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Stephen G Waxman - One of the best experts on this subject based on the ideXlab platform.
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status of peripheral sodium channel blockers for non addictive pain treatment
Nature Reviews Neurology, 2020Co-Authors: Matthew Alsaloum, Grant P. Higerd, Philip R. Effraim, Stephen G WaxmanAbstract:The effective and safe treatment of pain is an unmet health-care need. Current medications used for pain management are often only partially effective, carry dose-limiting adverse effects and are potentially addictive, highlighting the need for improved therapeutic agents. Most common pain conditions originate in the periphery, where dorsal root ganglion and trigeminal ganglion neurons feed pain information into the CNS. Voltage-gated sodium (NaV) channels drive neuronal excitability and three subtypes - NaV1.7, Nav1.8 and NaV1.9 - are preferentially expressed in the peripheral nervous system, suggesting that their inhibition might treat pain while avoiding central and cardiac adverse effects. Genetic and functional studies of human pain disorders have identified NaV1.7, Nav1.8 and NaV1.9 as mediators of pain and validated them as targets for pain treatment. Consequently, multiple NaV1.7-specific and Nav1.8-specific blockers have undergone clinical trials, with others in preclinical development, and the targeting of NaV1.9, although hampered by technical constraints, might also be moving ahead. In this Review, we summarize the clinical and preclinical literature describing compounds that target peripheral NaV channels and discuss the challenges and future prospects for the field. Although the potential of peripheral NaV channel inhibition for the treatment of pain has yet to be realized, this remains a promising strategy to achieve non-addictive analgesia for multiple pain conditions.
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the novel activity of carbamazepine as an activation modulator extends from nav1 7 mutations to the Nav1.8 s242t mutant channel from a patient with painful diabetic neuropathy
Molecular Pharmacology, 2018Co-Authors: Chongyang Han, Stephen G Waxman, Mark Estacion, Andreas C Themistocleous, Fadia B Dibhajj, Iulia Blesneac, Lawrence J Macala, C Fratter, David L H Bennett, Sulayman D DibhajjAbstract:Neuropathic pain in patients carrying sodium channel gain-of-function mutations is generally refractory to pharmacotherapy. However, we have shown that pretreatment of cells with clinically achievable concentration of carbamazepine (CBZ; 30 μM) depolarizes the voltage dependence of activation in some NaV1.7 mutations such as S241T, a novel CBZ mode of action of this drug. CBZ reduces the excitability of dorsal root ganglion (DRG) neurons expressing NaV1.7-S241T mutant channels, and individuals carrying the S241T mutation respond to treatment with CBZ. Whether the novel activation-modulating activity of CBZ is specific to NaV1.7, and whether this pharmacogenomic approach can be extended to other sodium channel subtypes, are not known. We report here the novel Nav1.8-S242T mutation, which corresponds to the NaV1.7-S241T mutation, in a patient with neuropathic pain and diabetic peripheral neuropathy. Voltage-clamp recordings demonstrated hyperpolarized and accelerated activation of Nav1.8-S242T. Current-clamp recordings showed that Nav1.8-S242T channels render DRG neurons hyperexcitable. Structural modeling shows that despite a substantial difference in the primary amino acid sequence of NaV1.7 and Nav1.8, the S242 (Nav1.8) and S241 (NaV1.7) residues have similar position and orientation in the domain I S4-S5 linker of the channel. Pretreatment with a clinically achievable concentration of CBZ corrected the voltage dependence of activation of Nav1.8-S242T channels and reduced DRG neuron excitability as predicted from our pharmacogenomic model. These findings extend the novel activation modulation mode of action of CBZ to a second sodium channel subtype, Nav1.8.
