48 results on '"Shlyonsky V"'
Search Results
2. Does NAD(P)H oxidase-derived H2O2 participate in hypotonicity-induced insulin release by activating VRAC in β-cells?
- Author
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Crutzen, R., Shlyonsky, V., Louchami, K., Virreira, M., Hupkens, E., Boom, A., Sener, A., Malaisse, W. J., and Beauwens, R.
- Published
- 2012
- Full Text
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3. Mechanosensitivity of gramicidin A channels in bulged bilayer membranes at constant tension
- Author
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Markin, V. S., Shlyonsky, V. Gh., Simon, S. A., Benos, D. J., and Ismailov, I. I.
- Published
- 2006
- Full Text
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4. The OpenPicoAmp-100k : an open-source high performance amplifier for single channel recording in planar lipid bilayers
- Author
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Shlyonsky, V, primary and Gall, D, additional
- Published
- 2019
- Full Text
- View/download PDF
5. Membrane-active properties of ferutinin
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Charishnikova, O., Dubis, A., Siergiejczyk, L., Shlyonsky, V., and Zamaraeva, M.
- Subjects
ТЕХНИЧЕСКИЕ И ПРИКЛАДНЫЕ НАУКИ. ОТРАСЛИ ЭКОНОМИКИ::Биотехнология [ЭБ БГУ] - Abstract
Мембранная биофизика
- Published
- 2016
6. Hydrogen Peroxide and Sodium Transport in the Lung and Kidney
- Author
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Shlyonsky, V., Boom, A., and Mies, F.
- Subjects
Article Subject ,urogenital system ,respiratory system - Abstract
Renal and lung epithelial cells are exposed to some significant concentrations of H2O2. In urine it may reach 100 μM, while in the epithelial lining fluid in the lung it is estimated to be in micromolar to tens-micromolar range. Hydrogen peroxide has a stimulatory action on the epithelial sodium channel (ENaC) single-channel activity. It also increases stability of the channel at the membrane and slows down the transcription of the ENaC subunits. The expression and the activity of the channel may be inhibited in some other, likely higher, oxidative states of the cell. This review discusses the role and the origin of H2O2 in the lung and kidney. Concentration-dependent effects of hydrogen peroxide on ENaC and the mechanisms of its action have been summarized. This review also describes outlooks for future investigations linking oxidative stress, epithelial sodium transport, and lung and kidney function.
- Published
- 2016
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7. Ferutinin as a Ca2+complexone: lipid bilayers, conductometry, FT-IR, NMR studies and DFT-B3LYP calculations
- Author
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Dubis, A., primary, Zamaraeva, M. V., additional, Siergiejczyk, L., additional, Charishnikova, O., additional, and Shlyonsky, V., additional
- Published
- 2015
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8. Does NAD(P)H oxidase-derived H2O2 participate in hypotonicity-induced insulin release by activating VRAC in β-cells?
- Author
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Crutzen, R., primary, Shlyonsky, V., additional, Louchami, K., additional, Virreira, M., additional, Hupkens, E., additional, Boom, A., additional, Sener, A., additional, Malaisse, W. J., additional, and Beauwens, R., additional
- Published
- 2011
- Full Text
- View/download PDF
9. Ferutinin as a Ca2+ complexone: lipid bilayers, conductometry, FT-IR, NMR studies and DFT-B3LYP calculations.
- Author
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Dubis, A., Zamaraeva, M. V., Siergiejczyk, L., Charishnikova, O., and Shlyonsky, V.
- Subjects
BILAYER lipid membranes ,CONDUCTOMETRIC analysis ,STOICHIOMETRY ,CALCIUM ions ,HYDROXYL group ,PHENOL - Abstract
Calcium ionophoretic properties of ferutinin were re-evaluated in solvent-containing bilayer lipid membranes. The slopes of conductance–concentration curves suggest that in the presence of a solvent in the membrane the majority of complexes appear to consist of a single terpenoid molecule bound to one Ca ion. By contrast, the stoichiometry of ferutinin–Ca
2+ complexes in acetone determined using the conductometric method was 2 : 1. While the cation–cation selectivity of ferutinin did not change, the cation–anion selectivity slightly decreased in solvent containing membranes. FT-IR and NMR data together with DFT calculations at the B3LYP/6-31G(d) level of theory indicate that in the absence of Ca ions ferutinin molecules are hydrogen-bonded at the phenol hydroxyl groups. The variations of absorption assigned to –OH and –C–O stretching mode suggest that ferutinin interacts strongly with Ca ions via the hydroxyl group of ferutinol and carboxyl oxygen of the complex ether bond. The coordination through the carbonyl group of ferutinin was demonstrated by theoretical calculations. Taken together, ferutinin molecules form H-bonded dimers, while complexation of Ca2+ by ferutinin ruptures this hydrogen bond due to spatial re-orientation of the ferutinin molecules from parallel to antiparallel alignment. [ABSTRACT FROM AUTHOR]- Published
- 2015
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10. Role of actin in regulation of epithelial sodium channels by CFTR
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Ismailov, I. I., primary, Berdiev, B. K., additional, Shlyonsky, V. G., additional, Fuller, C. M., additional, Prat, A. G., additional, Jovov, B., additional, Cantiello, H. F., additional, Ausiello, D. A., additional, and Benos, D. J., additional
- Published
- 1997
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11. Protein kinase A phosphorylation and G protein regulation of type II pneumocyte Na+ channels in lipid bilayers
- Author
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Berdiev, B. K., primary, Shlyonsky, V. G., additional, Senyk, O., additional, Keeton, D., additional, Guo, Y., additional, Matalon, S., additional, Cantiello, H. F., additional, Prat, A. G., additional, Ausiello, D. A., additional, Ismailov, I. I., additional, and Benos, D. J., additional
- Published
- 1997
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12. A biologic function for an "orphan" messenger: D-myo-inositol 3,4,5,6-tetrakisphosphate selectively blocks epithelial calcium-activated chloride channels.
- Author
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Ismailov, I I, primary, Fuller, C M, additional, Berdiev, B K, additional, Shlyonsky, V G, additional, Benos, D J, additional, and Barrett, K E, additional
- Published
- 1996
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13. Regulation of epithelial Na(+) channels by actin in planar lipid bilayers and in the Xenopus oocyte expression system.
- Author
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Jovov, B, Tousson, A, Ji, H L, Keeton, D, Shlyonsky, V, Ripoll, P J, Fuller, C M, and Benos, D J
- Abstract
The hypothesis that actin interactions account for the signature biophysical properties of cloned epithelial Na(+) channels (ENaC) (conductance, ion selectivity, and long mean open and closed times) was tested using planar lipid bilayer reconstitution and patch clamp techniques. We found the following. 1) In bilayers, actin produced a more than 2-fold decrease in single channel conductance, a 5-fold increase in Na(+) versus K(+) permselectivity, and a substantial increase in mean open and closed times of wild-type alphabetagamma-rENaC but had no effect on a mutant form of rENaC in which the majority of the C terminus of the alpha subunit was deleted (alpha(R613X)betagamma-rENaC). 2) When alpha(R613X)betagamma-rENaC was heterologously expressed in oocytes and single channels examined by patch clamp, 12.5-pS channels of relatively low cation permeability were recorded. These characteristics were identical to those recorded in bilayers for either alpha(R613X)betagamma-rENaC or wild-type alphabetagamma-rENaC in the absence of actin. Moreover, we show that rENaC subunits tightly associate, forming either homo- or heteromeric complexes when prepared by in vitro translation or when expressed in oocytes. Finally, we show that alpha-rENaC is properly assembled but retained in the endoplasmic reticulum compartment. We conclude that actin subserves an important regulatory function for ENaC and that planar bilayers are an appropriate system in which to study the biophysical and regulatory properties of these cloned channels.
- Published
- 1999
14. Identification of an amiloride binding domain within the alpha-subunit of the epithelial Na+ channel.
- Author
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Ismailov, I I, Kieber-Emmons, T, Lin, C, Berdiev, B K, Shlyonsky, V G, Patton, H K, Fuller, C M, Worrell, R, Zuckerman, J B, Sun, W, Eaton, D C, Benos, D J, and Kleyman, T R
- Abstract
Limited information is available regarding domains within the epithelial Na+ channel (ENaC) which participate in amiloride binding. We previously utilized the anti-amiloride antibody (BA7.1) as a surrogate amiloride receptor to delineate amino acid residues that contact amiloride, and identified a putative amiloride binding domain WYRFHY (residues 278-283) within the extracellular domain of alpharENaC. Mutations were generated to examine the role of this sequence in amiloride binding. Functional analyses of wild type (wt) and mutant alpharENaCs were performed by cRNA expression in Xenopus oocytes and by reconstitution into planar lipid bilayers. Wild type alpharENaC was inhibited by amiloride with a Ki of 169 nM. Deletion of the entire WYRFHY tract (alpharENaC Delta278-283) resulted in a loss of sensitivity of the channel to submicromolar concentrations of amiloride (Ki = 26.5 microM). Similar results were obtained when either alpharENaC or alpharENaC Delta278-283 were co-expressed with wt beta- and gammarENaC (Ki values of 155 nM and 22.8 microM, respectively). Moreover, alpharENaC H282D was insensitive to submicromolar concentrations of amiloride (Ki = 6.52 microM), whereas alpharENaC H282R was inhibited by amiloride with a Ki of 29 nM. These mutations do not alter ENaC Na+:K+ selectivity nor single-channel conductance. These data suggest that residues within the tract WYRFHY participate in amiloride binding. Our results, in conjunction with recent studies demonstrating that mutations within the membrane-spanning domains of alpharENaC and mutations preceding the second membrane-spanning domains of alpha-, beta-, and gammarENaC alters amiloride's Ki, suggest that selected regions of the extracellular loop of alpharENaC may be in close proximity to residues within the channel pore.
