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Francisco Bezanilla - One of the best experts on this subject based on the ideXlab platform.
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cut open oocyte Voltage Clamp Technique
Methods in Enzymology, 1998Co-Authors: Enrico Stefani, Francisco BezanillaAbstract:Publisher Summary The Xenopus oocyte is widely used as an expression system for ion channels. This chapter discusses the cut-open oocyte Voltage-Clamp Technique (COVG) that has relatively low current noise (ca. 1-nA rms at 5 kHz), can charge the membrane capacity in 20-40μs, and makes it possible to control the intracellular milieu by internal perfusion of the oocyte. Another feature is that stable recordings lasting for several hours can be easily obtained. These properties allow the adequate resolution of the time course of fast ionic and gating charge currents. This chapter describes the use and limitations of the COVG Technique.There are many advantages of the present method. High-frequency response and low noise recording (24-μs time constant and 1.2-nA rms at 5 kHz). This allows the accurate description of small gating currents and fast activation or deactivation of ionic currents to be adequately resolved. Currents up to 20-30μA can be adequately Clamped. Stable recording conditions lasting for several hours. Even though this Technique has internal access, rundown is minimized compared to excised patches. Access to the cell interior because of which the intracellular medium can be exchanged with various solutions is also an added advantage. This is of invaluable help in recording K + gating currents, an experimental condition that required the complete blockade of all ionic currents. This advantage makes this method suitable for the study of channel modulation by second messengers and drugs. In addition, channel selectivity properties can be defined by ion substitution experiments. Channel localization is also an advantage of the present method. A study of the membrane compartmentalization of channels can be easily obtained with this method, since it allows the Voltage Clamping of different regions of the oocyte surface.
Timothy Jegla - One of the best experts on this subject based on the ideXlab platform.
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characterization of kcnq5 q3 potassium channels expressed in mammalian cells
British Journal of Pharmacology, 2001Co-Authors: Alan D Wickenden, Anruo Zou, Kay P Wagoner, Timothy JeglaAbstract:Heteromeric KCNQ5/Q3 channels were stably expressed in Chinese Hamster ovary cells and characterized using the whole cell Voltage-Clamp Technique. KCNQ5/Q3 channels were activated by the novel anticonvulsant, retigabine (EC50 1.4 μM) by a mechanism that involved drug-induced, leftward shifts in the Voltage-dependence of channel activation (−31.8 mV by 30 μM retigabine). KCNQ5/Q3 channels were inhibited by linopirdine (IC50 7.7 μM) and barium (IC50 0.46 mM), at concentrations similar to those required to inhibit native M-currents. These findings identify KCNQ5/Q3 channels as a molecular target for retigabine and raise the possibility that activation of KCNQ5/Q3 channels may be responsible for some of the anti-convulsant activity of this agent. Furthermore, the sensitivity of KCNQ5/Q3 channels to linopirdine supports the possibility that potassium channels comprised of KCNQ5 and KCNQ3 may make a contribution to native M-currents. British Journal of Pharmacology (2001) 132, 381–384; doi:10.1038/sj.bjp.0703861
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Characterization of KCNQ5/Q3 potassium channels expressed in mammalian cells
British journal of pharmacology, 2001Co-Authors: Alan D Wickenden, P. Kay Wagoner, Anruo Zou, Timothy JeglaAbstract:Heteromeric KCNQ5/Q3 channels were stably expressed in Chinese Hamster ovary cells and characterized using the whole cell Voltage-Clamp Technique. KCNQ5/Q3 channels were activated by the novel anticonvulsant, retigabine (EC50 1.4 μM) by a mechanism that involved drug-induced, leftward shifts in the Voltage-dependence of channel activation (−31.8 mV by 30 μM retigabine). KCNQ5/Q3 channels were inhibited by linopirdine (IC50 7.7 μM) and barium (IC50 0.46 mM), at concentrations similar to those required to inhibit native M-currents. These findings identify KCNQ5/Q3 channels as a molecular target for retigabine and raise the possibility that activation of KCNQ5/Q3 channels may be responsible for some of the anti-convulsant activity of this agent. Furthermore, the sensitivity of KCNQ5/Q3 channels to linopirdine supports the possibility that potassium channels comprised of KCNQ5 and KCNQ3 may make a contribution to native M-currents. British Journal of Pharmacology (2001) 132, 381–384; doi:10.1038/sj.bjp.0703861
Alun D Hughes - One of the best experts on this subject based on the ideXlab platform.
