gallidermin, NVB302, and NAI107. Among the five peptides investigated, nisin is found to be the most efficient at forming water channels through a membrane, whereas gallidermin and MU1140 are found to be better at binding the lipid II molecules. Nisin's effectiveness in facilitating water transport across the membrane is due to the creation of several different water trajectories along with no significant water delay points along the paths. The shorter peptide deoxyactagardine B (NVB302) was found to not form a water channel. These detailed observations provide insights into the dual mechanisms of the action of lantibiotic peptides and can facilitate the design and development of novel lanthipeptides by strategic placement of different residues.

Effect of in-situ emulsification of surfactant on the enhanced oil recovery in low-permeability reservoirs

Surfactant flooding technology has become an important treatment for the enhance oil recovery (EOR) in low-permeability reservoirs, and which behavior of surfactant is more important for EOR, lowering interfacial tension (IFT) or in-situ emulsification, is still debatable. In this work, to ascertain the dominating behavior of surfactant, a series of comparative experiments were performed to observe their dynamic flooding performances in artificial cores over a permeability range between 5 mD and 50 mD. Besides, a 2D heterogeneous model was used to explore the effect of micro heterogeneity on surfactant flooding. At last, the adaptable limits, including heterogeneous degree and injective time, were further discussed. The results show, the overall oil recovery increases with permeability, while their flooding dynamics also changes with permeability. The results show, in-situ emulsification of surfactant plays the key role in oil recovery compared with IFT reduction, especially with the cores’ permeability decreasing. On the one hand, the strong emulsifying capability of surfactant is advantageous in dispersing the residual oil to reduce oil saturation. On the other hand, the formation of amounts of emulsion droplets is useful to block the pore and further contributes to establish pressure variation and delaying water-cut raising. Meanwhile, strong emulsifying capacity of surfactant is positive to improve the swept efficiency in micro heterogeneous distributions and avoid the formation of micro water channel. Lastly, the adaptable conditions tests indicate the extremely strong heterogeneity and low crude oil saturation are the crucial factor to affect the EOR performances owing to the ease of in-situ emulsification formation. Therefore, this finding of this study can help for better understanding of contribution of in-situ emulsification of surfactant for EOR and its limitations in low-permeability reservoirs, which could be a candidate with great potentials for field application and economic returns.

Formation of Water-Channel by Propylene Glycol into Polymer for Porous Materials.

In this study, a porous membrane with a cellulose acetate (CA) matrix was fabricated using propylene glycol with a water pressure treatment without a metal salt as an additive. The water pressure treatment of the fabricated CA membrane with propylene glycol yielded nanopores. The nanopores were formed as the additives in the CA chains led to plasticization. The weakened chains of the parts where the plasticization occurred were broken by the water pressure, which generated the pores. Compared to the previous study with glycerin as an additive, the size of the hydration region was controlled by the number of hydrophilic functional groups. When water pressure was applied to the CA membrane containing propylene glycol as an additive, the hydration area was small, so it was effective to control the pore size and the number of nano pores than glycerin. In addition, the number of nanopores and pore size could be easily adjusted by the water pressure. The porosity of the membrane was increased owing to the trace amount of propylene glycol, confirmed by scanning electron microscopy (SEM) and porosimetry. The interaction between the CA and propylene glycol was verified by Fourier-transform infrared spectroscopy (FT-IR) and thermogravimetric analysis (TGA). Consequently, it was the optimum composition to generate pores at the CA/propylene glycol 1:0.2 ratio, and porosity of 69.7% and average pore diameter of 300 nm was confirmed. Since it is a membrane with high porosity and nano sized pores, it is expected to be applied in various fields.

Efficient demulsification of ultralow-concentration crude oil-in-water emulsion by three-dimensional superhydrophilic channels

Efficient extraction of crude oil, the major energy resource of current concern and high demand worldwide, is of paramount importance in both energy and environmental fields. However, it remains a great challenge to separate the crude oil-in-water emulsions with an ultralow oil content of <200ppm. Here, the three-dimensional and superwetting channels are developed by coating titanium foams with anodic TiO2 nanotube arrays. The channels render superhydrophilic and underwater superoleophobic feature, which enables rapid formation of water channels that expel the oil droplets. A high separation efficiency of ∼96.8% and low total organic carbon content of ∼6 ppm are thus achieved for the ultralow-concentration crude oil-in-water emulsions. The pressure and time dependence of the separation process is systematically studied with a critical pressure of 12.25 kPa. Such a high performance is close to the theoretical limit imposed by the ultralow concentration, and shows obvious advances over either organic membranes or inorganic frameworks.

