The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Christian Bech Rosen - One of the best experts on this subject based on the ideXlab platform.
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single molecule site specific detection of protein phosphorylation with a nanopore
Nature Biotechnology, 2014Co-Authors: Christian Bech Rosen, David Rodriguezlarrea, Hagan BayleyAbstract:A protein nanopore can distinguish model proteins phosphorylated at adjacent sites. We demonstrate single-molecule, site-specific detection of protein phosphorylation with protein nanopore technology. A model protein, thioredoxin, was phosphorylated at two adjacent sites. Analysis of the ionic Current Amplitude and noise, as the protein unfolds and moves through an α-hemolysin pore, enables the distinction between unphosphorylated, monophosphorylated and diphosphorylated variants. Our results provide a step toward nanopore proteomics.
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single molecule site specific detection of protein phosphorylation with a nanopore
Nature Biotechnology, 2014Co-Authors: Christian Bech Rosen, David Rodriguezlarrea, Hagan BayleyAbstract:We demonstrate single-molecule, site-specific detection of protein phosphorylation with protein nanopore technology. A model protein, thioredoxin, was phosphorylated at two adjacent sites. Analysis of the ionic Current Amplitude and noise, as the protein unfolds and moves through an α-hemolysin pore, enables the distinction between unphosphorylated, monophosphorylated and diphosphorylated variants. Our results provide a step toward nanopore proteomics.
Hagan Bayley - One of the best experts on this subject based on the ideXlab platform.
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single molecule site specific detection of protein phosphorylation with a nanopore
Nature Biotechnology, 2014Co-Authors: Christian Bech Rosen, David Rodriguezlarrea, Hagan BayleyAbstract:A protein nanopore can distinguish model proteins phosphorylated at adjacent sites. We demonstrate single-molecule, site-specific detection of protein phosphorylation with protein nanopore technology. A model protein, thioredoxin, was phosphorylated at two adjacent sites. Analysis of the ionic Current Amplitude and noise, as the protein unfolds and moves through an α-hemolysin pore, enables the distinction between unphosphorylated, monophosphorylated and diphosphorylated variants. Our results provide a step toward nanopore proteomics.
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single molecule site specific detection of protein phosphorylation with a nanopore
Nature Biotechnology, 2014Co-Authors: Christian Bech Rosen, David Rodriguezlarrea, Hagan BayleyAbstract:We demonstrate single-molecule, site-specific detection of protein phosphorylation with protein nanopore technology. A model protein, thioredoxin, was phosphorylated at two adjacent sites. Analysis of the ionic Current Amplitude and noise, as the protein unfolds and moves through an α-hemolysin pore, enables the distinction between unphosphorylated, monophosphorylated and diphosphorylated variants. Our results provide a step toward nanopore proteomics.
Eleonore Moreau - One of the best experts on this subject based on the ideXlab platform.
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computer modeling of whole cell voltage clamp analyses to delineate guidelines for good practice of manual and automated patch clamp
Scientific Reports, 2021Co-Authors: Jerome Montnach, Maxime Lorenzini, Adrien Lesage, Isabelle Simon, Sebastien Nicolas, Eleonore Moreau, Celine Marionneau, Isabelle Baro, Michel De WaardAbstract:The patch-clamp technique and more recently the high throughput patch-clamp technique have contributed to major advances in the characterization of ion channels. However, the whole-cell voltage-clamp technique presents certain limits that need to be considered for robust data generation. One major caveat is that increasing Current Amplitude profoundly impacts the accuracy of the biophysical analyses of macroscopic ion Currents under study. Using mathematical kinetic models of a cardiac voltage-gated sodium channel and a cardiac voltage-gated potassium channel, we demonstrated how large Current Amplitude and series resistance artefacts induce an undetected alteration in the actual membrane potential and affect the characterization of voltage-dependent activation and inactivation processes. We also computed how dose-response curves are hindered by high Current Amplitudes. This is of high interest since stable cell lines frequently demonstrating high Current Amplitudes are used for safety pharmacology using the high throughput patch-clamp technique. It is therefore critical to set experimental limits for Current Amplitude recordings to prevent inaccuracy in the characterization of channel properties or drug activity, such limits being different from one channel type to another. Based on the predictions generated by the kinetic models, we draw simple guidelines for good practice of whole-cell voltage-clamp recordings.