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effect of inhibition of tyrosine phosphatases on Voltage operated calcium channel currents in rabbit isolated ear artery cells
British Journal of Pharmacology, 1998Co-Authors: S Wijetunge, Joanne S Lymn, Alun D HughesAbstract:1. The effect of increasing cellular tyrosine phosphorylation by inhibiting endogenous tyrosine phosphatases was examined on Voltage-operated calcium channel currents in vascular smooth muscle cells. 2. In single ear artery smooth muscle cells of the rabbit, studied by the whole cell Voltage Clamp Technique, intracellular application of the tyrosine phosphatase inhibitors, sodium orthovanadate (100 microM) and peroxyvanadate (100 microM orthovanadate + 1 mM H2O2) increased Voltage-operated calcium channel currents by 56% and 83%, respectively. 3. Bath application of two other membrane permeant tyrosine phosphatase inhibitors, phenylarsine oxide (100 microM) and dephostatin (50 microM) also increased Voltage-operated calcium channel currents by 48% and 52%, respectively. 4. The selective tyrosine kinase inhibitor, tyrphostin-23 (100 microM) reduced calcium channel currents by 41%. Pre-incubation with tyrphostin-23 abolished the effects of peroxyvanadate, phenylarsine oxide and dephostatin on calcium channels. 5. Western blot analysis of rabbit ear artery cell lysates showed increased tyrosine phosphorylation of several endogenous proteins following treatment with peroxyvanadate. 6. These results indicate that a number of structurally dissimilar inhibitors of tyrosine phosphatases increase Voltage-operated calcium channel currents in arterial smooth muscle cells presumably due to increased tyrosine phosphorylation.
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effect of platelet derived growth factor on Voltage operated calcium channels in rabbit isolated ear artery cells
British Journal of Pharmacology, 1995Co-Authors: S Wijetunge, Alun D HughesAbstract:1. Platelet derived growth factor (PDGF), AB and BB isoforms (100 pM) increased calcium channel currents measured by whole cell Voltage Clamp Technique in single vascular smooth muscle cells isolated from rabbit ear arteries. 2. Tyrphostin-23 (100 microM) a selective inhibitor of protein tyrosine kinases, reduced calcium channel currents. Pre-incubation with tyrphostin-23 prevented PDGF-AB induced increase in calcium channel currents. However, in these same cells 10 nM (+)-202791, a dihydropyridine calcium channel agonist, did increase calcium channel currents. 3. Bistyrphostin (10 microM), a selective inhibitor of epidermal growth factor (EGF)-kinase also reduced calcium channel currents and inhibited PDGF-AB-induced increases in calcium channel currents. 4. Genistein (100 microM) a selective inhibitor of tyrosine kinases, structurally unrelated to the tryphostins, also inhibited calcium channel currents and pre-incubation with genistein prevented the PDGF-AB-induced rise in calcium channel currents. 5. These results indicate that PDGF increases calcium channel currents in vascular smooth muscle. This action of PDGF probably involves a tyrosine kinase.
Enrico Stefani - One of the best experts on this subject based on the ideXlab platform.