- Published
- 1997
15. Kinetic interconversion of rat and bovine homologs of the alpha subunit of an amiloride-sensitive Na+ channel by C-terminal truncation of the bovine subunit.
- Author
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Fuller, C M, Ismailov, I I, Berdiev, B K, Shlyonsky, V G, and Benos, D J
- Abstract
We have recently cloned the alpha subunit of a bovine amiloride-sensitive Na+ channel (alphabENaC). This subunit shares extensive homology with both rat and human alphaENaC subunits but shows marked divergence at the C terminus beginning at amino acid 584 of the 697-residue sequence. When incorporated into planar lipid bilayers, alphabENaC almost exclusively exhibits a main transition to 39 picosiemens (pS) with very rare 13 pS step transitions to one of two subconductance states (26 and 13 pS). In contrast, the alpha subunit of the rat renal homolog of ENaC (alpharENaC) has a main transition step to 13 pS that is almost constituitively open, with a second stepwise transition of 26 to 39 pS. A deletion mutant of alphabENaC, encompassing the entire C-terminal region (R567X), converts the kinetic behavior of alphabENaC to that of alpharENaC, i. e. a transition to 13 pS followed by a second 26 pS transition to 39 pS. Chemical cross-linking of R567X restores the wild-type alphabENaC gating pattern, whereas treatment with the reducing agent dithiothreitol produced only 13 pS transitions. In contrast, an equivalent C-terminal truncation of alpharENaC (R613X) had no effect on the gating pattern of alpharENaC. These results are consistent with the hypothesis that interactions between the C termini of alphabENaC account for the different kinetic behavior of this member of the ENaC family of Na+ channels.
- Published
- 1996
16. Carboxylmethylation of the beta subunit of xENaC regulates channel activity.
- Author
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Rokaw, M D, Wang, J M, Edinger, R S, Weisz, O A, Hui, D, Middleton, P, Shlyonsky, V, Berdiev, B K, Ismailov, I, Eaton, D C, Benos, D J, and Johnson, J P
- Abstract
The action of aldosterone to increase apical membrane permeability in responsive epithelia is thought to be due to activation of sodium channels. Aldosterone stimulates methylation of a 95-kDa protein in apical membrane of A6 cells, and we have previously shown that methylation of a 95-kDa protein in the immunopurified Na+ channel complex increases open probability of these channels in planar lipid bilayers. We report here that aldosterone stimulates carboxylmethylation of the beta subunit of xENaC in A6 cells. In vitro translated beta subunit, but not alpha or gamma, serves as a substrate for carboxylmethylation. Carboxylmethylation of ENaC reconstituted in planar lipid bilayers leads to an increase in open probability only when beta subunit is present. When the channel complex is immunoprecipitated from A6 cells and analyzed by Western blot with antibodies to the three subunits of xENaC, all three subunits are recognized as constituents of the complex. The results suggest that Na+ channel activity in A6 cells is regulated, in part, by carboxylmethylation of the beta subunit of xENaC.
- Published
- 1998
17. Antitubercular potential and pH-driven mode of action of salicylic acid derivatives.
- Author
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Laudouze J, Francis T, Forest E, Mies F, Bolla JM, Crauste C, Canaan S, Shlyonsky V, Santucci P, and Cavalier JF
- Abstract
In the search for new antituberculosis drugs with novel mechanisms of action, we evaluated the antimycobacterial activity of a panel of eight phenolic acids against four pathogenic mycobacterial model species, including Mycobacterium tuberculosis. We demonstrated that salicylic acid (SA), as well as the iodinated derivatives 5-iodo-salicylic acid (5ISA) and 3,5-diiodo-salicylic acid (3,5diISA), displayed promising antitubercular activities. Remarkably, using a genetically encoded mycobacterial intrabacterial pH reporter, we describe for the first time that SA, 5ISA, 3,5diISA, and the anti-inflammatory drug aspirin (ASP) act by disrupting the intrabacterial pH homeostasis of M. tuberculosis in a dose-dependent manner under in vitro conditions mimicking the endolysosomal pH of macrophages. In contrast, the structurally related second-line anti-TB drug 4-aminosalicylic acid (PAS) had no pH-dependent activity and was strongly antagonized by l-methionine supplementation, thereby suggesting distinct modes of action. Finally, we propose that SA, ASP, and its two iodinated derivatives could restrict M. tuberculosis growth in a pH-dependent manner by acidifying the cytosol of the bacilli, therefore making such compounds very attractive for further development of antibacterial agents., (© 2024 The Author(s). FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)
- Published
- 2024
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18. Cell Type-Specific Anti- and Pro-Oxidative Effects of Punica granatum L. Ellagitannins.
- Author
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Olchowik-Grabarek E, Sekowski S, Mierzwinska I, Zukowska I, Abdulladjanova N, Shlyonsky V, and Zamaraeva M
- Abstract
Pomegranate and its by-products contain a broad spectrum of phytochemicals, such as flavonoids, phenolic acids and tannins, having pleiotropic preventive and prophylactic properties in health disorders related to oxidative stress and microbial contamination. Here, we examined the biological effects of a pomegranate peel ellagitannins-enriched (>90%) extract, PETE. In vitro studies revealed that PETE has a strong antiradical action towards synthetic radicals and biologically relevant ROS surpassing or comparable to that of Trolox. In cellular models, it showed concentration-dependent (25-100 µg/mL) yet opposing effects depending on the cell membrane type and exposure conditions. In erythrocytes, PETE protected membrane integrity in the presence of the strong oxidant HClO and restored reduced glutathione levels to up to 85% of the control value while having much weaker acute and long-term intrinsic effects. Such protection persisted even after the removal of the extract from cells, indicating strong membrane interaction. In HeLa cancer cells, and at concentrations lower than those used for red blood cells, PETE induced robust potentiation of ROS production and mitochondrial potential dissipation, leading to autophagy-like membrane morphology changes and cell death. In S. aureus , the growth arrest and bacterial death in the presence of PETE (with MIC = 31.25 µg/mL and MBC = 125 µg/mL, respectively) can be linked to the tripled ROS induction by the extract in the same concentration range. This study indicates a specificity of ROS production by the pomegranate extract depending on the type of cell, the concentration of the extract and the time of incubation. This specificity witnesses a strong potential of the extract components as candidates in antioxidant and pro-oxidant therapy.
- Published
- 2024
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19. Neurosimilator for Undergraduate Biophysics and Neurophysiology Courses.
- Author
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Dupuis F, Shlyonsky V, de Prelle B, and Gall D
- Abstract
Stringent animal welfare principles are forcing undergraduate instructors to avoid the use of animals. Therefore, many hands-on lab sessions using laboratory animals are progressively replaced by computer simulations. These versatile software simulations permit the observation of the behavior of biological systems under a great variety of experimental conditions. While this versatility is important, computer simulations often work even when a student makes wrong assumptions, a situation that poses its own pedagogical problem. Hands-on learning provides pupils with the opportunity to safely make mistakes and learn organically through trial and error and should therefore still be promoted. We propose an electronic model of an excitable cell composed of different modules representing different parts of a neuron - dendrites, soma, axon and node of Ranvier. We describe a series of experiments that allow students to better understand differences between passive and active cell responses and differences between myelinated and demyelinated axons. These circuits can also be used to demonstrate temporal and spatial summation of signals coming to the neuron via dendrites, as well as the neuron coding by firing frequency. Finally, they permit experimental determination along with theoretical calculations of important biophysical properties of excitable cells, such as rheobase, chronaxie and space constant. This open-source model has been successfully integrated into an undergraduate course of the physiology of excitable cells and student feedback assessment reveals that it helped students to understand important notions of the course. Thus, this neuromorphic circuit could be a valuable tool for biophysics and neuroscience courses in other universities., (Copyright © 2024 Faculty for Undergraduate Neuroscience.)
- Published
- 2024
- Full Text
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20. Electrophysiological and spectroscopic investigation of hydrolysable tannins interaction with α-hemolysin of S. aureus.
- Author
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Olchowik-Grabarek E, Sekowski S, Mies F, Bitiucki M, Swiecicka I, Abdulladjanova N, Shlyonsky V, and Zamaraeva M
- Subjects
- Hemolysin Proteins, Staphylococcus aureus, Tannins pharmacology, Tannins chemistry, Glucose, Hydrolyzable Tannins pharmacology, Methicillin-Resistant Staphylococcus aureus
- Abstract
In this study, using bilayer lipid membrane technique, we report a novel facet of antihemolytic activity of two tannins (1,2,3,4,5-penta-O-galloyl-β-D-glucose (PGG) and 1,2-di-O-galloyl-4,6-valoneoyl-β-D-glucose (dGVG)), which consists in inhibiting the formation of α-hemolysin channels and blocking the conductivity of already formed channels. These effects were observed at tannin concentrations well below minimal inhibitory concentration values for S. aureus growth. Using spectroscopic methods, we show that these two tannins differing in molecular structure but having the same number of -OH groups and aromatic rings form firm complexes with hemolysin in aqueous solutions, which may underlie the disruption of its subsequent interaction with the membrane, thus preventing hemolysis of erythrocytes. In all experimental settings, PGG was the more active compound compared to dGVG, that indicates the important role of the flexibility of the tannin molecule in interaction with the toxin. In addition, we found that PGG, but not dGVG, was able to block the release of the toxin by bacterial cells. This toxin is a strong pathogenic factor causing a number of diseases and therefore is considered as a virulence target for treatment of S. aureus infection, so the data obtained suggest that PGG and possibly other tannins of similar structure have therapeutic potential in fighting the virulence of S. aureus., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)
- Published
- 2023
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21. The Structural Changes in the Membranes of Staphylococcus aureus Caused by Hydrolysable Tannins Witness Their Antibacterial Activity.