Solvothermal self assembly of three lanthanide(III)-succinates: Crystal structure, topological analysis and DFT calculations on water channel

Three new coordination polymers have been assembled by self-assembly of lanthanide(III) ions and succinic acid in dimethylformamide–water (DMF-H2O) mixed solvent having formulae; {[Ln(suc)1.5(H2O)2]•xH2O}n; (1), (2) and (3) [Where Ln = La (1), Pr (2) and Nd (3), suc = succinate dianion; x = 2 for CP 1, x = 1 for CP 2 and x = 4 for CP 3]. In all three coordination polymers (CPs) 1-3, the Ln(III) centres are ten coordinated with bicapped square antiprismatic geometry (SAPRS-10). Crystallographic analysis reveals that CPs 1-3 crystallize in triclinic system P¯1 space group. The linker succinic acid displays two different bonding modes: (μ2-κ4, η2:η2) and (μ4-κ6, η1:η2:η2:η1). All CPs 1-3 are isostructural and isomorphous. The two Ln centres in CPs 1-3 are bridged together through succinate anions via (μ4-κ6, η1:η2:η2:η1) mode forming dinuclear (Ln2O18) Secondary building unit (SBU) which are extended infinitely to generate one-dimensional chains. These 1D chains are further extended along the b-axis to form two-dimensional layered structures via (μ2-κ4, η2:η2) mode and finally three-dimensional network. In case of Nd, the quality of the crystal allows to locate a major number of lattice water molecules (x = 4) than in the other two complexes and, remarkably, the formation of water channels can be realized and studied. It has been analyzed using DFT calculations combined with the molecular electrostatic potential (MEP) surfaces, the quantum theory of “atoms-in-molecules” QTAIM and non–covalent interaction index (NCIplot) analyses.

Aquaporins in regulation of plant protective responses to drought

Plasmolemma permeability is an integral indicator of the functional state of plant cells under stress. Aquaporins (AQPs), specialized transmembrane proteins that form water channels and play an important role in the adaptation of plants to adverse conditions and, in particular, to lack or excess of water, are involved in the formation of the response to drought. The main function of AQPs is to facilitate the movement of water across cell membranes and maintain aqueous cell homeostasis. Under stressful conditions, there is both an increase and decrease in the expression of individual aquaporin genes. Analysis of the data revealed differences in the expression of AQPs genes in stable and sensitive plant genotypes. It turned out that aquaporins in different stress-resistant varieties of the same species also respond differently to drought. The review provides brief information on the history of the discovery of aquaporins, the structure and function of these proteins, summarizes the latest information on the role of aquaporins in the regulation of metabolism and the response of plants to stressors, with particular emphasis on aquaporins in drought protection. The discovery and study of AQPs expands the possibilities of using genetic engineering methods for the selection of new plant species, in particular, more resistant to drought and salinization of the soil, as well as to increase their productivity. The use of aquaporins in biotechnology to improve drought resistance of various species has many prospects.

Polyphenol etched ZIF-8 modified graphene oxide nanofiltration membrane for efficient removal of salts and organic molecules

Multi-layer graphene oxide (GO) membranes show great perspective in vast fields due to special two-dimensional structure. However, pure GO membranes generally face main obstacles of low flux, weak interlayer interaction and poor stability, which hinder their wide application in separation. Introduction of nano-porous crystals into interlayer of GO nanosheets to form 3D/2D structure is a potential strategy to overcome these issues. In this study, novel polyphenol etched hollow MOF/GO composite membrane was first synthesized employing modified hollow metal organic frameworks with strong hydrophilicity and hollow structure by one-step etching-aided cross-linking method. MOF nanoparticles with hollow structure expanded interlayer spacing of GO nanosheets and brought about formation of additional water channels, significantly reducing water transfer resistance. Meanwhile, nano-porous crystal MOFs modified by phenolic materials were able to cross-link adjacent GO nanosheets via π–π interfacial interactions to achieve stable structure and increase anti-swelling property of membranes. The pores with abundant hydroxyl groups provided strong free energy barrier for ions at medium and low concentrations of ionic strength, therefore not only greatly improved membrane permeaselectivity, but also enhanced anti-fouling performance. The prepared eZIF-8/GO nanofiltration membrane exhibited excellent permeate flux of 16.4 L m−2 h−1 bar−1, 382% higher than pure GO membrane, and competitive Na2SO4 rejection of 86.3%. By simply adjusting GO loading to further reduce membrane thickness, the permeate flux reached up to 45.4 L m−2 h−1 bar−1 with high rejection more than 99% for organic molecules. These results indicate that novel MOF/GO composite membranes have broad application prospects in desalination and water treatment.