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cut open oocyte Voltage Clamp Technique
Methods in Enzymology, 1998Co-Authors: Enrico Stefani, Francisco BezanillaAbstract:Publisher Summary The Xenopus oocyte is widely used as an expression system for ion channels. This chapter discusses the cut-open oocyte Voltage-Clamp Technique (COVG) that has relatively low current noise (ca. 1-nA rms at 5 kHz), can charge the membrane capacity in 20-40μs, and makes it possible to control the intracellular milieu by internal perfusion of the oocyte. Another feature is that stable recordings lasting for several hours can be easily obtained. These properties allow the adequate resolution of the time course of fast ionic and gating charge currents. This chapter describes the use and limitations of the COVG Technique.There are many advantages of the present method. High-frequency response and low noise recording (24-μs time constant and 1.2-nA rms at 5 kHz). This allows the accurate description of small gating currents and fast activation or deactivation of ionic currents to be adequately resolved. Currents up to 20-30μA can be adequately Clamped. Stable recording conditions lasting for several hours. Even though this Technique has internal access, rundown is minimized compared to excised patches. Access to the cell interior because of which the intracellular medium can be exchanged with various solutions is also an added advantage. This is of invaluable help in recording K + gating currents, an experimental condition that required the complete blockade of all ionic currents. This advantage makes this method suitable for the study of channel modulation by second messengers and drugs. In addition, channel selectivity properties can be defined by ion substitution experiments. Channel localization is also an advantage of the present method. A study of the membrane compartmentalization of channels can be easily obtained with this method, since it allows the Voltage Clamping of different regions of the oocyte surface.
Sophia Khom - One of the best experts on this subject based on the ideXlab platform.
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Design, Synthesis, and Pharmacological Evaluation of Novel β2/3 Subunit-Selective γ‑Aminobutyric Acid Type A (GABAA) Receptor Modulators
2018Co-Authors: Marco Stadler, Ernst Urban, Serena Monticelli, Thomas Seidel, Denise Luger, Isabella Salzer, Stefan Boehm, Wolfgang Holzer, Christoph Schwarzer, Sophia KhomAbstract:Subunit-selective modulation of γ-aminobutyric acid type A receptors (GABAAR) is considered to exert fewer side effects compared to unselective clinically used drugs. Here, the β2/3 subunit-selective GABAAR modulators valerenic acid (VA) and loreclezole (LOR) guided the synthesis of novel subunit-selective ligands with simplified structures. We studied their effects on GABAARs expressed in Xenopus laevis oocytes using two-microelectrode Voltage Clamp Technique. Five compounds showed significantly more efficacious modulation of GABA-evoked currents than VA and LOR with retained potency and selectivity. Compound 18 [(E)-2–Cyano-3-(2,4-dichlorophenyl)but-2-enamide] induced the highest maximal modulation of GABA-induced chloride currents (Emax: 3114 ± 242%), while 12 [(Z)-3-(2,4-dichlorophenyl)but-2-enenitrile] displayed the highest potency (EC50: 13 ± 2 μM). Furthermore, in hippocampal neurons 12 facilitated phasic and tonic GABAergic inhibition, and in vivo studies revealed significantly more potent protection against pentylenetetrazole (PTZ)-induced seizures compared to VA and LOR. Collectively, compound 12 constitutes a novel, simplified, and subunit-selective GABAAR modulator with low-dose anticonvulsant activity
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efficient modulation of γ aminobutyric acid type a receptors by piperine derivatives
Journal of Medicinal Chemistry, 2014Co-Authors: Angela Schoffmann, Sophia Khom, Igor Baburin, Laurin Wimmer, Daria Goldmann, Juliane Hintersteiner, T Schwarz, Michael Hintersteininger, Peter Pakfeifer, Mouhssin OufirAbstract:Piperine activates TRPV1 (transient receptor potential vanilloid type 1 receptor) receptors and modulates γ-aminobutyric acid type A receptors (GABAAR). We have synthesized a library of 76 piperine analogues and analyzed their effects on GABAAR by means of a two-microelectrode Voltage-Clamp Technique. GABAAR were expressed in Xenopus laevis oocytes. Structure–activity relationships (SARs) were established to identify structural elements essential for efficiency and potency. Efficiency of piperine derivatives was significantly increased by exchanging the piperidine moiety with either N,N-dipropyl, N,N-diisopropyl, N,N-dibutyl, p-methylpiperidine, or N,N-bis(trifluoroethyl) groups. Potency was enhanced by replacing the piperidine moiety by N,N-dibutyl, N,N-diisobutyl, or N,N-bistrifluoroethyl groups. Linker modifications did not substantially enhance the effect on GABAAR. Compound 23 [(2E,4E)-5-(1,3-benzodioxol-5-yl)-N,N-dipropyl-2,4-pentadienamide] induced the strongest modulation of GABAA (maximal GABA-in...