- Author
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Olchowik-Grabarek E, Sękowski S, Kwiatek A, Płaczkiewicz J, Abdulladjanova N, Shlyonsky V, Swiecicka I, and Zamaraeva M
- Abstract
Polyphenols, including tannins, are phytochemicals with pronounced antimicrobial properties. We studied the activity of two hydrolysable tannins, (i) gallotannin-1,2,3,4,5-penta-O-galloyl-β-D-glucose (PGG) and (ii) ellagitannin-1,2-di-O-galloyl-4,6-valoneoyl-β-D-glucose (dGVG), applied alone and in combination with antibiotics against Staphylococcus aureus strain 8324-4. We also evaluated the effect of these tannins on bacterial membrane integrity and fluidity and studied their interaction with membrane proteins and lipids. A correlation between the antimicrobial activity of the tannins and their membranotropic action depending on the tannin molecular structure has been demonstrated. We found that the antibacterial activity of PGG was stronger than dGVG, which can be associated with its larger flexibility, dipole moment, and hydrophobicity. In addition, we also noted the membrane effects of the tannins observed as an increase in the size of released bacterial membrane vesicles.
- Published
- 2022
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22. Enzymatic synthesis and characterization of aryl iodides of some phenolic acids with enhanced antibacterial properties.
- Author
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Olchowik-Grabarek E, Mies F, Sekowski S, Dubis AT, Laurent P, Zamaraeva M, Swiecicka I, and Shlyonsky V
- Subjects
- Anti-Bacterial Agents pharmacology, Microbial Sensitivity Tests, Staphylococcus aureus, Hemolysin Proteins, Iodides pharmacology
- Abstract
Phenolic acids represent a class of drugs with mild antibacterial properties. We have synthesized iodinated gallic and ferulic acids and together with commercially available iodinated forms of salicylic acids studied their cytotoxicity, bacteriostatic and anti-virulence action. Out of these, iodogallic acid had lowest minimal inhibitory concentration (MIC) against Staphylococcus aureus (MIC = 0.4 mM/118.8 μg/ml). Yet, it had strong effect on erythrocyte membrane lipid ordering and on α-hemolysin secretion by the bacteria at lower non-bacteriostatic and non-cytotoxic concentrations (<0.1 mM). Iodogallic acid formed static complexes with α-hemolysin in solutions (logK
b = 4.69 ± 0.07) and inhibited its nano-pore conduction in artificial lipid bilayers (IC50 = 37.9 ± 5.3 μM). These effects of iodogallic acid converged on prevention of hemolysis induced by α-hemolysin (IC50 = 41.5 ± 4.2 μM) and pointed to enhanced and diverse anti-virulence properties of some aryl iodides. The analysis of molecular surface electrostatic charge distribution, molecular hydrophilicity, electronegativity, and dipole moment of studied compounds suggested the importance of the number of hydroxyl groups and their proximity to iodine in anti-virulence activity manifestation. In iodogallic acid, charge redistribution resulted in higher hydrophilicity without concomitant change in overall molecular electronegativity and dipole moment compared to non-iodinated gallic acid. This study shows new directions for the development of antibacterial/antivirulence therapeutics., (Copyright © 2022 Elsevier B.V. All rights reserved.)- Published
- 2022
- Full Text
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23. Inhibition of interaction between Staphylococcus aureus α-hemolysin and erythrocytes membrane by hydrolysable tannins: structure-related activity study.
- Author
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Olchowik-Grabarek E, Sekowski S, Bitiucki M, Dobrzynska I, Shlyonsky V, Ionov M, Burzynski P, Roszkowska A, Swiecicka I, Abdulladjanova N, and Zamaraeva M
- Subjects
- Animals, Erythrocyte Membrane drug effects, Euphorbiaceae chemistry, Gallic Acid analogs & derivatives, Glucose analogs & derivatives, Plant Extracts chemistry, Sheep, Staphylococcus aureus enzymology, Tannins chemistry, Hemolysin Proteins pharmacology, Hemolysis drug effects, Hemolytic Agents pharmacology, Plant Extracts pharmacology, Tannins pharmacology
- Abstract
The objective of the study was a comparative analysis of the antihemolytic activity against two Staphylococcus aureus strains (8325-4 and NCTC 5655) as well as α-hemolysin and of the membrane modifying action of four hydrolysable tannins with different molecular mass and flexibility: 3,6-bis-O-di-O-galloyl-1,2,4-tri-O-galloyl-β-D-glucose (T1), 1,2,3,4,5-penta-O-galloyl-β-D-glucose (T2), 3-O-galloyl-1,2-valoneoyl-β-D-glucose (T3) and 1,2-di-O-galloyl-4,6-valoneoyl-β-D-glucose (T4). We showed that all the compounds studied manifested antihemolytic effects in the range of 5-50 µM concentrations. However, the degree of the reduction of hemolysis by the investigated tannins was not uniform. A valoneoyl group-containing compounds (T3 and T4) were less active. Inhibition of the hemolysis induced by α-hemolysin was also noticed on preincubated with the tannins and subsequently washed erythrocytes. In this case the efficiency again depended on the tannin structure and could be represented by the following order: T1 > T2 > T4 > T3. We also found a relationship between the degree of antihemolytic activity of the tannins studied and their capacity to increase the ordering parameter of the erythrocyte membrane outer layer and to change zeta potential. Overall, our study showed a potential of the T1 and T2 tannins as anti-virulence agents. The results of this study using tannins with different combinations of molecular mass and flexibility shed additional light on the role of tannin structure in activity manifestation.
- Published
- 2020
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24. The OpenPicoAmp-100k: an open-source high-performance amplifier for single channel recording in planar lipid bilayers.
- Author
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Shlyonsky V and Gall D
- Subjects
- Amplifiers, Electronic, Ion Channels metabolism, Electrophysiology methods, Lipid Bilayers metabolism
- Abstract
We propose an upgraded version of our previously designed open-source lipid bilayer amplifier. This improved amplifier is now suitable both for the use in introductory courses in biophysics and neurosciences at the undergraduate level and for scientific research. Similar to its predecessor, the OpenPicoAmp-100k is designed using the common lithographic printed circuit board fabrication process and off-the-shelf electronic components. It consists of the high-speed headstage, followed by voltage-gain amplifier with built-in 6-order Bessel filter. The amplifier has a bandwidth of 100 kHz in the presence of 100-pF input membrane capacitance and is capable of measuring ion channel current with amplitudes from sub-pA and up to ± 4 nA. At the full bandwidth and with a 1 GΩ transimpedance gain, the amplifier shows 12 pA
rms noise with an open input and 112 pArms noise in the presence of 100-pF input capacitance, while at the 5-kHz bandwidth (typical in single-channel experiments), noise amounts to 0.45 pArms and 2.11 pArms , respectively. Using an optocoupler circuit producing TTL-controlled current impulses and using 50% threshold analysis, we show that at full bandwidth, the amplifier has deadtimes of 3.5 μs and 5 μs at signal-to-noise ratios (SNR) of 9 and 1.7, respectively. Near 100% of true current impulses longer than 5 μs and 6.6 μs are detected at these two respective SNRs, while false event detection rate remains acceptably low. The wide bandwidth of the amplifier was confirmed in bilayer experiments with alamethicin, for which open ion channel current events shorter that 10 μs could be resolved.- Published
- 2019
- Full Text
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25. DUOX1-mediated hydrogen peroxide release regulates sodium transport in H441 bronchiolar epithelial cells.
- Author
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Mies F, Virreira M, Goolaerts A, Djerbib S, Beauwens R, Shlyonsky V, and Boom A
- Subjects
- Anti-Inflammatory Agents pharmacology, Biological Transport drug effects, Cell Line, Dexamethasone pharmacology, Dual Oxidases genetics, Electrophysiological Phenomena, Gene Expression Regulation drug effects, Humans, Dual Oxidases metabolism, Epithelial Cells drug effects, Epithelial Cells metabolism, Hydrogen Peroxide metabolism, Sodium metabolism
- Abstract
Aim: Dexamethasone has been shown to induce the formation of epithelial domes by bronchiolar H441 cells. It stimulates the expression of both amiloride inhibitable epithelial sodium channels (ENaC) and dual oxidase-1 (DUOX1). We therefore ask the question whether DUOX1 expression and production of submillimolar amounts of H
2 O2 is instrumental for the sodium channel upregulation observed in H441 cells., Methods: In vitro cell culture, nystatin-perforated whole-cell patch-clamp technique, immunocytochemistry and RT-PCR methods have been used., Results: Cells forming epithelial domes induced by dexamethasone (0.1 μmol L-1 , 24 hours) and by 5-aza-2'-deoxytidine (1 μmol L-1 , 48 hours) expressed more DUOX1 protein compared with other cells in the monolayer. Dome formation could be inhibited by exogenous catalase in a concentration-dependent manner and by the NADPH oxidase inhibitor diphenyliodonium, which suggested the involvement of H2 O2 . While single application of 0.2 mmol L-1 H2 O2 induced transient dome formation, lower doses were ineffective and higher doses disrupted the cell monolayer. Hydrogen peroxide (0.1 mmol L-1 ) activated acutely amiloride-sensitive whole-cell currents from 3.91 ± 0.79 pA pF-1 to 4.76 ± 0.98 pA pF-1 in dome-forming cells and had no effect in cells outside of domes. ENaC but not DUOX1 transcription was potentiated by catalase in the presence of dexamethasone, which suggested negative feedback of H2 O2 on ENaC gene expression., Conclusion: Our observations suggest that tonic production of H2 O2 by DUOX1 participates in maintaining the level of vectorial sodium transport by lung epithelial cells. Moreover, the system appears to be well tuned as it would allow H2 O2 -dependent innate immunity without inducing airway/alveolar sodium and fluid hyperabsorption., (© 2018 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)- Published
- 2019
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26. Prophylactic and curative effect of rosemary leaves extract in a bleomycin model of pulmonary fibrosis.
