Enrico Leipold - Academia.edu (original) (raw)

Papers by Enrico Leipold

Research paper thumbnail of Isolation and transfection of myenteric neurons from mice for patch-clamp applications

Frontiers in Molecular Neuroscience

The enteric nervous system (ENS) is a complex neuronal network organized in ganglionated plexuses... more The enteric nervous system (ENS) is a complex neuronal network organized in ganglionated plexuses that extend along the entire length of the gastrointestinal tract. Largely independent of the central nervous system, the ENS coordinates motility and peristalsis of the digestive tract, regulates secretion and absorption, and is involved in immunological processes. Electrophysiological methods such as the patch-clamp technique are particularly suitable to study the function of neurons as well as the biophysical parameters of the underlying ion channels under both physiological and pathophysiological conditions. However, application of the patch-clamp method to ENS neurons remained difficult because they are embedded in substantial tissue layers that limit access to and targeted manipulation of these cells. Here, we present a robust step-by-step protocol that involves isolation of ENS neurons from adult mice, culturing of the cells, their transfection with plasmid DNA, and subsequent el...

Research paper thumbnail of Structural insights into the interaction of antifungal peptides and ergosterol containing fungal membrane

Biochimica et Biophysica Acta (BBA) - Biomembranes

Research paper thumbnail of A novel m-conopeptide , CnIIIC , exerts potent and preferential inhibition of NaV 1 . 2 / 1 . 4 channels and blocks neuronal nicotinic acetylcholine receptors

Atheris Laboratories, Bernex-Geneva, Switzerland, CNRS, Institut de Neurobiologie Alfred Fessard,... more Atheris Laboratories, Bernex-Geneva, Switzerland, CNRS, Institut de Neurobiologie Alfred Fessard, Laboratoire de Neurobiologie et Développement, Gif sur Yvette cedex, France, Bijvoet Center for Biomolecular Research, Padualaan, Utrecht, the Netherlands, Department of Neuroscience, Geneva, Switzerland, Center for Molecular Biomedicine, Department of Biophysics, Friedrich Schiller University of Jena and Jena University Hospital, Jena, Germany, Laboratory of Marine Toxins, Program of Physiology and Biophysics, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile, Santiago, Chile, Department of Structural Biology and Bioinformatics, Faculty of Medicine, University of Geneva, Switzerland, and Laboratorium voor Toxicologie, Campus Gasthuisberg, Onderwijs en Navorsing, Herestraat, Leuven, Belgium Correspondence Dr Jordi Molgó, Laboratoire de Neurobiologie et Développement, CNRS, bâtiments 32-33, 1 avenue de la Terrasse, 91198 Gif sur Yvette cedex, France. E-mail: Jord...

Research paper thumbnail of Where cone snails and spiders meet: design of small cyclic sodium‐channel inhibitors

The FASEB Journal, 2018

A 13 aa residue voltage-gated sodium (Na V) channel inhibitor peptide, Pn, containing 2 disulfide... more A 13 aa residue voltage-gated sodium (Na V) channel inhibitor peptide, Pn, containing 2 disulfide bridges was designed by using a chimeric approach. This approach was based on a common pharmacophore deduced from sequence and secondary structural homology of 2 Na V inhibitors: Conus kinoshitai toxin IIIA, a 14 residue cone snail peptide with 3 disulfide bonds, and Phoneutria nigriventer toxin 1, a 78 residue spider toxin with 7 disulfide bonds. As with the parent peptides, this novel Na V channel inhibitor was active on Na V 1.2. Through the generation of 3 series of peptide mutants, we investigated the role of key residues and cyclization and their influence on Na V inhibition and subtype selectivity. Cyclic PnCS1, a 10 residue peptide cyclized via a disulfide bond, exhibited increased inhibitory activity toward therapeutically relevant Na V channel subtypes, including Na V 1.7 and Na V 1.9, while displaying remarkable serum stability. These peptides represent the first and the smallest cyclic peptide Na V modulators to date and are promising templates for the development of toxin-based therapeutic agents.-Peigneur,

Research paper thumbnail of Pain insensitivity: distal S6-segment mutations in NaV1.9 emerge as critical hotspot

Neurogenetics, Jan 13, 2017

Research paper thumbnail of Solution structure of the skeletal muscle and neuronal voltage gated sodium channel antagonist mu-conotoxin CnIIIC

The following versions of software and data (see references i ○) were used in the production of t... more The following versions of software and data (see references i ○) were used in the production of this report:

Research paper thumbnail of Photonic modulation of membrane potential by flash-light illumination of metallic beads