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GABAA receptor modulation by piperine and a non-TRPV1 activating derivative
Biochemical Pharmacology, 2013Co-Authors: Sophia Khom, Igor Baburin, Angela Schoffmann, Juliane Hintersteiner, Barbara Strommer, Thomas Erker, Thomas Schwarz, Christooph Schwarzer, Janine Zaugg, Matthias HamburgerAbstract:The action of piperine (the pungent component of pepper) and its derivative SCT-66 ((2E,4E)-5-(1,3-benzodioxol-5-yl))-N,N-diisobutyl-2,4-pentadienamide) on different gamma-aminobutyric acid (GABA) type A (GABAA) receptors, transient-receptor-potential-vanilloid-1 (TRPV1) receptors and behavioural effects were investigated. GABAA receptor subtypes and TRPV1 receptors were expressed in Xenopus laevis oocytes. Modulation of GABA-induced chloride currents (IGABA) by piperine and SCT-66 and activation of TRPV1 was studied using the two-microelectrode-Voltage-Clamp Technique and fast perfusion. Their effects on explorative behaviour, thermoregulation and seizure threshold were analysed in mice. Piperine acted with similar potency on all GABAA receptor subtypes (EC50 range: 42.8 ± 7.6 μM (α2β2)–59.6 ± 12.3 μM (α3β2)). IGABA modulation by piperine did not require the presence of a γ2S-subunit, suggesting a binding site involving only α and β subunits. IGABA activation was slightly more efficacious on receptors formed from β2/3 subunits (maximal IGABA stimulation through α1β3 receptors: 332 ± 64% and α1β2: 271 ± 36% vs. α1β1: 171 ± 22%, p
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gabaa receptor modulators from the chinese herbal drug junci medulla the pith of juncus effusus
Planta Medica, 2012Co-Authors: Judith Singhuber, Sophia Khom, Steffen Hering, Igor Baburin, Martin Zehl, Ernst Urban, Brigitte KoppAbstract:: The gamma-amino butyric acid (GABA) type A (GABA(A)) receptor represents a crucial target for clinical agents in the treatment of anxiety and insomnia. Using the two-microelectrode Voltage Clamp Technique on recombinant α₁β₂γ(2S) GABA (A) receptors, effusol (1) and dehydroeffusol (2) were isolated in a bioactivity-guided approach from the pith of Juncus effusus L. Both compounds concentration-dependently enhanced GABA induced chloride currents (I(GABA)) by a maximum 188 ± 20 (1) and 239 ± 18 % (2), independent of the benzodiazepine (BZ) binding site. This activity on the GABA (A) receptor may explain the traditional use of J. effusus as a sedative and anxiolytic agent in Chinese medicine.
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Bioactivity-Guided Isolation of GABAA Receptor Modulating Constituents from the Rhizomes of Actaea racemosa
Journal of natural products, 2010Co-Authors: Serhat Sezai Çiçek, Sophia Khom, Barbara Taferner, Steffen Hering, Hermann StuppnerAbstract:Black cohosh (Actaea racemosa) is a frequently used herbal remedy for the treatment of mild climacteric symptoms. In the present study, the modulation of γ-aminobutryic acid (GABA)-induced chloride currents (IGABA) through GABA type A (GABAA) receptors by black cohosh extracts and isolated compounds was investigated. GABAA receptors, consisting of α1, β2, and γ2S subunits, were expressed in Xenopus laevis oocytes, and potentiation of IGABA was measured using the two-microelectrode Voltage Clamp Technique. In a bioactivity-guided isolation procedure the positive modulation of IGABA could be restricted to the plant terpenoid fractions, resulting in the isolation of 11 cycloartane glycosides, of which four significantly (p < 0.05) enhanced IGABA. The most efficient effect was observed for 23-O-acetylshengmanol 3-O-β-d-xylopyranoside (4, 100 μM), enhancing IGABA by 1692 ± 201%, while actein (1), cimigenol 3-O-β-d-xylopyranoside (6), and 25-O-acetylcimigenol 3-O-α-l-arabinopyranoside (8) were significantly les...