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modulation of low voltage activated inward Current permeable to sodium and calcium by darpp 32 drives spontaneous firing of insect octopaminergic neurosecretory cells
Frontiers in Systems Neuroscience, 2017Co-Authors: Bruno Lapied, Eleonore Moreau, Antoine Defaix, Maria Stankiewicz, Valérie RaymondAbstract:Identification of the different intracellular pathways that control phosphorylation/dephosphorylation process of ionic channels represents an exciting alternative approach for studying the ionic mechanisms underlying neuronal pacemaker activity. In the central nervous system of the cockroach Periplaneta americana, octopaminergic neurons, called dorsal unpaired median (DUM neurons), generate spontaneous repetitive action potentials. Short-term cultured adult DUM neurons isolated from the terminal abdominal ganglion of the nerve cord were used to study the regulation of the low-voltage-activated channel permeable to sodium and calcium (Na/Ca), involved in the pre-depolarization, under whole cell voltage- and Current-clamp conditions. A bell-shaped curve illustrating the regulation of the Amplitude of the maintained Current versus [ATP]i was observed. This suggested the existence of phosphorylation mechanisms. The PKA inhibitor, H89 and elevating [cAMP]i, increased and decreased the Current Amplitude, respectively. These results together with the effects of forskoline indicated a regulation of the Current via a cAMP/PKA cascade. Furthermore, intracellular application of PP2B inhibitors, cyclosporine A, FK506 and PP1/2A inhibitor, okadaic acid decreased the Current Amplitude. From these results and because octopamine regulates DUM neuron electrical activity via an elevation of [cAMP]i, we wanted to know if, like in vertebrate dopaminergic neurons, octopamine receptor stimulation could indirectly affect the Current via PKA-mediated phosphorylation of Dopamine- and cAMP-regulated Phosphoprotein-32 (DARPP-32) known to inhibit PP1/2A. Experiments were performed using intracellular application of phospho-DARPP-32 and non-phospho-DARPP-32. Phospho-DARPP-32 strongly reduced the Current Amplitude whereas non-phospho-DARPP-32 did not affect the Current. All together, these results confirm that DARPP-32-mediated inhibition of PP1/2A regulates the maintained Na/Ca Current, which contributes to the development of the pre-depolarizing phase of the DUM neuron pacemaker activity.
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Modulation of Low-Voltage-Activated Inward Current Permeable to Sodium and Calcium by DARPP-32 Drives Spontaneous Firing of Insect Octopaminergic Neurosecretory Cells
Frontiers in Systems Neuroscience, 2017Co-Authors: Bruno Lapied, Eleonore Moreau, Antoine Defaix, Maria Stankiewicz, Valérie RaymondAbstract:Identification of the different intracellular pathways that control phosphorylation/dephosphorylation process of ionic channels represents an exciting alternative approach for studying the ionic mechanisms underlying neuronal pacemaker activity. In the central nervous system of the cockroach Periplaneta americana, octopaminergic neurons, called dorsal unpaired median (DUM; DUM neurons), generate spontaneous repetitive action potentials. Short-term cultured adult DUM neurons isolated from the terminal abdominal ganglion (TAG) of the nerve cord were used to study the regulation of a tetrodotoxin-sensitive low-voltage-activated (LVA) channel permeable to sodium and calcium (Na/Ca), under whole cell voltage-and Current-clamp conditions. A bell-shaped curve illustrating the regulation of the Amplitude of the maintained Current vs. [ATP]i was observed. This suggested the existence of phosphorylation mechanisms. The protein kinase A (PKA)inhibitor, H89 and elevating [ cyclic adenosine 3 0, 5 0 monophosphate, cAMP] i, increased and decreased the Current Amplitude, respectively. This indicated a regulation of the Current via a cAMP/PKA cascade. Furthermore, intracellular application of PP2B inhibitors, cyclosporine A, FK506 and PP1/2A inhibitor, okadaic acid decreased the Current Amplitude. From these results and because octopamine (OA) regulates DUM neuron electrical activity via an elevation of [cAMP]i, we wanted to know if, like in vertebrate dopaminergic neurons, OA receptor (OAR) stimulation could indirectly affect the Current via PKA-mediated phosphorylation of Dopamine-and cAMP-regulated Phosphoprotein-32 (DARPP-32) known to inhibit PP1/2A. Experiments were performed using intracellular application of phospho-DARPP-32 and non-phospho-DARPP-32. Phospho-DARPP-32 strongly reduced the Current Amplitude whereas non-phospho-DARPP-32 did not affect the Current. All together, these results confirm that DARPP- 32-mediated inhibition of PP1/2A regulates the maintained sodium/calcium Current, which contributes to the development of the pre-depolarizing phase of the DUM neuron pacemaker activity.
Valérie Raymond - One of the best experts on this subject based on the ideXlab platform.