- Author
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Bahri S, Ben Ali R, Gasmi K, Mlika M, Fazaa S, Ksouri R, Serairi R, Jameleddine S, and Shlyonsky V
- Subjects
- Animals, Antioxidants pharmacology, Body Weight drug effects, Glutathione metabolism, Lipid Peroxidation drug effects, Lung drug effects, Lung metabolism, Lung pathology, Male, Plant Extracts pharmacology, Plant Leaves chemistry, Pulmonary Fibrosis chemically induced, Pulmonary Fibrosis prevention & control, Rats, Rats, Wistar, Bleomycin toxicity, Plant Extracts therapeutic use, Pulmonary Fibrosis drug therapy, Rosmarinus chemistry
- Abstract
Context: Pulmonary fibrosis is a devastating disease without effective treatment. Rosemary is appreciated since ancient times for its medicinal properties, while biomolecules originated from the plant have an antioxidant and antifibrotic effect., Objective: The effects of Rosmarinus officinalis L. (Lamiaceae) leaves extract (RO) on bleomycin-induced lung fibrosis were investigated., Materials and Methods: Male Wistar rats were given a single dose of bleomycin (BLM, 4 mg/kg, intratracheal), while RO (75 mg/kg, intraperitoneal) was administered 3 days later and continued for 4 weeks (BLM/RO1-curative group). Alternatively, RO was administered 2 weeks before BLM and continued 15 days thereafter (BLM/RO2-prophylactic group). Antioxidant activities of RO and lung tissues were studied by standard methods. Histological staining revealed lung architecture and collagen deposition. RO was characterized for its polyphenol content and by high-performance liquid chromatography., Results: RO polyphenol content was 60.52 mg/g of dry weight, carnosic and rosmarinic acids being major components (6.886 and 2.351 mg/g). Antioxidant effect of RO (DPPH and FRAP assay) expressed as IC
50 values were 2.23 μg/mL and 0.074 μg/mL, respectively. In BLM/RO1 and BLM/RO2 lung architecture was less compromised compared to BLM, which was reflected in lower fibrosis score (2.33 ± 0.33 and 1.8 ± 0.32 vs 3.7 ± 0.3). Malondialdehyde levels were attenuated (141% and 108% vs 258% of normal value). Catalase and glutathione-S-transferase activities were normalized (103% and 117% vs 59%, 85% and 69% vs 23%, respectively)., Discussion and Conclusion: RO has a protective effect against BLM-induced oxidative stress and lung fibrosis due to its phenolic compounds.- Published
- 2017
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27. Rosmarinic acid potentiates carnosic acid induced apoptosis in lung fibroblasts.
- Author
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Bahri S, Mies F, Ben Ali R, Mlika M, Jameleddine S, Mc Entee K, and Shlyonsky V
- Subjects
- Animals, Bleomycin, Catalase metabolism, Cell Cycle, Cell Line, Cell Movement, Cell Proliferation drug effects, Collagen metabolism, Humans, Hydroxyproline metabolism, Inhibitory Concentration 50, Lipid Peroxidation, Lung cytology, Male, Mice, Oxidative Stress, Plant Extracts pharmacology, Pulmonary Alveoli metabolism, Pulmonary Fibrosis drug therapy, Rats, Rats, Wistar, Signal Transduction, Superoxide Dismutase metabolism, Vitamin E metabolism, p38 Mitogen-Activated Protein Kinases metabolism, Rosmarinic Acid, Abietanes pharmacology, Apoptosis, Cinnamates pharmacology, Depsides pharmacology, Fibroblasts drug effects
- Abstract
Pulmonary fibrosis is characterized by over-population and excessive activation of fibroblasts and myofibroblasts disrupting normal lung structure and functioning. Rosemary extract rich in carnosic acid (CA) and rosmarinic acid (RA) was reported to cure bleomycin-(BLM)-induced pulmonary fibrosis. We demonstrate that CA decreased human lung fibroblast (HLF) viability with IC50 value of 17.13±1.06 μM, while RA had no cytotoxic effect. In the presence of 50 μM of RA, dose-response for CA shifted to IC50 value of 11.70±1.46 μM, indicating synergic action. TGFβ-transformed HLF, rat lung fibroblasts and L929 cells presented similar sensitivity to CA and CA+RA (20μM+100μM, respectively) treatment. Rat alveolar epithelial cells died only under CA+RA treatment, while A549 cells were not affected. Annexin V staining and DNA quantification suggested that HLF are arrested in G0/G1 cell cycle phase and undergo apoptosis. CA caused sustained activation of phospho-Akt and phospho-p38 expression and inhibition of p21 protein.Addition of RA potentiated these effects, while RA added alone had no action.Only triple combination of inhibitors (MAPK-p38, pan-caspase, PI3K/Akt/autophagy) partially attenuated apoptosis; this suggests that cytotoxicity of CA+RA treatment has a complex mechanism involving several parallel signaling pathways. The in vivo antifibrotic effect of CA and RA was compared with that of Vitamine-E in BLM-induced fibrosis model in rats. We found comparable reduction in fibrosis score by CA, RA and CA+RA, attenuation of collagen deposition and normalization of oxidative stress markers. In conclusion, antifibrotic effect of CA+RA is due to synergistic pro-apoptotic action on lung fibroblasts and myofibroblasts.
- Published
- 2017
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28. Relevance of carnosic acid to the treatment of several health disorders: Molecular targets and mechanisms.
- Author
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Bahri S, Jameleddine S, and Shlyonsky V
- Subjects
- Abietanes adverse effects, Abietanes pharmacokinetics, Animals, Anti-Obesity Agents adverse effects, Anti-Obesity Agents pharmacokinetics, Antineoplastic Agents, Phytogenic adverse effects, Antineoplastic Agents, Phytogenic pharmacokinetics, Biological Availability, Dermatologic Agents adverse effects, Dermatologic Agents pharmacokinetics, Disease Models, Animal, Gastrointestinal Agents adverse effects, Gastrointestinal Agents pharmacokinetics, Humans, Neuroprotective Agents adverse effects, Neuroprotective Agents pharmacokinetics, Abietanes therapeutic use, Anti-Obesity Agents therapeutic use, Antineoplastic Agents, Phytogenic therapeutic use, Dermatologic Agents therapeutic use, Gastrointestinal Agents therapeutic use, Neuroprotective Agents therapeutic use
- Abstract
Carnosic acid is a phenolic diterperne compound found in abundance in sage and rosemary, which are both widely used in traditional medicine. Research over the past decade indicates that carnosic acid has multiple bioactive properties including antioxidant, anti-inflammatory and anticancer activities among others. This review summarizes the current in vitro and in vivo data about the efficacy of carnosic acid in the prevention or treatment of various experimental health disorders. The analysis of the literature allows an insight into the participation of numerous signaling pathways modulated by carnosic acid, into its synergistic potential and, thus, into the divergence in cellular mechanisms of action of this molecule., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)
- Published
- 2016
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29. Anoctamin 1 (Ano1) is required for glucose-induced membrane potential oscillations and insulin secretion by murine β-cells.
- Author
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Crutzen R, Virreira M, Markadieu N, Shlyonsky V, Sener A, Malaisse WJ, Beauwens R, Boom A, and Golstein PE
- Subjects
- Animals, Anoctamin-1, Calcium metabolism, Cells, Cultured, Chloride Channels antagonists & inhibitors, Chlorides metabolism, Exocytosis, Humans, Insulin-Secreting Cells physiology, Mice, Mice, Inbred C57BL, Rats, Rats, Wistar, Chloride Channels metabolism, Glucose metabolism, Insulin metabolism, Insulin-Secreting Cells metabolism, Membrane Potentials
- Abstract
Anions such as Cl(-) and HCO3 (-) are well known to play an important role in glucose-stimulated insulin secretion (GSIS). In this study, we demonstrate that glucose-induced Cl(-) efflux from β-cells is mediated by the Ca(2+)-activated Cl(-) channel anoctamin 1 (Ano1). Ano1 expression in rat β-cells is demonstrated by reverse transcriptase-polymerase chain reaction, western blotting, and immunohistochemistry. Typical Ano1 currents are observed in whole-cell and inside-out patches in the presence of intracellular Ca(++): at 1 μM, the Cl(-) current is outwardly rectifying, and at 2 μM, it becomes almost linear. The relative permeabilities of monovalent anions are NO3 (-) (1.83 ± 0.10) > Br(-) (1.42 ± 0.07) > Cl(-) (1.0). A linear single-channel current-voltage relationship shows a conductance of 8.37 pS. These currents are nearly abolished by blocking Ano1 antibodies or by the inhibitors 2-(5-ethyl-4-hydroxy-6-methylpyrimidin-2-ylthio)-N-(4-(4-methoxyphenyl)thiazol-2-yl)acetamide (T-AO1) and tannic acid (TA). These inhibitors induce a strong decrease of 16.7-mM glucose-stimulated action potential rate (at least 87 % on dispersed cells) and a partial membrane repolarization with T-AO1. They abolish or strongly inhibit the GSIS increment at 8.3 mM and at 16.7 mM glucose. Blocking Ano1 antibodies also abolish the 16.7-mM GSIS increment. Combined treatment with bumetanide and acetazolamide in low Cl(-) and HCO3 (-) media provokes a 65 % reduction in action potential (AP) amplitude and a 15-mV AP peak repolarization. Although the mechanism triggering Ano1 opening remains to be established, the present data demonstrate that Ano1 is required to sustain glucose-stimulated membrane potential oscillations and insulin secretion.