The electrical membrane potential (Vm) is a crucial parameter determining the physiological prope... more The electrical membrane potential (Vm) is a crucial parameter determining the physiological properties of cells – in particular of excitable cells. For studying cell function it is mandatory to manipulate Vm. Approaches involving genetically encoded proteins (optogenetics) are now widely used to manipulate Vm with optical means. Despite the success of such methods there are some shortcomings, such as the need to genetically modify cells or a limited time resolution, which ask for alternative/additional methods. Here we evaluated the use of transient illumination of metallic beads. We use superparamagnetic beads (polystyrene-coated iron-core beads, Dynabeads® 4.5 μm diameter, Life technologies AS, Oslo), targeted to the cell membrane by CD8-antibodies, to modulate Vm. Flash-light excitation via the epifluorescence channel of an inverted microscope (Xenon flash lamp, about 500 μs, >1015 photon/m2, up to 470 nm), was combined with whole-cell patch-clamp recordings. CD8 was expressed in HEK 293 cells. Flash illumination of a single cell-associated bead resulted in transient inward current of about 500 pA at -70 mV using a 40x objective; the waveform of the current reflected the time course of the excitation light. With a 100x objective inward currents of up to 20 nA were observed. Currents reversed at about 0 mV indicating a transient non-selective conductance. The current approximately doubled with two beads bound to the cell. Flash-light-induced currents decreased about exponentially with the distance to the cell membrane when the beads were placed using a separate patch pipette (half-width about 2-5 μm). Silver particles of approximately the same size resulted in similar signals. Measuring Vm of murine dorsal root ganglia neurons in the current-clamp mode, single-bead illumination was sufficient to elicit action potentials within 500 μs. Thus, transient illumination of superparamagnetic beads, targeted to specific cellular sites via antibody coating or direct placement, may provide means of rapid and spatially confined electrical cell stimulation.

Research paper thumbnail of Sodium channels as gateable non-photonic sensors for membrane-delimited reactive species

Biochimica et Biophysica Acta (BBA) - Biomembranes, 2014

Reactive oxygen species (ROS) and reactive oxygen intermediates (ROI) play crucial roles in physi... more Reactive oxygen species (ROS) and reactive oxygen intermediates (ROI) play crucial roles in physiological processes. While excessive ROS damages cells, small fluctuations in ROS levels represent physiological signals important for vital functions. Despite the physiological importance of ROS, many fundamental questions remain unanswered, such as which types of ROS occur in cells, how they distribute inside cells, and how long they remain in an active form. The current study presents a ratiometric sensor of intracellular ROS levels based on genetically engineered voltage-gated sodium channels (roNa V). roNa V can be used for detecting oxidative modification that occurs near the plasma membrane with a sensitivity similar to existing fluorescence-based ROS sensors. Moreover, roNa V has several advantages over traditional sensors because it does not need excitation light for sensing, and thus, can be used to detect phototoxic cellular modifications. In addition, the ROS dynamic range of roNa V is easily manipulated in real time by means of the endogenous channel inactivation mechanism. Measurements on ROS liberated from intracellular Lucifer Yellow and genetically encoded KillerRed has revealed an assessment of ROS lifetime in individual mammalian cells. Flashlight-induced ROS concentration decayed with two major time constants of about 10 and 1000 ms.

Research paper thumbnail of Application of Room-Temperature Aprotic and Protic Ionic Liquids for Oxidative Folding of Cysteine-Rich Peptides

ChemBioChem, 2014

Scheme 1. Structures of the ionic liquids used to synthesize conotoxin m-SIIIA by the oxidative s... more Scheme 1. Structures of the ionic liquids used to synthesize conotoxin m-SIIIA by the oxidative self-folding strategy, together with abbreviations.

Research paper thumbnail of Electrophysiological Properties of Induced Pluripotent Stem Cell-Derived Midbrain Dopaminergic Neurons Correlate With Expression of Tyrosine Hydroxylase

Frontiers in Cellular Neuroscience, 2022

Induced pluripotent stem cell (iPSC)-based generation of tyrosine hydroxylase-positive (TH+) dopa... more Induced pluripotent stem cell (iPSC)-based generation of tyrosine hydroxylase-positive (TH+) dopaminergic neurons (DNs) is a powerful method for creating patient-specific in vitro models to elucidate mechanisms underlying Parkinson’s disease (PD) at the cellular and molecular level and to perform drug screening. However, currently available differentiation paradigms result in highly heterogeneous cell populations, often yielding a disappointing fraction (<50%) of the PD-relevant TH+ DNs. To facilitate the targeted analysis of this cell population and to characterize their electrophysiological properties, we employed CRISPR/Cas9 technology and generated an mCherry-based human TH reporter iPSC line. Subsequently, reporter iPSCs were subjected to dopaminergic differentiation using either a “floor plate protocol” generating DNs directly from iPSCs or an alternative method involving iPSC-derived neuronal precursors (NPC-derived DNs). To identify the strategy with the highest conversio...