Molecular dynamic simulation study on formation of water channel in oil film detachment process controlled by surfactant polarity

Adopting molecular dynamic simulations method, four kinds of surfactant with different head groups and same alkyl chain, sodium dodecyl sulfonate (SDSn), sodium dodecyl sulfate (SDS), sodium dodecyl benzene sulfonate (SDBS), and sodium dodecyl-dioxyethylene ether sulfate (AES) were employed to study the polarity effect on oil film detachment from silica surface. Our results indicates that the formation of water channel is crucial for oil film detachment and was discussed in detail, and the size of water channel is adopted to evaluate the detachment of oil film. Our simulation results indicated that the stronger the surfactant polarity is, the easier the water channels form, and gave the order of oil detachment capability: AES > SDBS > SDS > SDSn. Our study is helpful to deeply understand the oil film detachment behavior and the polarity effect from molecular and atomic insight.

Mirror twin boundaries in MoSe2 monolayers as one dimensional nanotemplates for selective water adsorption.

Water adsorption on transition metal dichalcogenides and other 2D materials is generally governed by weak van der Waals interactions. This results in a hydrophobic character of the basal planes, and defects may play a significant role in water adsorption and water cluster nucleation. However, there is a lack of detailed experimental investigations on water adsorption on defective 2D materials. Here, by combining low-temperature scanning tunneling microscopy (STM) experiments and density functional theory (DFT) calculations, we study in that context the well-defined mirror twin boundary (MTB) networks separating mirror-grains in 2D MoSe2. These MTBs are dangling bond-free extended crystal modifications with metallic electronic states embedded in the 2D semiconducting matrix of MoSe2. Our DFT calculations indicate that molecular water also interacts similarly weak with these MTBs as with the defect-free basal plane of MoSe2. However, in low temperature STM experiments, nanoscopic water structures are observed that selectively decorate the MTB network. This localized adsorption of water is facilitated by functionalization of the MTBs by hydroxyls formed by dissociated water. Hydroxyls may form by dissociating of water at undercoordinated defects or adsorbing of radicals from the gas phase in the UHV chamber. Our DFT analysis indicates that the metallic MTBs adsorb these radicals much stronger than on the basal plane due to charge transfer from the metallic states into the molecular orbitals of the OH groups. Once the MTBs are functionalized with hydroxyls, molecular water can attach to them, forming water channels along the MTBs. This study demonstrates the role metallic defect states play in the adsorption of water even in the absence of unsaturated bonds that have been so far considered to be crucial for adsorption of hydroxyls or water.

Genetic Algorithm Approach for the Optimization of Protein Antifreeze Activity Using Molecular Simulations

Antifreeze proteins (AFPs) are of much interest for their ability to inhibit ice growth at low concentrations. In this work, we present a genetic algorithm for the in silico design of AFP mutants with improved antifreeze activity, measured as the predicted thermal hysteresis at a fixed concentration, ΔTC. Central to the algorithm is our recently developed neural network method for predicting ΔTC from molecular simulations [Kozuch et al., PNAS, 115, 13252 (2018)]. Applying the algorithm to three structurally diverse AFPs, wfAFP, rQAE, and RiAFP, we find that significantly improved mutants are discovered for rQAE and RiAFP. Testing of the optimized mutants shows an increase in ΔTC of 0.572 ± 0.11 K (262 ± 50.6%) and 1.33 ± 0.14 K (39.9 ± 4.19%) over the native structures for rQAE and RiAFP, respectively. Structural analysis of the optimized mutants reveals that the algorithm is able to exploit two pathways for enhancing the predicted antifreeze activity of the mutants: (1) increasing the local order of surface waters by encouraging the formation of internal water channels in the protein and (2) increasing the total ice-binding area by improving the planar structure of the ice-binding surface. Additionally, analysis of all mutants explored by the algorithm reveals that a subset of residues, mainly nonpolar, are particularly helpful in improving antifreeze activity at the ice-binding surface.

The Synaptotagmin-1 C2B Domain Is a Key Regulator in the Stabilization of the Fusion Pore.