- Published
- 2016
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30. Ferutinin as a Ca(2+) complexone: lipid bilayers, conductometry, FT-IR, NMR studies and DFT-B3LYP calculations.
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Dubis A, Zamaraeva MV, Siergiejczyk L, Charishnikova O, and Shlyonsky V
- Subjects
- Bridged Bicyclo Compounds chemistry, Conductometry, Hydrogen Bonding, Ions chemistry, Magnetic Resonance Spectroscopy, Spectroscopy, Fourier Transform Infrared, Benzoates chemistry, Calcium chemistry, Coordination Complexes chemistry, Cycloheptanes chemistry, Lipid Bilayers chemistry, Sesquiterpenes chemistry
- Abstract
Calcium ionophoretic properties of ferutinin were re-evaluated in solvent-containing bilayer lipid membranes. The slopes of conductance-concentration curves suggest that in the presence of a solvent in the membrane the majority of complexes appear to consist of a single terpenoid molecule bound to one Ca ion. By contrast, the stoichiometry of ferutinin-Ca(2+) complexes in acetone determined using the conductometric method was 2 : 1. While the cation-cation selectivity of ferutinin did not change, the cation-anion selectivity slightly decreased in solvent containing membranes. FT-IR and NMR data together with DFT calculations at the B3LYP/6-31G(d) level of theory indicate that in the absence of Ca ions ferutinin molecules are hydrogen-bonded at the phenol hydroxyl groups. The variations of absorption assigned to -OH and -C-O stretching mode suggest that ferutinin interacts strongly with Ca ions via the hydroxyl group of ferutinol and carboxyl oxygen of the complex ether bond. The coordination through the carbonyl group of ferutinin was demonstrated by theoretical calculations. Taken together, ferutinin molecules form H-bonded dimers, while complexation of Ca(2+) by ferutinin ruptures this hydrogen bond due to spatial re-orientation of the ferutinin molecules from parallel to antiparallel alignment.
- Published
- 2015
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31. The OpenPicoAmp: an open-source planar lipid bilayer amplifier for hands-on learning of neuroscience.
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Shlyonsky V, Dupuis F, and Gall D
- Subjects
- Anti-Bacterial Agents pharmacology, Biophysics education, Electric Capacitance, Equipment Design, Gramicidin pharmacology, Humans, Ion Channels metabolism, Lipid Bilayers metabolism, Neurosciences education, Amplifiers, Electronic, Biophysics instrumentation, Lipid Bilayers chemistry, Neurosciences instrumentation
- Abstract
Understanding the electrical biophysical properties of the cell membrane can be difficult for neuroscience students as it relies solely on lectures of theoretical models without practical hands on experiments. To address this issue, we developed an open-source lipid bilayer amplifier, the OpenPicoAmp, which is appropriate for use in introductory courses in biophysics or neurosciences at the undergraduate level, dealing with the electrical properties of the cell membrane. The amplifier is designed using the common lithographic printed circuit board fabrication process and off-the-shelf electronic components. In addition, we propose a specific design for experimental chambers allowing the insertion of a commercially available polytetrafluoroethylene film. We provide a complete documentation allowing to build the amplifier and the experimental chamber. The students hand-out giving step-by step instructions to perform a recording is also included. Our experimental setup can be used in basic experiments in which students monitor the bilayer formation by capacitance measurement and record unitary currents produced by ionic channels like gramicidin A dimers. Used in combination with a low-cost data acquisition board this system provides a complete solution for hands-on lessons, therefore improving the effectiveness in teaching basic neurosciences or biophysics.
- Published
- 2014
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32. Possible role of lysophosphatidic acid in rat model of hypoxic pulmonary vascular remodeling.
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Shlyonsky V, Naeije R, and Mies F
- Abstract
Pulmonary hypertension is characterized by cellular and structural changes in the vascular wall of pulmonary arteries. We hypothesized that lysophosphatidic acid (LPA), a bioactive lipid, is implicated in this vascular remodeling in a rat model of hypoxic pulmonary hypertension. Exposure of Wistar rats to 10% O2 for 3 weeks induced an increase in the mean serum levels of LPA, to 40.9 (log-detransformed standard deviations: 23.4-71.7) μM versus 21.6 (11.0-42.3) μM in a matched control animal group (P = 0.037). We also observed perivascular LPA immunohistochemical staining in lungs of hypoxic rats colocalized with the secreted lysophospholipase D autotaxin (ATX). Moreover, ATX colocalized with mast cell tryptase, suggesting implication of these cells in perivascular LPA production. Hypoxic rat lungs expressed more ATX transcripts (2.4-fold) and more transcripts of proteins implicated in cell migration: β2 integrin (1.74-fold), intracellular adhesion molecule 1 (ICAM-1; 1.84-fold), and αM integrin (2.70-fold). Serum from the hypoxic group of animals had significantly higher chemoattractant properties toward rat primary lung fibroblasts, and this increase in cell migration could be prevented by the LPA receptor 1 and 3 antagonists. LPA also increased adhesive properties of human pulmonary artery endothelial cells as well as those of human peripheral blood mononuclear cells, via the activation of LPA receptor 1 or 3 followed by the stimulation of gene expression of ICAM-1, β-1, E-selectin, and vascular cell adhesion molecule integrins. In conclusion, chronic hypoxia increases circulating and tissue levels of LPA, which might induce fibroblast migration and recruitment of mononuclear cells in pulmonary vasculature, both of which contribute to pulmonary vascular remodeling.
- Published
- 2014
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33. Ion permeability of artificial membranes evaluated by diffusion potential and electrical resistance measurements.
- Author
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Shlyonsky V
- Subjects
- Diffusion, Models, Theoretical, Polycarboxylate Cement chemistry, Potassium Chloride chemistry, Sodium Chloride chemistry, Electric Impedance, Ions, Membranes, Artificial
- Abstract
In the present article, a novel model of artificial membranes that provides efficient assistance in teaching the origins of diffusion potentials is proposed. These membranes are made of polycarbonate filters fixed to 12-mm plastic rings and then saturated with a mixture of creosol and n-decane. The electrical resistance and potential difference across these membranes can be easily measured using a low-cost volt-ohm meter and home-made Ag/AgCl electrodes. The advantage of the model is the lack of ionic selectivity of the membrane, which can be modified by the introduction of different ionophores to the organic liquid mixture. A membrane treated with the mixture containing valinomycin generates voltages from -53 to -25 mV in the presence of a 10-fold KCl gradient (in to out) and from -79 to -53 mV in the presence of a bi-ionic KCl/NaCl gradient (in to out). This latter bi-ionic gradient potential reverses to a value from +9 to +20 mV when monensin is present in the organic liquid mixture. Thus, the model can be build stepwise, i.e., all factors leading to the development of diffusion potentials can be introduced sequentially, helping students to understand the quantitative relationships of ionic gradients and differential membrane permeability in the generation of cell electrical signals.
- Published
- 2013
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34. Melatonin down-regulates volume-sensitive chloride channels in fibroblasts.
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Ben Soussia I, Mies F, Naeije R, and Shlyonsky V
- Subjects
- Animals, Cell Movement, Cell Proliferation, Cell Size, Chloride Channels antagonists & inhibitors, Fibroblasts cytology, Indoles pharmacology, Maleimides pharmacology, Patch-Clamp Techniques, Protein Kinase C antagonists & inhibitors, Protein Kinase C metabolism, Rats, Receptor, Melatonin, MT2 antagonists & inhibitors, Receptor, Melatonin, MT2 metabolism, Tryptamines pharmacology, Chloride Channels metabolism, Fibroblasts physiology, Melatonin pharmacology
- Abstract
Melatonin has been reported to present with vasorelaxant and anti-fibrotic properties. We hypothesized that melatonin may down-regulate volume-regulated anion channels (VRAC) in fibroblasts to limit their migration and proliferation. While acute exposure of L929 fibroblasts to melatonin did not result in a significant decrease in VRAC current, pretreatment with 100 μM melatonin for 1 h decreased swelling-dependent activation of anion currents by 83% as measured by whole-cell perforated patch-clamp technique. This down-regulation of VRAC currents was dose-dependent with a half-maximal inhibition of 3.02 ± 0.48 μM. Overnight treatment of cells with 100 nM melatonin had the same inhibitory potency as a 1-h treatment with 100 μM. A similar down-regulatory effect of melatonin on VRAC was observed in primary rat lung fibroblasts. The effect of melatonin was prevented by luzindole and K185 that suggests implication of MT2 receptor. GF109203X, a protein kinase C inhibitor, blocked melatonin's action on VRAC, indicating that MT2 receptor activation results in stimulation of PKC. Consequently, melatonin inhibited regulatory volume decrease following hypotonic swelling of cells. Melatonin also decreased the migration of L929 fibroblasts through the same pathways that blocked VRAC. There was no significant inhibition of cell proliferation. Our study suggests that the attenuation of fibrosis and vascular remodeling by melatonin seen in animal models of hypertension and pulmonary fibrosis might be, in part, related to blunted fibroblast migration possibly through protein kinase C-mediated decrease in chloride channel activity.