Research paper thumbnail of The μO-conotoxin MrVIA inhibits voltage-gated sodium channels by associating with domain-3

Several families of peptide toxins from cone snails affect voltage-gated sodium (Na(V)) channels:... more Several families of peptide toxins from cone snails affect voltage-gated sodium (Na(V)) channels: mu-conotoxins block the pore, delta-conotoxins inhibit channel inactivation, and muO-conotoxins inhibit Na(V) channels by an unknown mechanism. The only currently known muO-conotoxins MrVIA and MrVIB from Conus marmoreus were applied to cloned rat skeletal muscle (Na(V)1.4) and brain (Na(V)1.2) sodium channels in mammalian cells. A systematic domain-swapping strategy identified the C-terminal pore loop of domain-3 as the major determinant for Na(V)1.4 being more potently blocked than Na(V)1.2 channels. muO-conotoxins therefore show an interaction pattern with Na(V) channels that is clearly different from the related mu- and delta-conotoxins, indicative of a distinct molecular mechanism of channel inhibition.

Research paper thumbnail of Mechanosensitivity of NaV1.5 sodium channels is regulated by specific β-subunits

Pflügers Archiv - European Journal of Physiology

Research paper thumbnail of {"__content__"=>"Na1.9 Potentiates Oxidized Phospholipid-Induced TRP Responses Only under Inflammatory Conditions.", "sub"=>{"__content__"=>"V"}}

Frontiers in molecular neuroscience, 2018

Oxidized phospholipids (OxPL) like oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholin... more Oxidized phospholipids (OxPL) like oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) were recently identified as novel proalgesic targets in acute and chronic inflammatory pain. These endogenous chemical irritants are generated in inflamed tissue and mediate their pain-inducing function by activating the transient receptor potential channels TRPA1 and TRPV1 expressed in sensory neurons. Notably, prototypical therapeutics interfering with OxPL were shown to inhibit TRP channel activation and pain behavior. Here, we asked how OxPL excite primary sensory neurons of dorsal root ganglia (DRG neurons from mice of either sex). Acute stimulation of sensory neurons with the prototypical OxPL 1-palmitoyl-2-glutaryl--glycero-3-phosphocholine (PGPC) evoked repetitive calcium spikes in small-diameter neurons. As Na1.9, a voltage-gated sodium channel involved in nociceptor excitability, was previously shown to be essential for the generation of calcium spikes in motoneurons...

Research paper thumbnail of Subtype-specific block of voltage-gated K+ channels by μ-conopeptides

Biochemical and Biophysical Research Communications

Research paper thumbnail of Voltage-gated sodium channels and pain

e-Neuroforum

Painful stimuli are detected by specialized neurons, nociceptors, and are translated into action ... more Painful stimuli are detected by specialized neurons, nociceptors, and are translated into action potentials, that are conducted along afferent pathways into the central nervous system, where they are conceived as pain. Voltage-gated sodium channels (Na

Research paper thumbnail of Spannungsgesteuerte Natriumkanäle und Schmerz

e-Neuroforum

ZusammenfassungSchmerzhafte Reize werden von spezialisierten Nervenzellen, den sog. Nozizeptoren,... more ZusammenfassungSchmerzhafte Reize werden von spezialisierten Nervenzellen, den sog. Nozizeptoren, registriert und in Aktionspotenziale übersetzt, die entlang afferenter Bahnen in das zentrale Nervensystem gelangen und dort als Schmerz interpretiert werden. Spannungsgesteuerte Natriumkanäle (Na

Research paper thumbnail of Non-photonic sensing of membrane-delimited reactive species with a Na+ channel protein containing selenocysteine