Fusion pores serve as an effective mechanism to connect intracellular organelles and release vesicle contents during exocytosis. A complex lipid rearrangement takes place as membranes approximate, bend, fuse, and establish a traversing water channel to define the fusion pore, linking initially isolated chambers. Thermodynamically, the process is unfavorable and thought to be mediated by specialized proteins. In this work, we have developed a reaction coordinate to induce fusion pores from initially flat and parallel lipid bilayers and we have used it to describe the effects of the synaptotagmin-1 C2B domain during the process. We have obtained free-energy profiles of the whole lipid reorganization in biologically realistic membranes, going from planar and parallel bilayers through stalk hemifusion to water channel formation. Our results point to a lysine-rich polybasic region on synaptotagmin-1 C2B as the key to lipid reorganization control through the formation of phosphatidylinositol bisphosphate clusters that stabilize the fusion pore.

Down-regulation of Aquaporin-1 mediates a microglial phenotype switch affecting glioma growth

Aquaporin 1 (AQP1), a transmembrane protein that forms water channels, has previously been shown to facilitate growth and progression of many types of tumors by modulating tumor cell migration, proliferation and angiogenesis. Here, we determined the impact of AQP1 expression in the tumor environment on the progression of brain tumors. Primary microglia from wild type(WT) and AQP1 knockout(KO) mice were used to test AQP1 effect on microglia function by using Western blot, quantative PCR, in an experimental in vivo mouse glioma model and organotypic brain slice culture. Deletion of AQP1 in the host tissue significantly reduced the survival of the mice implanted with GL261 glioma cells. The density of glioma-associated microglia/macrophages was almost doubled in AQP1KO mice. We found that factors secreted from GL261 cells decrease microglial AQP1 expression via the MEK/ERK pathway, and that inhibition of this pathway with Trametinib reduced tumor growth and prolonged the survival of tumor bearing mice, an effect which required the presence of microglia. Deletion of AQP1 in cultured microglia resulted in an increase in migratory activity and a decrease in TLR4-dependent innate immune responses. Our study demonstrates a functional relevance of AQP1 expression in microglia and hints to AQP1 as a potential novel target for glioma therapy.

Electrolyte nanofluid performance on the oil detachment from an oil-wetted carbonate surface: Water channel formation using molecular dynamics simulation

In this research, molecular dynamics simulation is used to provide a molecular-scale insight into the working mechanisms of the silica nanoparticles in oil detachment from carbonate surface in absence and presence of ionic compounds. The oil model comprises of heptane, decane and toluene mixture. The contents of aqueous phase are variated in different cases: (I) water, (II) water and ions of Na+, Cl−, Mg2+and SO42−, (III) water and silica nanoparticles and (IV) water, silica nanoparticles and individual ions. In each simulation, an oil layer with a thickness of 15°A were placed between the calcite surface and the water phase to cut the interactions between calcite surface and oil phase and form an oil-wet surface. According to the results, in the absence of nanoparticles, the aqueous layer could not change the conformation of the oil layer. However, in presence of nanoparticles, water molecules could diffuse into the oil phase and move toward calcite surface. Contact angle measurement showed oil contact angle reaching to 112.5° and 120.5° for the systems of (III) and (IV), respectively. Radial distribution function calculation showed more nanoparticles approaching the calcite surface in the presence of ions yielding to more detachment of the oil molecules and also showed the Na+ and Cl− ions more effects than Mg2+ and SO42−.

Internal water channel formation in CXCR4 is crucial for Gi-protein coupling upon activation by CXCL12

Chemokine receptor CXCR4 is a major drug target for numerous diseases because of its involvement in the regulation of cell migration and the developmental process. In this study, atomic-level molecular dynamics simulations were used to determine the activation mechanism and internal water formation of CXCR4 in complex with chemokine CXCL12 and Gi-protein. The results indicated that CXCL12-bound CXCR4 underwent transmembrane 6 (TM6) outward movement and a decrease in tyrosine toggle switch by eliciting the breakage of hydrophobic layer to form a continuous internal water channel. In the GDP-bound Gαi-protein state, the rotation and translation of the α5-helix of Gαi-protein toward the cytoplasmic pocket of CXCR4 induced an increase in interdomain distance for GDP leaving. Finally, an internal water channel formation model was proposed based on our simulations for CXCL12-bound CXCR4 in complex with Gαi-protein upon activation for downstream signaling. This model could be useful in anticancer drug development.