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modulation of low voltage activated inward Current permeable to sodium and calcium by darpp 32 drives spontaneous firing of insect octopaminergic neurosecretory cells
Frontiers in Systems Neuroscience, 2017Co-Authors: Bruno Lapied, Eleonore Moreau, Antoine Defaix, Maria Stankiewicz, Valérie RaymondAbstract:Identification of the different intracellular pathways that control phosphorylation/dephosphorylation process of ionic channels represents an exciting alternative approach for studying the ionic mechanisms underlying neuronal pacemaker activity. In the central nervous system of the cockroach Periplaneta americana, octopaminergic neurons, called dorsal unpaired median (DUM neurons), generate spontaneous repetitive action potentials. Short-term cultured adult DUM neurons isolated from the terminal abdominal ganglion of the nerve cord were used to study the regulation of the low-voltage-activated channel permeable to sodium and calcium (Na/Ca), involved in the pre-depolarization, under whole cell voltage- and Current-clamp conditions. A bell-shaped curve illustrating the regulation of the Amplitude of the maintained Current versus [ATP]i was observed. This suggested the existence of phosphorylation mechanisms. The PKA inhibitor, H89 and elevating [cAMP]i, increased and decreased the Current Amplitude, respectively. These results together with the effects of forskoline indicated a regulation of the Current via a cAMP/PKA cascade. Furthermore, intracellular application of PP2B inhibitors, cyclosporine A, FK506 and PP1/2A inhibitor, okadaic acid decreased the Current Amplitude. From these results and because octopamine regulates DUM neuron electrical activity via an elevation of [cAMP]i, we wanted to know if, like in vertebrate dopaminergic neurons, octopamine receptor stimulation could indirectly affect the Current via PKA-mediated phosphorylation of Dopamine- and cAMP-regulated Phosphoprotein-32 (DARPP-32) known to inhibit PP1/2A. Experiments were performed using intracellular application of phospho-DARPP-32 and non-phospho-DARPP-32. Phospho-DARPP-32 strongly reduced the Current Amplitude whereas non-phospho-DARPP-32 did not affect the Current. All together, these results confirm that DARPP-32-mediated inhibition of PP1/2A regulates the maintained Na/Ca Current, which contributes to the development of the pre-depolarizing phase of the DUM neuron pacemaker activity.
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Modulation of Low-Voltage-Activated Inward Current Permeable to Sodium and Calcium by DARPP-32 Drives Spontaneous Firing of Insect Octopaminergic Neurosecretory Cells
Frontiers in Systems Neuroscience, 2017Co-Authors: Bruno Lapied, Eleonore Moreau, Antoine Defaix, Maria Stankiewicz, Valérie RaymondAbstract:Identification of the different intracellular pathways that control phosphorylation/dephosphorylation process of ionic channels represents an exciting alternative approach for studying the ionic mechanisms underlying neuronal pacemaker activity. In the central nervous system of the cockroach Periplaneta americana, octopaminergic neurons, called dorsal unpaired median (DUM; DUM neurons), generate spontaneous repetitive action potentials. Short-term cultured adult DUM neurons isolated from the terminal abdominal ganglion (TAG) of the nerve cord were used to study the regulation of a tetrodotoxin-sensitive low-voltage-activated (LVA) channel permeable to sodium and calcium (Na/Ca), under whole cell voltage-and Current-clamp conditions. A bell-shaped curve illustrating the regulation of the Amplitude of the maintained Current vs. [ATP]i was observed. This suggested the existence of phosphorylation mechanisms. The protein kinase A (PKA)inhibitor, H89 and elevating [ cyclic adenosine 3 0, 5 0 monophosphate, cAMP] i, increased and decreased the Current Amplitude, respectively. This indicated a regulation of the Current via a cAMP/PKA cascade. Furthermore, intracellular application of PP2B inhibitors, cyclosporine A, FK506 and PP1/2A inhibitor, okadaic acid decreased the Current Amplitude. From these results and because octopamine (OA) regulates DUM neuron electrical activity via an elevation of [cAMP]i, we wanted to know if, like in vertebrate dopaminergic neurons, OA receptor (OAR) stimulation could indirectly affect the Current via PKA-mediated phosphorylation of Dopamine-and cAMP-regulated Phosphoprotein-32 (DARPP-32) known to inhibit PP1/2A. Experiments were performed using intracellular application of phospho-DARPP-32 and non-phospho-DARPP-32. Phospho-DARPP-32 strongly reduced the Current Amplitude whereas non-phospho-DARPP-32 did not affect the Current. All together, these results confirm that DARPP- 32-mediated inhibition of PP1/2A regulates the maintained sodium/calcium Current, which contributes to the development of the pre-depolarizing phase of the DUM neuron pacemaker activity.
David Rodriguezlarrea - One of the best experts on this subject based on the ideXlab platform.
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single molecule site specific detection of protein phosphorylation with a nanopore
Nature Biotechnology, 2014Co-Authors: Christian Bech Rosen, David Rodriguezlarrea, Hagan BayleyAbstract:A protein nanopore can distinguish model proteins phosphorylated at adjacent sites. We demonstrate single-molecule, site-specific detection of protein phosphorylation with protein nanopore technology. A model protein, thioredoxin, was phosphorylated at two adjacent sites. Analysis of the ionic Current Amplitude and noise, as the protein unfolds and moves through an α-hemolysin pore, enables the distinction between unphosphorylated, monophosphorylated and diphosphorylated variants. Our results provide a step toward nanopore proteomics.
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single molecule site specific detection of protein phosphorylation with a nanopore
Nature Biotechnology, 2014Co-Authors: Christian Bech Rosen, David Rodriguezlarrea, Hagan BayleyAbstract:We demonstrate single-molecule, site-specific detection of protein phosphorylation with protein nanopore technology. A model protein, thioredoxin, was phosphorylated at two adjacent sites. Analysis of the ionic Current Amplitude and noise, as the protein unfolds and moves through an α-hemolysin pore, enables the distinction between unphosphorylated, monophosphorylated and diphosphorylated variants. Our results provide a step toward nanopore proteomics.