Free Radical Biology and Medicine

Photonic experiments are of key importance in life sciences but light-induced side effects are se... more Photonic experiments are of key importance in life sciences but light-induced side effects are serious confounding factors. Here we introduce roNa V 2, an engineered voltage-gated Na + channel harboring a selenocysteine in its inactivation motif, as a non-photonic, sensitive, gateable, and reversible sensor for membrane-delimited reactive species. roNa V 2 allows for the assessment of chemical modification induced in fluorescence microscopy settings with high sensitivity and time resolution and it demonstrates the usefulness of ion channels as highly sensitive reporters of membrane processes. Cellular reactive species (RS), e.g., superoxide (O 2 −⋅), hydroxide (HO ⋅), hydrogen peroxide (H 2 O 2), and hypochlorous acid (HClO) play crucial roles in physiological processes, such as signal transduction, activation of nuclear transcription factors, gene expression, and immune regulation 1-3. However, excess RS modify lipids, proteins, and nucleic acids, and are often associated with adverse consequences such as neurodegeneration, atherosclerosis, diabetes, sepsis, and cancer 4. RS differ in their production, concentration, distribution, lifetime, molecular targets, and biological functions. Therefore, there is a clear demand for sensitive probes to precisely monitor cellular RS with spatio-temporal resolution to understand the multifaceted role of cellular RS in cell physiology. Recently, mutants and fusions of genetically encoded GFP (green fluorescent protein), for example, reduction and oxidation sensitive variants roGFP2 5 and Grx1-roGFP2 (Grx = glutaredoxin) 6 , received much attention for cellular RS monitoring. roGFP2 is a genetically engineered fluorescent redox reporter, which was developed by inserting a dithiol-disulfide pair into GFP. The breakage and the formation of the disulfide bridge in roGFP2 leads to a differential change in fluorescence at 400 nm and 470 nm excitation wavelength, thus yielding a ratiometric signal that reports on oxidative changes in its local environment. Fusion of human glutaredoxin-1 to roGFP2 provides strongly accelerated formation of a disulfide bridge and, hence, increases the sensitivity of Grx1-roGFP2 in the physiologically relevant range for glutathione-mediated RS modifications 6. These fluorescent reporters offer several useful features, such as the choice of localized sub-cellular targeting, real-time RS detection, and ratiometric observation that overcome artifacts arising due to photo-bleaching and inhomogeneous distribution of the sensors. However, there is the inherent problem of phototoxicity that can lead to lasting irreversible destruction of cellular structures and may even result in immediate alteration of molecular function. Despite the obvious detrimental consequences of visible light in cell physiological studies, the issue of phototoxicity has been only infrequently considered in life science research 7. The presence of intracellular flavin-containing oxidases, the cytochrome system, heme-containing proteins, and tryptophan-rich proteins not only confound with fluorescence techniques, they may also act as cellular photosensitizers and constitute the major source of light-induced RS 8-10. Moreover, photo-activated GFP disturbs cellular redox homeostasis either by producing endogenous superoxide or altering the glutathione homeostasis 11-13. Therefore, the potential interference of blue light as used for GFP-based imaging with the cellular redox system clearly demands for alternative tools or probes for monitoring cellular RS. Previously, we have reported a genetically engineered voltage-gated Na + (Na V) channel with a cysteine residue in its inactivation domain (roNa V 1; rNa V 1.4 mutant M1305C, also termed IFC) as a non-photonic RS sensor delimited to the cell membrane 14. Na V channels activate (open) and inactivate (close) rapidly in response to

Research paper thumbnail of New Insight in Cold Pain: Role of Ion Channels, Modulation, and Clinical Perspectives

Journal of Neuroscience, 2016

Cold temperature detection involves the process of sensory transduction in cutaneous primary sens... more Cold temperature detection involves the process of sensory transduction in cutaneous primary sensory nerve terminals, which converts thermal stimuli into depolarizations of the membrane. This transformation into electrical signals is followed by the subsequent propagation of action potentials in cold-sensitive afferent nerve fibers. A large array of ion channels shapes this process; however, the precise contribution of specific ion channel subtypes to cold perception and cold pain remains elusive. This review aims at giving an update on our current understanding of the role played by TRPs, leak K ϩ and voltage-gated Na ϩ and K ϩ channels in the transduction of cold by nociceptors and in cold-induced pain.

Research paper thumbnail of Molecular interaction of δ-conopeptide EVIA with voltage-gated Na(+) channels

Biochimica et biophysica acta, 2016

Research paper thumbnail of A Subtle Alternative Splicing Event of the NaV1.8 Voltage-Gated Sodium Channel is Conserved in Human, Rat, and Mouse

Journal of Molecular Neuroscience, 2010

The voltage-gated sodium channel subtype Na(V)1.8 (SCN10A) is exclusively expressed in dorsal roo... more The voltage-gated sodium channel subtype Na(V)1.8 (SCN10A) is exclusively expressed in dorsal root ganglia (DRG) and plays a critical role in pain perception. We isolated mRNA from human, rat, and mouse DRGs and screened for alternatively spliced isoforms of the SCN10A mRNA using 454 sequencing. In all three species, we found an event of subtle alternative splicing at a NAGNAG tandem acceptor that results in isoforms including or lacking glutamine 1030 (Na(V)1.8+Q and Na(V)1.8-Q, respectively) within the cytoplasmic loop between domains II and III. The relative amount of Na(V)1.8-Q mRNA in adult DRG was measured with 14.1 +/- 0.1% in humans and 11.2 +/- 0.2% in rats. This is in contrast to an abundance of 64.3 +/- 0.3% in mouse DRG. Thus, the NAGNAG tandem acceptor in SCN10A is conserved among rodents and humans but its alternative usage apparently occurs with species-specific abundance. Analysis of human Na(V)1.8+Q and -Q isoforms in whole-cell patch-clamp experiments after heterologous expression in the neuroblastoma cell line Neuro-2A revealed no obvious impact of the splicing event on channel function.