- Published
- 2012
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35. Opposing effects of bone morphogenetic protein-2 and endothelin-1 on lung fibroblast chloride currents.
- Author
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Shlyonsky V, Soussia IB, Naeije R, and Mies F
- Subjects
- Actins biosynthesis, Antigens, Differentiation biosynthesis, Bone Morphogenetic Protein 2 pharmacology, Cell Line, Endothelin-1 pharmacology, Fibroblasts pathology, Gene Expression Regulation drug effects, Humans, Ion Transport drug effects, Protein Kinase C metabolism, Pulmonary Fibrosis pathology, Signal Transduction drug effects, Bone Morphogenetic Protein 2 metabolism, Chlorides metabolism, Endothelin-1 metabolism, Fibroblasts metabolism, Pulmonary Fibrosis metabolism
- Abstract
Alteration in the control of bone morphogenetic protein (BMP)-regulated genes and increased expression of endothelin (ET)-1 are both believed to play important roles in the still incompletely understood pathobiology of pulmonary vascular remodeling and fibrosis. Recent studies have drawn attention to the contribution of adventitial fibroblast activation in these phenomena. Because chloride channels are involved in the control of physiological function of fibroblasts, we hypothesized that these channels are differentially regulated by BMPs and ET. We measured chloride ion currents by whole-cell path-clamping in cultured primary human pulmonary fibroblasts. The application of BMP2 prevented activation of these currents by hypotonic challenge in a time- and dose-dependent manner, partially via protein kinase C signaling. Maximal inhibition was observed after 45-minute incubation of cells in the presence of 10 ng/ml of BMP2. ET-1 did not activate chloride channels acutely; however, prolonged treatment of cells with ET-1 (100 nM, 2 h) induced the appearance of lysophosphatidic acid-activated chloride currents (a marker of differentiated myofibroblasts), and this induction could be effectively blocked by BMP2 pretreatment (10 ng/ml). BMP2 also prevented stimulation of α-smooth muscle actin gene expression and cell migration of fibroblasts induced by ET-1. We conclude that ET-1 and BMP2 have opposing effects on chloride channel activity in human fibroblasts. This is a potentially relevant mechanism involved in pulmonary vascular remodeling and fibrosis.
- Published
- 2011
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36. Serotonin decreases alveolar epithelial fluid transport via a direct inhibition of the epithelial sodium channel.
- Author
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Goolaerts A, Roux J, Ganter MT, Shlyonsky V, Chraibi A, Stéphane R, Mies F, Matthay MA, Naeije R, Sariban-Sohraby S, Howard M, and Pittet JF
- Subjects
- Amiloride pharmacology, Animals, Cell Line, Cell Line, Tumor, Epithelium pathology, Humans, Hypoxia, Ions metabolism, Lung metabolism, Mice, Mice, Inbred C57BL, Oocytes metabolism, Patch-Clamp Techniques, Rats, Trachea metabolism, Xenopus, Epithelial Sodium Channels metabolism, Pulmonary Alveoli metabolism, Serotonin metabolism
- Abstract
Hypoxia and epithelial stretch that are commonly observed in patients with acute lung injury have been shown to promote the release of serotonin (5-hydroxytryptamine, 5-HT) in vitro. However, whether 5-HT contributes to the decrease of alveolar epithelial fluid transport, which is a hallmark of lung injury, is unknown. Thus, we investigated the effect of 5-HT on ion and fluid transport across the alveolar epithelium. 5-HT caused a dose-dependent inhibition of the amiloride-sensitive current across primary rat and human alveolar epithelial type II cell monolayers, but did not affect Na(+)/K(+) ATPase function. Furthermore, we found that the 5-HT induced inhibition of ion transport across the lung epithelium was receptor independent, as it was not prevented by the blockade of 5-HT2R (5-HT receptor 2), 5-HT3R (5-HT receptor 3), or by pretreatment with an intracellular calcium-chelating agent, BAPTA-AM (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester). In addition, the stimulation of 5-HT1R (5-HT receptor 1), 5-HT2R (5-HT receptor 2), 5-HT4R (5-HT receptor 4), and 5-HT7R (5-HT receptor 7) failed to reproduce the 5-HT effect on amiloride-sensitive sodium transport. We ascertained that 5-HT directly inhibited the function of rat alphabetagamma epithelial sodium channel (ENaC), as determined by heterologous expression of rat ENaC in Xenopus oocytes that do not express endogenous ENaC nor 5-HT receptors (5-HTR). Exposure of mice to hypoxia for 1 hour induced a 30% increase of 5-HT secretion into the distal airways of mice. Finally, the intratracheal instillation of 5-HT inhibited the amiloride-sensitive fraction of alveolar fluid clearance in mice. Together, these results indicate that 5-HT inhibits the amiloride-sensitive fraction of the alveolar epithelial fluid transport via a direct interaction with ENaC, and thus can be an endogenous inhibitor of this ion channel.
- Published
- 2010
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37. Electrophysiological characterization of rat type II pneumocytes in situ.
- Author
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Shlyonsky V, Goolaerts A, Mies F, and Naeije R
- Subjects
- Animals, Cell Survival, Dexamethasone pharmacology, Electrophysiology, Male, Membrane Potentials drug effects, Membrane Potentials physiology, Nystatin pharmacology, Patch-Clamp Techniques, Pulmonary Alveoli cytology, Pulmonary Alveoli drug effects, Pulmonary Circulation, Rats, Rats, Wistar, Chlorides physiology, Pulmonary Alveoli physiology, Sodium physiology
- Abstract
Optimal aeration of the lungs is dependent on an alveolar fluid clearance, a process that is governed by Na+ and Cl- transport. However, the specific contribution of various ion channels in different alveolar cell types under basal or stimulated conditions is not exactly known. We established a novel functional model of rat lung slices suitable for nystatin-perforated whole-cell patch-clamp experiments. Lung slices retained a majority of live cells for up to 72 hours. Type II pneumocytes in situ had a mean capacitance of 8.8 +/- 2.5 pF and a resting membrane potential of -4.4 +/- 1.9 mV. Bath replacement of Na+ with NMDG+ decreased inward whole-cell currents by 70%, 21% and 52% of which were sensitive to 10 microM and 1 mM of amiloride, respectively. Exposure of slices to 0.5 microM dexamethasone for 1 hour did not affect ion currents, while chronic exposure (0.5 microM, 24-72 h) induced an increase in both total Na+-entry currents and amiloride-sensitive currents. Under acute exposure to 100 microM cpt-cAMP, Type II cells in situ rapidly hyperpolarized by 25-30 mV, due to activation of whole-cell Cl- currents sensitive to 0.1 mM of 5-Nitro-2-(3-phenylpropylamino)benzoic acid. In addition, in the presence of cpt-cAMP, total sodium currents and currents sensitive to 10 microM amiloride increased by 32% and 70%, respectively. Thus, in Type II pneumocytes in situ: (1) amiloride-sensitive sodium channels contribute to only half of total Na+-entry and are stimulated by chronic exposure to glucocorticoids; (2) acute increase in cellular cAMP content simultaneously stimulates the entry of Cl- and Na+ ions.
- Published
- 2008
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38. Role of membrane curvature in mechanoelectrical transduction: ion carriers nonactin and valinomycin sense changes in integral bending energy.
- Author
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Shlyonsky VG, Markin VS, Andreeva I, Pedersen SE, Simon SA, Benos DJ, and Ismailov II
- Subjects
- Biological Transport, Active, Cations, Monovalent, Chlorpromazine pharmacology, Hydrostatic Pressure, Iontophoresis methods, Kinetics, Macrolides pharmacology, Mathematics, Membrane Potentials, Models, Biological, Thermodynamics, Anti-Bacterial Agents pharmacology, Cell Membrane Permeability drug effects, Electric Conductivity, Lipid Bilayers metabolism, Valinomycin pharmacology
- Abstract
We describe the phenomenon of mechanoelectrical transduction in macroscopic lipid bilayer membranes modified by two cation-selective ionophores, valinomycin and nonactin. We found that bulging these membranes, while maintaining the membrane tension constant, produced a marked supralinear increase in specific carrier-mediated conductance. Analyses of the mechanisms involved in mechanoelectrical transduction induced by the imposition of a hydrostatic pressure gradient or by an amphipathic compound chlorpromazine reveal similar changes in the charge carrier motility and carrier reaction rates at the interface(s). Furthermore, the relative change in membrane conductance was independent of membrane diameter, but was directly proportional to the square of membrane curvature, thus relating the observed phenomena to the bilayer bending energy. Extrapolated to biological membranes, these findings indicate that ion transport in cells can be influenced simply by changing shape of the membrane, without a change in membrane tension.
- Published
- 2006
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39. Halothane directly modifies Na+ and K+ channel activities in cultured human alveolar epithelial cells.