High-flux anti-fouling nanofibrous composite ultrafiltration membranes containing negatively charged water channels

High-flux nanofibrous composite ultrafiltration (UF) membranes consist of nanocellulose integrated polyacrylonitrile (PAN) as the barrier layer and electrospun PAN nanofibers deposited on a polyethylene terephthalate (PET) non-woven mat as the supporting layer were fabricated by solution coating approach. The chemical composition of nanofibrous composite membranes was investigated with ATR-FTIR, TGA, and EDX measurements, while the top and cross-sectional morphology of the membranes were observed via SEM and AFM scans. An interpenetrating nanofiber-polymer network was created in the barrier layer which drastically enhanced the mechanical strength of the composite membrane. The hydrophilicity of the nanofibrous composite membranes increased with nanocellulose component evidenced by water contact angle measurements, which could enhance the anti-fouling properties of the membranes. The as-prepared membranes were investigated for protein separation and wastewater treatment. The ultrafiltration performance of nanofibrous composite membranes with different composition ratios of nanocellulose to PAN was demonstrated using a cross-flow filtration system, the highest pure water permeability of 1508 L/(m2h·MPa) was achieved with a coating solution of 0.10 wt% of nanocellulose and 0.05 wt% of PAN where it created optimized water channels in the barrier layer of the composite membranes. The molecular weight cut-off (MWCO) of these membranes was determined with dextran as a marker, where the maximum pore size of the membranes varied in the range of 20.8–46.0 nm. Moreover, proteins and polypeptides with different molecular weights were employed to challenge the nanofiber composite membrane performance. High permeation flux of 147.1 L/m2h and high rejection ratio of 98.4% were accomplished; meanwhile, the high selectivity against protein and polypeptides was attained, indicating the membrane's applicability for the selective separation. The anti-fouling properties of the nanofibrous composite membranes were also intensely investigated and the filtration efficiency, in terms of permeation flux and rejection ratio, was evaluated and compared with that of commercially available UF membranes (UN100, US050, and UE050) that possess similar membrane pore size. The permeation flux of the nanofibrous composite membrane was 2–3 times higher than that of commercial membranes, due to the formation of water channels in the barrier layer, while the rejection ratio remained the same as 99.6%, indicating that the nanofibrous composite membrane could be a good candidate for wastewater treatment, in addition to the highly selective separation of proteins.

Cyclic γ-Peptides With Transmembrane Water Channel Properties.

Self-assembling peptides can be used to design new materials for medical and biological applications. Here we synthesized and characterized two novel cyclic γ-peptides (γ-CPs) with hydrophobic inner surfaces. The NMR and FT-IR studies confirmed that the CPs could self-assemble into parallel stacking structures via intermolecular H-bonds and π-π interactions. The morphologies of the self-assembly CPs showed bundles of nanotubes via transmission electron microscopy (TEM); these nanotubes form water channels to transport water across the lipid membrane. The properties of blocking the transport of protons like natural water channels showed that the hydrophobic inner surfaces are important in artificial transmembrane water channel designs. These studies also showed that water transport was a function of pore size and length of the assemblies.

Contribution of Tricellulin to Paracellular Water Permeability in Two Epithelial Cell Lines of Different Tightness

Large amounts of water molecules pass through epithelia driven by osmotic gradients along two pathways, paracellular, transcellular, or both. The transcellular pathway comprises aquaporin (AQP) water channels and some carriers like SGLT1. The existence of a paracellular pathway for water through the tight junctions (TJ) was discussed controversially for many years until molecular components of the paracellular water transport, claudin‐2 and claudin‐15, were identified. Recently, an effect of angulin‐2 (ILDR1) on water transport was shown, suggesting that the tricellular tight junction (tTJ) is also involved, but the role of its barrier‐forming protein, tricellulin, was not elucidated so far.For analyzing this, two epithelial cell lines featuring properties of tight epithelium and intermediate‐tight epithelium, Madin‐Darby canine kidney cells clone 7 (MDCK C7) and human colon cells (HT‐29/B6), respectively, were stably transfected with shRNA targeting tricellulin. Standard molecular biological techniques were applied to analyze expression, localization and function of transfected/regulated TJ proteins. Transepithelial water transport was measured in clones with unchanged expression of other transport‐relevant proteins before and after TJ modification using mannitol or 4‐kDa dextran as an osmotic gradient.After shRNA transfection into MDCK C7 cells, two knockdown (KD) clones with lowest expression of tricellulin were chosen. Tricellulin KD downregulated the expression of occludin and claudin‐4 and ‐8 but upregulated that of claudin‐1, thus roughly balancing expression of the tightening proteins. TER was reduced in both tricellulin KD clones; however, the permeability to 4‐kDa FITC‐dextran was increased only in the clone exhibiting the highest reduction of tricellulin. Nonetheless, water fluxes induced by a gradient of mannitol or 4‐kDa dextran were greatly increased in both tricellulin KD clones compared with their respective controls. In the intermediate‐tight epithelial cell line, HT‐29/B6, again two tricellulin KD clones were chosen. Tricellulin KD upregulated the expression of tightening claudins (‐1, ‐3 and ‐8) and angulin‐1, but also upregulated the expression of the claudin‐2 and AQP‐4, both forming water channels. On the other hand, AQP‐3 was downregulated. Comparable to what was found in MDCK C7 cells, tricellulin KD caused TER to decrease and permeability to 4‐kDa FITC‐dextran to increase compared to controls. Remarkably, water fluxes were not significantly different between the tricellulin KD clones and their controls.We conclude that in MDCK C7 cells, a cell line devoid of claudin‐2‐based water channels, tricellulin increases water permeability at reduced expression. In contrast, in HT‐29/B6 cells, tricellulin KD does not significantly alter transepithelial water transport, most probably concealed by the presence of larger transport rates mediated by other proteins. All in all, the contribution of tricellulin to overall water transport becomes significant if no other major transport pathways for water are present.Support or Funding InformationSupported by DFG GRK 2318 A3