Research paper thumbnail of Isolation and transfection of myenteric neurons from mice for patch-clamp applications

Frontiers in Molecular Neuroscience

The enteric nervous system (ENS) is a complex neuronal network organized in ganglionated plexuses... more The enteric nervous system (ENS) is a complex neuronal network organized in ganglionated plexuses that extend along the entire length of the gastrointestinal tract. Largely independent of the central nervous system, the ENS coordinates motility and peristalsis of the digestive tract, regulates secretion and absorption, and is involved in immunological processes. Electrophysiological methods such as the patch-clamp technique are particularly suitable to study the function of neurons as well as the biophysical parameters of the underlying ion channels under both physiological and pathophysiological conditions. However, application of the patch-clamp method to ENS neurons remained difficult because they are embedded in substantial tissue layers that limit access to and targeted manipulation of these cells. Here, we present a robust step-by-step protocol that involves isolation of ENS neurons from adult mice, culturing of the cells, their transfection with plasmid DNA, and subsequent el...

Research paper thumbnail of Structural insights into the interaction of antifungal peptides and ergosterol containing fungal membrane

Biochimica et Biophysica Acta (BBA) - Biomembranes

Research paper thumbnail of A novel m-conopeptide , CnIIIC , exerts potent and preferential inhibition of NaV 1 . 2 / 1 . 4 channels and blocks neuronal nicotinic acetylcholine receptors

Atheris Laboratories, Bernex-Geneva, Switzerland, CNRS, Institut de Neurobiologie Alfred Fessard,... more Atheris Laboratories, Bernex-Geneva, Switzerland, CNRS, Institut de Neurobiologie Alfred Fessard, Laboratoire de Neurobiologie et Développement, Gif sur Yvette cedex, France, Bijvoet Center for Biomolecular Research, Padualaan, Utrecht, the Netherlands, Department of Neuroscience, Geneva, Switzerland, Center for Molecular Biomedicine, Department of Biophysics, Friedrich Schiller University of Jena and Jena University Hospital, Jena, Germany, Laboratory of Marine Toxins, Program of Physiology and Biophysics, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile, Santiago, Chile, Department of Structural Biology and Bioinformatics, Faculty of Medicine, University of Geneva, Switzerland, and Laboratorium voor Toxicologie, Campus Gasthuisberg, Onderwijs en Navorsing, Herestraat, Leuven, Belgium Correspondence Dr Jordi Molgó, Laboratoire de Neurobiologie et Développement, CNRS, bâtiments 32-33, 1 avenue de la Terrasse, 91198 Gif sur Yvette cedex, France. E-mail: Jord...

Research paper thumbnail of Where cone snails and spiders meet: design of small cyclic sodium‐channel inhibitors

The FASEB Journal, 2018

A 13 aa residue voltage-gated sodium (Na V) channel inhibitor peptide, Pn, containing 2 disulfide... more A 13 aa residue voltage-gated sodium (Na V) channel inhibitor peptide, Pn, containing 2 disulfide bridges was designed by using a chimeric approach. This approach was based on a common pharmacophore deduced from sequence and secondary structural homology of 2 Na V inhibitors: Conus kinoshitai toxin IIIA, a 14 residue cone snail peptide with 3 disulfide bonds, and Phoneutria nigriventer toxin 1, a 78 residue spider toxin with 7 disulfide bonds. As with the parent peptides, this novel Na V channel inhibitor was active on Na V 1.2. Through the generation of 3 series of peptide mutants, we investigated the role of key residues and cyclization and their influence on Na V inhibition and subtype selectivity. Cyclic PnCS1, a 10 residue peptide cyclized via a disulfide bond, exhibited increased inhibitory activity toward therapeutically relevant Na V channel subtypes, including Na V 1.7 and Na V 1.9, while displaying remarkable serum stability. These peptides represent the first and the smallest cyclic peptide Na V modulators to date and are promising templates for the development of toxin-based therapeutic agents.-Peigneur,

Research paper thumbnail of Pain insensitivity: distal S6-segment mutations in NaV1.9 emerge as critical hotspot

Neurogenetics, Jan 13, 2017

Research paper thumbnail of Solution structure of the skeletal muscle and neuronal voltage gated sodium channel antagonist mu-conotoxin CnIIIC

The following versions of software and data (see references i ○) were used in the production of t... more The following versions of software and data (see references i ○) were used in the production of this report:

Research paper thumbnail of Photonic modulation of membrane potential by flash-light illumination of metallic beads