- Author
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Roch A, Shlyonsky V, Goolaerts A, Mies F, and Sariban-Sohraby S
- Subjects
- Adenocarcinoma, Cell Line, Tumor, Cells, Cultured, Gases, Halogens pharmacology, Humans, Lung Neoplasms, Membrane Potentials drug effects, Membrane Potentials physiology, Potassium Channels drug effects, Pulmonary Alveoli cytology, Pulmonary Alveoli drug effects, Respiratory Mucosa drug effects, Sodium Channels drug effects, Halothane pharmacology, Potassium Channels physiology, Pulmonary Alveoli physiology, Respiratory Mucosa physiology, Sodium Channels physiology
- Abstract
During inhalational anesthesia, halogenated gases are in direct contact with the alveolar epithelium, in which they may affect transepithelial ion and fluid transport. The effects of halogenated gases in vivo on epithelial Na+ and K+ channels, which participate in alveolar liquid clearance, remain unclear. In the present study, the effects of halothane (1, 2, and 4% atm) on ion-channel function in cultured human alveolar cells were investigated using the patch-clamp technique. After exposure to 4% halothane, amiloride-sensitive whole-cell inward currents increased by 84+/-22%, whereas tetraethylammonium-sensitive outward currents decreased by 63+/-7%. These effects, which occurred within 30 s, remained for 30-min periods of exposure to the gas, were concentration-dependent, and were reversible upon washout. Pretreatment with amiloride prevented 90+/-7% of the increase in inward currents without change in outward currents, consistent with an activation of amiloride-sensitive epithelial sodium channels. Tetraethylammonium obliterated 90+/-9% of the effect of halothane on outward currents, without change in inward currents, indicating inhibition of Ca2+-activated K+ channels. These channels were identified in excised patches to be small-conductance Ca2+-activated K+ channels. These effects of halothane were not modified after the inhibition of cytosolic phospholipase A2 by aristolochic acid. Exposure of the cells to either trypsin or to low Na+ completely prevented the increase in amiloride-sensitive currents induced by halothane, suggesting a release of Na+ channels self-inhibition. Thus, halothane modifies differentially and independently Na+ and K+ permeabilities in human alveolar cells.
- Published
- 2006
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40. Differentiation of epithelial Na+ channel function. An in vitro model.
- Author
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Shlyonsky V, Goolaerts A, Van Beneden R, and Sariban-Sohraby S
- Subjects
- Base Sequence, Cell Line, Tumor, DNA Primers, Epithelial Sodium Channels, Fluorescent Dyes metabolism, Humans, Immunohistochemistry, In Vitro Techniques, Polymerase Chain Reaction, Sodium Channels metabolism, Cell Differentiation, Models, Biological, Sodium Channels physiology
- Abstract
Confluent monolayers of epithelial cells grown on nonporous support form fluid-filled hemicysts called domes, which reflect active ion transport across the epithelium. Clara-like H441 lung adenocarcinoma cells grown on glass supports and exposed to 50 nM dexamethasone developed domes in a time-dependent fashion. Uplifting of small groups of cells occurred within 6-12 h, well formed domes appeared between 24 and 48 h, and after 7 days, individual domes started to merge. Cells inside of domes compared with those outside domes, or with monolayers not exposed to dexamethasone, differed by higher surfactant production, an increased cytokeratin expression, and the localization of claudin-4 proteins to the plasma membrane. In patch clamp studies, amiloride-blockable sodium currents were detected exclusively in cells inside domes, whereas in cells outside of domes, sodium crossed the membrane through La3+-sensitive nonspecific cation channels. Cells grown on permeable support without dexamethasone expressed amiloride-sensitive currents only after tight electrical coupling was achieved (transepithelial electrical resistance (R(t)) > 1 kilohm). In real-time quantitative PCR experiments, the addition of dexamethasone increased the content of claudin-4, occludin, and Na+ channel gamma-subunit (gamma-ENaC) mRNAs by 1.34-, 1.32-, and 1.80-fold, respectively, after 1 h and was followed by an increase at 6 h in the content of mRNA of alpha- and beta-ENaC and of alpha1- and beta1-Na,K-ATPase. In the absence of dexamethasone, neither change in gene expression nor cell uplifting was observed. Our data suggest that during epithelial differentiation, coordinated expression of tight junction proteins precedes the development of vectorial transport of sodium, which in turn leads to the fluid accumulation in basolateral spaces that is responsible for dome formation.
- Published
- 2005
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41. Modulation of epithelial Na+ channel activity by long-chain n-3 fatty acids.
- Author
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Mies F, Shlyonsky V, Goolaerts A, and Sariban-Sohraby S
- Subjects
- Amiloride pharmacology, Animals, Cell Line, Cell Polarity physiology, Cyclic AMP-Dependent Protein Kinases metabolism, Diuretics pharmacology, Epithelial Sodium Channels, Fatty Acids, Omega-6 pharmacology, Kidney metabolism, Membrane Potentials drug effects, Membrane Potentials physiology, Patch-Clamp Techniques, Sodium metabolism, Sodium Channels physiology, Xenopus laevis, Fatty Acids, Omega-3 pharmacology, Kidney cytology, Sodium Channels metabolism
- Abstract
The epithelial sodium channel is found in apical membranes of a variety of native epithelial tissues, where it regulates sodium and fluid balance. In vivo, a number of hormones and other endogenous factors, including polyunsaturated fatty acids (PUFAs), regulate these channels. We tested the effects of essential n-3 and n-6 PUFAs on amiloride-sensitive sodium transport in A6 epithelial cells. Eicosapentaenoic acid [EPA; C20:5(n-3)] transiently stimulated amiloride-sensitive open-circuit current (I(Na)) from 4.0 +/- 0.3 to 7.7 +/- 0.3 microA/cm2 within 30 min (P < 0.001). No activation was seen in the presence of 10 microM amiloride. In cell-attached but not in cell-excised patches, EPA acutely increased the open probability of sodium channels from 0.45 +/- 0.08 to 0.63 +/- 0.10 (P = 0.02, paired t-test). n-6 PUFAs, including linoleic acid (C18:2), eicosatetraynoic acid (C20:4), and docosapentanoic acid (C22:5) had no effect, whereas n-3 docosahexanoic acid (C22:6) activated amiloride-sensitive I(Na) in a manner similar to EPA. Activation of I(Na) by EPA was prevented by H-89, a PKA inhibitor. Similarly, PKA activity was stimulated by EPA. Nonspecific stimulation of phosphodiesterase activity by CoCl2 completely prevented the effect of EPA on sodium transport. We conclude that n-3 PUFAs activate epithelial sodium channels downstream of cAMP in a cAMP-dependent pathway also involving PKA.
- Published
- 2004
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42. Gating of amiloride-sensitive Na(+) channels: subunit-subunit interactions and inhibition by the cystic fibrosis transmembrane conductance regulator.
- Author
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Berdiev BK, Shlyonsky VG, Karlson KH, Stanton BA, and Ismailov II
- Subjects
- Amiloride pharmacology, Animals, Base Sequence, Biophysical Phenomena, Biophysics, DNA Primers genetics, In Vitro Techniques, Ion Channel Gating drug effects, Kinetics, Protein Structure, Tertiary, Rats, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Sodium Channels genetics, Cystic Fibrosis Transmembrane Conductance Regulator pharmacology, Sodium Channels chemistry, Sodium Channels metabolism
- Abstract
In search of the structural basis for gating of amiloride-sensitive Na(+) channels, kinetic properties of single homo and heterooligomeric ENaCs formed by the subunits with individual truncated cytoplasmic domains were studied in a cell-free planar lipid bilayer reconstitution system. Our results identify the N-terminus of the alpha-subunit as a major determinant of kinetic behavior of both homooligomeric and heterooligomeric ENaCs, although the carboxy-terminal domains of beta- and gamma-ENaC subunits play important role(s) in modulation of the kinetics of heterooligomeric channels. We also found that the cystic fibrosis transmembrane conductance regulator (CFTR) inhibits amiloride-sensitive channels, at least in part, by modulating their gating. Comparison of these data suggests that the modulatory effects of the beta- and gamma-ENaC subunits, and of the CFTR, may involve the same, or closely related, mechanism(s); namely, "locking" the heterooligomeric channels in their closed state. These mechanisms, however, do not completely override the gating mechanism of the alpha-channel.
- Published
- 2000
- Full Text
- View/download PDF
43. Peptide inhibition of ENaC.
- Author
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Ismailov II, Shlyonsky VG, Serpersu EH, Fuller CM, Cheung HC, Muccio D, Berdiev BK, and Benos DJ
- Subjects
- Amino Acid Sequence, Circular Dichroism, Epithelial Sodium Channels, Lipid Bilayers metabolism, Models, Chemical, Molecular Sequence Data, Peptide Fragments chemistry, Peptide Fragments metabolism, Peptide Fragments pharmacology, Peptides chemical synthesis, Sodium Channels metabolism, Solutions, Peptides pharmacology, Sodium Channel Blockers
- Abstract
Liddle's disease is an autosomal dominant form of human hypertension resulting from a basal activation of amiloride-sensitive Na+ channels (ENaC). This channel activation is produced by mutations in the beta- and/or gamma-carboxy-terminal cytoplasmic tails, in many cases causing a truncation of the last 45-76 amino acids. In this study, we tested two hypotheses; first, beta- and gamma-ENaC C-terminal truncation mutants (beta DeltaC and gamma DeltaC), in combination with the wild-type alpha-ENaC subunit, reproduce the Liddle's phenotype at the single channel level, i.e., an increase in open probability (Po), and second, these C-terminal regions of beta- and gamma-ENaC act as intrinsic blockers of this channel. Our results indicate that alpha beta DeltaC gamma DeltaC-rENaC, incorporated into planar lipid bilayers, has a significantly higher single channel Po compared to the wild-type channel (0.85 vs 0.60, respectively), and that 30-mer synthetic peptides corresponding to the C-terminal region of either beta- or gamma-ENaC block the basal-activated channel in a concentration-dependent fashion. Moreover, there was a synergy between the peptides for channel inhibition when added together. We conclude that the increase in macroscopic Na+ reabsorption that occurs in Liddle's disease is at least in part due to an increase in single channel Po and that the cytoplasmic tails of the beta- and gamma-ENaC subunits are important in the modulation of ENaC activity.