Involvement of PDZ‐SAP97 Interactions in Regulating AQP2 Translocation in Response to Vasopressin in LLC‐PK1 cells

Arginine‐vasopressin‐ (AVP) mediated translocation of aquaporin‐2 (AQP2) protein forming water channels from storage vesicles to the membrane of renal collecting ducts is critical for renal conservation of water. The type‐1 PDZ‐binding motif (PBM) in AQP2 “GTKA” is a critical barcode for its translocation, but its precise role and that of its interacting protein partners in this process remains obscure. We determined that SAP97, a MAGUK protein involved in establishing epithelial cell polarity was an avid binding partner to the PBM of AQP2. The role of the PBM and SAP97 on AQP2 redistribution in response to AVP was assessed in LLC‐PK1 renal collecting cells by confocal microscopy and by cell surface biotinylation techniques. These studies indicated that the distribution of AQP2 and SAP97 overlapped in the kidneys and in LLC‐PK1 and that knockdown of SAP97 inhibited the translocation of AQP2 in response to AVP. The binding between AQP2 and SAP97 was mediated by specific interactions between the second PDZ of SAP97 and the PBM of AQP2. Mechanistically, inactivation of the PBM of AQP2, global delocalization of protein kinase A (PKA) or knockdown of SAP97, inhibited AQP2 translocation as well as AVP‐ and forskolin‐mediated phosphorylation of serine256 in AQP2, which serves as the major translocation barcode of AQP2. These results suggest that targeting of PKA to the AQP2 microdomain via SAP97‐AQP2 interactions in association with cross‐talk between two barcodes in AQP2, namely the PBM and phospho‐Ser256, play an important role in the translocation of AQP2 forming channels in the kidney.Support or Funding InformationThis work was supported by the National Institutes of Health grant: HL‐085848 and by Bridge Funding support by the University of Tennessee‐HSC.

Development and Characterization of the Shortest Anti-Adhesion Peptide Analogue of B49Mod1.

Inhibition of cancer cell adhesion is an effective approach to killing adherent cancer cells. B49 and its analog B49Mod1 peptides, derived from the extracellular domain (ECD) of bone marrow stromal antigen 2 (BST-2), display anti-adhesion activity on breast cancer cells. However, the minimal sequence required for this anti-adhesion activity is unknown. Here, we further characterized the anti-adhesion activity of B49Mod1. We show that the anti-adhesion activity of B49Mod1 may require cysteine-linked disulfide bond and that the peptide is susceptible to proteolytic deactivation. Using structure-activity relationship studies, we identified an 18-Mer sequence (B18) as the minimal peptide sequence mediating the anti-adhesion activity of B49Mod1. Atomistic molecular dynamic (MD) simulations reveal that B18 forms a stable complex with the ECD of BST-2 in aqueous solution. MD simulations further reveal that B18 may cause membrane defects that facilitates peptide translocation across the bilayer. Placement of four B18 chains as a transmembrane bundle results in water channel formation, indicating that B18 may impair membrane integrity and form pores. We hereby identify B18 as the minimal peptide sequence required for the anti-adhesion activity of B49Mod1 and provide atomistic insight into the interaction of B18 with BST-2 and the cell membrane.