The electrical membrane potential (Vm) is a crucial parameter determining the physiological prope... more The electrical membrane potential (Vm) is a crucial parameter determining the physiological properties of cells – in particular of excitable cells. For studying cell function it is mandatory to manipulate Vm. Approaches involving genetically encoded proteins (optogenetics) are now widely used to manipulate Vm with optical means. Despite the success of such methods there are some shortcomings, such as the need to genetically modify cells or a limited time resolution, which ask for alternative/additional methods. Here we evaluated the use of transient illumination of metallic beads. We use superparamagnetic beads (polystyrene-coated iron-core beads, Dynabeads® 4.5 μm diameter, Life technologies AS, Oslo), targeted to the cell membrane by CD8-antibodies, to modulate Vm. Flash-light excitation via the epifluorescence channel of an inverted microscope (Xenon flash lamp, about 500 μs, >1015 photon/m2, up to 470 nm), was combined with whole-cell patch-clamp recordings. CD8 was expressed in HEK 293 cells. Flash illumination of a single cell-associated bead resulted in transient inward current of about 500 pA at -70 mV using a 40x objective; the waveform of the current reflected the time course of the excitation light. With a 100x objective inward currents of up to 20 nA were observed. Currents reversed at about 0 mV indicating a transient non-selective conductance. The current approximately doubled with two beads bound to the cell. Flash-light-induced currents decreased about exponentially with the distance to the cell membrane when the beads were placed using a separate patch pipette (half-width about 2-5 μm). Silver particles of approximately the same size resulted in similar signals. Measuring Vm of murine dorsal root ganglia neurons in the current-clamp mode, single-bead illumination was sufficient to elicit action potentials within 500 μs. Thus, transient illumination of superparamagnetic beads, targeted to specific cellular sites via antibody coating or direct placement, may provide means of rapid and spatially confined electrical cell stimulation.

Research paper thumbnail of Sodium channels as gateable non-photonic sensors for membrane-delimited reactive species

Biochimica et Biophysica Acta (BBA) - Biomembranes, 2014

Reactive oxygen species (ROS) and reactive oxygen intermediates (ROI) play crucial roles in physi... more Reactive oxygen species (ROS) and reactive oxygen intermediates (ROI) play crucial roles in physiological processes. While excessive ROS damages cells, small fluctuations in ROS levels represent physiological signals important for vital functions. Despite the physiological importance of ROS, many fundamental questions remain unanswered, such as which types of ROS occur in cells, how they distribute inside cells, and how long they remain in an active form. The current study presents a ratiometric sensor of intracellular ROS levels based on genetically engineered voltage-gated sodium channels (roNa V). roNa V can be used for detecting oxidative modification that occurs near the plasma membrane with a sensitivity similar to existing fluorescence-based ROS sensors. Moreover, roNa V has several advantages over traditional sensors because it does not need excitation light for sensing, and thus, can be used to detect phototoxic cellular modifications. In addition, the ROS dynamic range of roNa V is easily manipulated in real time by means of the endogenous channel inactivation mechanism. Measurements on ROS liberated from intracellular Lucifer Yellow and genetically encoded KillerRed has revealed an assessment of ROS lifetime in individual mammalian cells. Flashlight-induced ROS concentration decayed with two major time constants of about 10 and 1000 ms.

Research paper thumbnail of Application of Room-Temperature Aprotic and Protic Ionic Liquids for Oxidative Folding of Cysteine-Rich Peptides

ChemBioChem, 2014

Scheme 1. Structures of the ionic liquids used to synthesize conotoxin m-SIIIA by the oxidative s... more Scheme 1. Structures of the ionic liquids used to synthesize conotoxin m-SIIIA by the oxidative self-folding strategy, together with abbreviations.

Research paper thumbnail of Electrophysiological Properties of Induced Pluripotent Stem Cell-Derived Midbrain Dopaminergic Neurons Correlate With Expression of Tyrosine Hydroxylase

Frontiers in Cellular Neuroscience, 2022

Induced pluripotent stem cell (iPSC)-based generation of tyrosine hydroxylase-positive (TH+) dopa... more Induced pluripotent stem cell (iPSC)-based generation of tyrosine hydroxylase-positive (TH+) dopaminergic neurons (DNs) is a powerful method for creating patient-specific in vitro models to elucidate mechanisms underlying Parkinson’s disease (PD) at the cellular and molecular level and to perform drug screening. However, currently available differentiation paradigms result in highly heterogeneous cell populations, often yielding a disappointing fraction (<50%) of the PD-relevant TH+ DNs. To facilitate the targeted analysis of this cell population and to characterize their electrophysiological properties, we employed CRISPR/Cas9 technology and generated an mCherry-based human TH reporter iPSC line. Subsequently, reporter iPSCs were subjected to dopaminergic differentiation using either a “floor plate protocol” generating DNs directly from iPSCs or an alternative method involving iPSC-derived neuronal precursors (NPC-derived DNs). To identify the strategy with the highest conversio...