- Published
- 1999
- Full Text
- View/download PDF
44. Purification and reconstitution of an outwardly rectified Cl- channel from tracheal epithelia.
- Author
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Jovov B, Shlyonsky VG, Berdiev BK, Ismailov II, and Benos DJ
- Subjects
- 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid pharmacology, Animals, Antibodies, Blotting, Western, Calcium Channels biosynthesis, Calcium Channels isolation & purification, Cattle, Chlorides metabolism, Chromatography, Ion Exchange, Electric Conductivity, Electrophoresis, Polyacrylamide Gel, Epithelial Cells cytology, GTP-Binding Proteins metabolism, Lipid Bilayers, Macromolecular Substances, Membrane Potentials, Molecular Weight, Trachea cytology, Calcium Channels physiology, Epithelial Cells physiology, Trachea physiology
- Abstract
We reported the identification of three outwardly rectified Cl- channel (ORCC) candidate proteins (115, 85, and 52 kDa) from bovine tracheal epithelia. We have raised polyclonal antibodies against these isolated proteins. Incorporation into planar lipid bilayers of material partly purified from bovine tracheal apical membranes with one of these antibodies as a ligand (anti-p115) resulted in the incorporation of an ORCC identical in biophysical characteristics to one we previously described. We developed a new purification procedure to increase the yield and purity of this polypeptide. The purification scheme that gave the best results in terms of overall protein yield and purity was a combination of anion- and cation-exchange chromatography followed by immunopurification. By use of this purification scheme, 7 microg of the 115-kDa protein were purified from 20 mg of tracheal apical membrane proteins. Incorporation of this highly purified material into planar lipid bilayers revealed a DIDS-inhibitable channel with the following properties: linear conductance of 87 +/- 9 pS in symmetrical Cl- solutions, halide selectivity sequence of I- > Cl- > Br-, and lack of sensitivity to protein kinase A, Ca2+, or dithiothreitol. Using anti-Galphai antibodies to precipitate Galphai protein(s) from the partly purified preparations, we demonstrated that the loss of rectification of the ORCC was due to uncoupling of Galphai protein(s) from the ORCC protein and that the 115-kDa polypeptide is an ORCC.
- Published
- 1998
- Full Text
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45. Cation permeability of a cloned rat epithelial amiloride-sensitive Na+ channel.
- Author
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Ismailov II, Shlyonsky VG, Alvarez O, and Benos DJ
- Subjects
- Animals, Binding Sites, Computer Simulation, Electric Conductivity, Epithelial Sodium Channels, Lipid Bilayers metabolism, Lithium pharmacokinetics, Models, Biological, Permeability, Phospholipids chemistry, Phospholipids metabolism, Potassium metabolism, Rats, Sodium metabolism, Cations metabolism, Sodium Channels metabolism
- Abstract
1. Conductance of heterotrimeric rat epithelial Na+ channels (alpha, beta, gamma-rENaCs) for Li+ and Na+ in planar lipid bilayers was a non-linear function of ion concentration, with a maximum of 30.4 +/- 2.9 pS and 18.5 +/- 1.9 pS at 1 M Li+ and Na+, respectively. 2. The alpha, beta, gamma-rENaC conductance measured in symmetrical mixtures of Na(+)-Li+ (1 M) exhibited an anomalous mole fraction dependence, with a minimum at 4:1 Li+ to Na+ molar ratio. 3. Permeability ratios PK/PNa and PLi/PNa of the channel calculated from the bionic reversal potentials were dependent on ion concentration: PK/PNa was 0.11 +/- 0.01, and PLi/PNa was 1.6 +/- 0.3 at 50 mM; PK/PNa was 0.04 +/- 0.01 and PLi/PNa was 2.5 +/- 0.4 at 3 M, but differed from the ratios of single-channel conductances in symmetrical Li+, Na+ or K+ solutions. The permeability sequence determined by either method was Li+ > Na+ > K+ >> Rb+ Cs+. 4. Predictions of a model featuring two binding sites and three energy barriers (2S3B), and allowing double occupancy, developed on the basis of single ion current-voltage relationships, are in agreement with the observed conductance maximum in single ion experiments, conductance minimum in the mole fraction experiments, non-linearity of the current-voltage curves in bionic experiments, and the concentration dependence of permeability ratios. 5. Computer simulations using the 2S3B model recreate the ion concentration dependencies of single-channel conductance observed for the immunopurified bovine renal amiloride-sensitive Na+ channel, and short-circuit current in frog skin, thus supporting the hypothesis that ENaCs form a core conduction unit of epithelial Na+ channels.
- Published
- 1997
- Full Text
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46. Streaming potential measurements in alphabetagamma-rat epithelial Na+ channel in planar lipid bilayers.
- Author
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Ismailov II, Shlyonsky VG, and Benos DJ
- Subjects
- Animals, Electrophysiology, Epithelium metabolism, Rats, Water metabolism, Lipid Bilayers metabolism, Sodium Channels physiology
- Abstract
Streaming potentials across cloned epithelial Na+ channels (ENaC) incorporated into planar lipid bilayers were measured. We found that the establishment of an osmotic pressure gradient (Deltapi) across a channel-containing membrane mimicked the activation effects of a hydrostatic pressure differential (DeltaP) on alphabetagamma-rENaC, although with a quantitative difference in the magnitude of the driving forces. Moreover, the imposition of a Deltapi negates channel activation by DeltaP when the Deltapi was directed against DeltaP. A streaming potential of 2.0 +/- 0.7 mV was measured across alphabetagamma-rat ENaC (rENaC)-containing bilayers at 100 mM symmetrical [Na+] in the presence of a 2 Osmol/kg sucrose gradient. Assuming single file movement of ions and water within the conduction pathway, we conclude that between two and three water molecules are translocated together with a single Na+ ion. A minimal effective pore diameter of 3 A that could accommodate two water molecules even in single file is in contrast with the 2-A diameter predicted from the selectivity properties of alphabetagamma-rENaC. The fact that activation of alphabetagamma-rENaC by DeltaP can be reproduced by the imposition of Deltapi suggests that water movement through the channel is also an important determinant of channel activity.
- Published
- 1997
- Full Text
- View/download PDF
47. Point mutations in alpha bENaC regulate channel gating, ion selectivity, and sensitivity to amiloride.
- Author
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Fuller CM, Berdiev BK, Shlyonsky VG, Ismailov II, and Benos DJ
- Subjects
- Amino Acid Sequence, Animals, Cattle, Diuretics pharmacology, Electrophysiology, Epithelial Sodium Channels, Gene Expression, Ion Channel Gating drug effects, Kinetics, Lipid Bilayers, Liposomes metabolism, Membrane Proteins metabolism, Molecular Sequence Data, Mutagenesis, Site-Directed, Oocytes metabolism, Point Mutation, Potassium metabolism, Sodium Channels chemistry, Xenopus, Amiloride pharmacology, Ion Channel Gating physiology, Sodium Channels genetics, Sodium Channels metabolism
- Abstract
We have generated two site-directed mutants, K504E and K515E, in the alpha subunit of an amiloride-sensitive bovine epithelial Na+ channel, alpha bENaC. The region in which these mutations lie is in the large extracellular loop immediately before the second membrane-spanning domain (M2) of the protein. We have found that when membrane vesicles prepared from Xenopus oocytes expressing either K504E or K515E alpha bENaC are incorporated into planar lipid bilayers, the gating pattern, cation selectivity, and amiloride sensitivity of the resultant channel are all altered as compared to the wild-type protein. The mutated channels exhibit either a reduction or a complete lack of its characteristic burst-type behavior, significantly reduced Na+:K+ selectivity, and an approximately 10-fold decrease in the apparent inhibitory equilibrium dissociation constant (Ki) for amiloride. Single-channel conductance for Na+ was not affected by either mutation. On the other hand, both K504E and K515E alpha bENaC mutants were significantly more permeable to K+, as compared to wild type. These observations identify a lysine-rich region between amino acid residues 495 and 516 of alpha bENaC as being important to the regulation of fundamental channel properties.
- Published
- 1997
- Full Text
- View/download PDF
48. Mechanosensitivity of an epithelial Na+ channel in planar lipid bilayers: release from Ca2+ block.
- Author
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Ismailov II, Berdiev BK, Shlyonsky VG, and Benos DJ
- Subjects
- Amiloride pharmacology, Animals, Dogs, Electric Conductivity, Epithelium physiology, Ion Channel Gating, Kinetics, Macromolecular Substances, Membrane Potentials drug effects, Microsomes metabolism, Models, Chemical, Patch-Clamp Techniques, Protein Biosynthesis, Rabbits, Reticulocytes metabolism, Sodium Channels biosynthesis, Sodium Channels chemistry, Calcium pharmacology, Lipid Bilayers, Sodium Channels physiology
- Abstract
A family of novel epithelial Na+ channels (ENaCs) have recently been cloned from several different tissues. Three homologous subunits (alpha, beta, gamma-ENaCs) from the core conductive unit of Na(+)-selective, amiloride-sensitive channels that are found in epithelia. We here report the results of a study assessing the regulation of alpha,beta,gamma-rENaC by Ca2+ in planar lipid bilayers. Buffering of the bilayer bathing solutions to [Ca2+] < 1 nM increased single-channel open probability by fivefold. Further investigation of this phenomenon revealed that Ca2+ ions produced a voltage-dependent block, affecting open probability but not the unitary conductance of ENaC. Imposing a hydrostatic pressure gradient across bilayers containing alpha,beta,gamma-rENaC markedly reduced the sensitivity of these channels to inhibition by [Ca2+]. Conversely, in the nominal absence of Ca2+, the channels lost their sensitivity to mechanical stimulation. These results suggest that the previously observed mechanical activation of ENaCs reflects a release of the channels from block by Ca2+.
- Published
- 1997
- Full Text
- View/download PDF
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