Research paper thumbnail of The μO-conotoxin MrVIA inhibits voltage-gated sodium channels by associating with domain-3

Several families of peptide toxins from cone snails affect voltage-gated sodium (Na(V)) channels:... more Several families of peptide toxins from cone snails affect voltage-gated sodium (Na(V)) channels: mu-conotoxins block the pore, delta-conotoxins inhibit channel inactivation, and muO-conotoxins inhibit Na(V) channels by an unknown mechanism. The only currently known muO-conotoxins MrVIA and MrVIB from Conus marmoreus were applied to cloned rat skeletal muscle (Na(V)1.4) and brain (Na(V)1.2) sodium channels in mammalian cells. A systematic domain-swapping strategy identified the C-terminal pore loop of domain-3 as the major determinant for Na(V)1.4 being more potently blocked than Na(V)1.2 channels. muO-conotoxins therefore show an interaction pattern with Na(V) channels that is clearly different from the related mu- and delta-conotoxins, indicative of a distinct molecular mechanism of channel inhibition.

Research paper thumbnail of Mechanosensitivity of NaV1.5 sodium channels is regulated by specific β-subunits

Pflügers Archiv - European Journal of Physiology

Research paper thumbnail of {"__content__"=>"Na1.9 Potentiates Oxidized Phospholipid-Induced TRP Responses Only under Inflammatory Conditions.", "sub"=>{"__content__"=>"V"}}

Frontiers in molecular neuroscience, 2018

Oxidized phospholipids (OxPL) like oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholin... more Oxidized phospholipids (OxPL) like oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) were recently identified as novel proalgesic targets in acute and chronic inflammatory pain. These endogenous chemical irritants are generated in inflamed tissue and mediate their pain-inducing function by activating the transient receptor potential channels TRPA1 and TRPV1 expressed in sensory neurons. Notably, prototypical therapeutics interfering with OxPL were shown to inhibit TRP channel activation and pain behavior. Here, we asked how OxPL excite primary sensory neurons of dorsal root ganglia (DRG neurons from mice of either sex). Acute stimulation of sensory neurons with the prototypical OxPL 1-palmitoyl-2-glutaryl--glycero-3-phosphocholine (PGPC) evoked repetitive calcium spikes in small-diameter neurons. As Na1.9, a voltage-gated sodium channel involved in nociceptor excitability, was previously shown to be essential for the generation of calcium spikes in motoneurons...

Research paper thumbnail of Subtype-specific block of voltage-gated K+ channels by μ-conopeptides

Biochemical and Biophysical Research Communications

Research paper thumbnail of Voltage-gated sodium channels and pain

e-Neuroforum

Painful stimuli are detected by specialized neurons, nociceptors, and are translated into action ... more Painful stimuli are detected by specialized neurons, nociceptors, and are translated into action potentials, that are conducted along afferent pathways into the central nervous system, where they are conceived as pain. Voltage-gated sodium channels (Na

Research paper thumbnail of Spannungsgesteuerte Natriumkanäle und Schmerz

e-Neuroforum

ZusammenfassungSchmerzhafte Reize werden von spezialisierten Nervenzellen, den sog. Nozizeptoren,... more ZusammenfassungSchmerzhafte Reize werden von spezialisierten Nervenzellen, den sog. Nozizeptoren, registriert und in Aktionspotenziale übersetzt, die entlang afferenter Bahnen in das zentrale Nervensystem gelangen und dort als Schmerz interpretiert werden. Spannungsgesteuerte Natriumkanäle (Na

Research paper thumbnail of Non-photonic sensing of membrane-delimited reactive species with a Na+ channel protein containing selenocysteine

Free Radical Biology and Medicine

Photonic experiments are of key importance in life sciences but light-induced side effects are se... more Photonic experiments are of key importance in life sciences but light-induced side effects are serious confounding factors. Here we introduce roNa V 2, an engineered voltage-gated Na + channel harboring a selenocysteine in its inactivation motif, as a non-photonic, sensitive, gateable, and reversible sensor for membrane-delimited reactive species. roNa V 2 allows for the assessment of chemical modification induced in fluorescence microscopy settings with high sensitivity and time resolution and it demonstrates the usefulness of ion channels as highly sensitive reporters of membrane processes. Cellular reactive species (RS), e.g., superoxide (O 2 −⋅), hydroxide (HO ⋅), hydrogen peroxide (H 2 O 2), and hypochlorous acid (HClO) play crucial roles in physiological processes, such as signal transduction, activation of nuclear transcription factors, gene expression, and immune regulation 1-3. However, excess RS modify lipids, proteins, and nucleic acids, and are often associated with adverse consequences such as neurodegeneration, atherosclerosis, diabetes, sepsis, and cancer 4. RS differ in their production, concentration, distribution, lifetime, molecular targets, and biological functions. Therefore, there is a clear demand for sensitive probes to precisely monitor cellular RS with spatio-temporal resolution to understand the multifaceted role of cellular RS in cell physiology. Recently, mutants and fusions of genetically encoded GFP (green fluorescent protein), for example, reduction and oxidation sensitive variants roGFP2 5 and Grx1-roGFP2 (Grx = glutaredoxin) 6 , received much attention for cellular RS monitoring. roGFP2 is a genetically engineered fluorescent redox reporter, which was developed by inserting a dithiol-disulfide pair into GFP. The breakage and the formation of the disulfide bridge in roGFP2 leads to a differential change in fluorescence at 400 nm and 470 nm excitation wavelength, thus yielding a ratiometric signal that reports on oxidative changes in its local environment. Fusion of human glutaredoxin-1 to roGFP2 provides strongly accelerated formation of a disulfide bridge and, hence, increases the sensitivity of Grx1-roGFP2 in the physiologically relevant range for glutathione-mediated RS modifications 6. These fluorescent reporters offer several useful features, such as the choice of localized sub-cellular targeting, real-time RS detection, and ratiometric observation that overcome artifacts arising due to photo-bleaching and inhomogeneous distribution of the sensors. However, there is the inherent problem of phototoxicity that can lead to lasting irreversible destruction of cellular structures and may even result in immediate alteration of molecular function. Despite the obvious detrimental consequences of visible light in cell physiological studies, the issue of phototoxicity has been only infrequently considered in life science research 7. The presence of intracellular flavin-containing oxidases, the cytochrome system, heme-containing proteins, and tryptophan-rich proteins not only confound with fluorescence techniques, they may also act as cellular photosensitizers and constitute the major source of light-induced RS 8-10. Moreover, photo-activated GFP disturbs cellular redox homeostasis either by producing endogenous superoxide or altering the glutathione homeostasis 11-13. Therefore, the potential interference of blue light as used for GFP-based imaging with the cellular redox system clearly demands for alternative tools or probes for monitoring cellular RS. Previously, we have reported a genetically engineered voltage-gated Na + (Na V) channel with a cysteine residue in its inactivation domain (roNa V 1; rNa V 1.4 mutant M1305C, also termed IFC) as a non-photonic RS sensor delimited to the cell membrane 14. Na V channels activate (open) and inactivate (close) rapidly in response to

Research paper thumbnail of New Insight in Cold Pain: Role of Ion Channels, Modulation, and Clinical Perspectives

Journal of Neuroscience, 2016

Cold temperature detection involves the process of sensory transduction in cutaneous primary sens... more Cold temperature detection involves the process of sensory transduction in cutaneous primary sensory nerve terminals, which converts thermal stimuli into depolarizations of the membrane. This transformation into electrical signals is followed by the subsequent propagation of action potentials in cold-sensitive afferent nerve fibers. A large array of ion channels shapes this process; however, the precise contribution of specific ion channel subtypes to cold perception and cold pain remains elusive. This review aims at giving an update on our current understanding of the role played by TRPs, leak K ϩ and voltage-gated Na ϩ and K ϩ channels in the transduction of cold by nociceptors and in cold-induced pain.

Research paper thumbnail of Molecular interaction of δ-conopeptide EVIA with voltage-gated Na(+) channels

Biochimica et biophysica acta, 2016

Research paper thumbnail of A Subtle Alternative Splicing Event of the NaV1.8 Voltage-Gated Sodium Channel is Conserved in Human, Rat, and Mouse

Journal of Molecular Neuroscience, 2010

The voltage-gated sodium channel subtype Na(V)1.8 (SCN10A) is exclusively expressed in dorsal roo... more The voltage-gated sodium channel subtype Na(V)1.8 (SCN10A) is exclusively expressed in dorsal root ganglia (DRG) and plays a critical role in pain perception. We isolated mRNA from human, rat, and mouse DRGs and screened for alternatively spliced isoforms of the SCN10A mRNA using 454 sequencing. In all three species, we found an event of subtle alternative splicing at a NAGNAG tandem acceptor that results in isoforms including or lacking glutamine 1030 (Na(V)1.8+Q and Na(V)1.8-Q, respectively) within the cytoplasmic loop between domains II and III. The relative amount of Na(V)1.8-Q mRNA in adult DRG was measured with 14.1 +/- 0.1% in humans and 11.2 +/- 0.2% in rats. This is in contrast to an abundance of 64.3 +/- 0.3% in mouse DRG. Thus, the NAGNAG tandem acceptor in SCN10A is conserved among rodents and humans but its alternative usage apparently occurs with species-specific abundance. Analysis of human Na(V)1.8+Q and -Q isoforms in whole-cell patch-clamp experiments after heterologous expression in the neuroblastoma cell line Neuro-2A revealed no obvious impact of the splicing event on channel function.