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Nav1.9 expression in magnocellular neurosecretory cells of supraoptic nucleus
Experimental Neurology, 2014Co-Authors: Joel A Black, Dymtro Vasylyev, Sulayman D Dib-hajj, Stephen G WaxmanAbstract:Abstract Osmoregulation in mammals is tightly controlled by the release of vasopressin and oxytocin from magnocellular neurosecretory cells (MSC) of the supraoptic nucleus (SON). The release of vasopressin and oxytocin in the neurohypophysis by axons of MSC is regulated by bursting activity of these neurons, which is influenced by multiple sources, including intrinsic membrane properties, paracrine contributions of glial cells, and extrinsic synaptic inputs. Previous work has shown that bursting activity of MSC is tetrodotoxin (TTX)-sensitive, and that TTX-S sodium channels Nav1.2, Nav1.6 and Nav1.7 are expressed by MSC and upregulated in response to osmotic challenge in rats. The TTX-resistant sodium channels, Nav1.8 and Nav1.9, are preferentially expressed, at relatively high levels, in peripheral neurons, where their properties are linked to repetitive firing and subthreshold electrogenesis, respectively, and are often referred to as “peripheral” sodium channels. Both sodium channels have been implicated in pain pathways, and are under study as potential therapeutic targets for pain medications which might be expected to have minimal CNS side effects. We show here, however, that Nav1.9 is expressed by vasopressin- and oxytocin-producing MSC of the rat supraoptic nucleus (SON). We also show that cultured MSC exhibit sodium currents that have characteristics of Nav1.9 channels. In contrast, Nav1.8 is not detectable in the SON. These results suggest that Nav1.9 may contribute to the firing pattern of MSC of the SON, and that careful assessment of hypothalamic function be performed as NaV1.9 blocking agents are studied as potential pain therapies.
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nav1 7 stress induced changes in immunoreactivity within magnocellular neurosecretory neurons of the supraoptic nucleus
Molecular Pain, 2013Co-Authors: Joel A Black, Janneke G J Hoeijmakers, Catharina G Faber, Ingemar S J Merkies, Stephen G WaxmanAbstract:Background: NaV1.7 is preferentially expressed, at relatively high levels, in peripheral neurons, and is often referred to as a “peripheral” sodium channel, and NaV1.7-specific blockers are under study as potential pain therapeutics which might be expected to have minimal CNS side effects. However, occasional reports of patients with NaV1.7 gain-of-function mutations and apparent hypothalamic dysfunction have appeared. The two sodium channels previously studied within the rat hypothalamic supraoptic nucleus, NaV1.2 and NaV1.6, display up-regulated expression in response to osmotic stress. Results: Here we show that NaV1.7 is present within vasopressin-producing neurons and oxytocin-producing neurons within the rat hypothalamus, and demonstrate that the level of Nav1.7 immunoreactivity is increased in these cells in response to osmotic stress. Conclusions: NaV1.7 is present within neurosecretory neurons of rat supraoptic nucleus, where the level of immunoreactivity is dynamic, increasing in response to osmotic stress. Whether NaV1.7 levels are up-regulated within the human hypothalamus in response to environmental factors or stress, and whether NaV1.7 plays a functional role in human hypothalamus, is not yet known. Until these questions are resolved, the present findings suggest the need for careful assessment of hypothalamic function in patients with NaV1.7 mutations, especially when subjected to stress, and for monitoring of hypothalamic function as NaV1.7 blocking agents are studied.
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a sodium channel mutation linked to epilepsy increases ramp and persistent current of nav1 3 and induces hyperexcitability in hippocampal neurons
Experimental Neurology, 2010Co-Authors: Mark Estacion, Andreas Gasser, Sulayman D Dibhajj, Stephen G WaxmanAbstract:article i nfo Voltage-gated sodium channelopathies underlie many excitability disorders. Genes SCN1A, SCN2A and SCN9A, which encode pore-forming α-subunits NaV1.1, NaV1.2 and NaV1.7, are clustered on human chromosome 2, and mutations in these genes have been shown to underlie epilepsy, migraine, and somatic pain disorders. SCN3A, the gene which encodes NaV1.3, is part of this cluster, but until recently was not associated with any mutation. A charge-neutralizing mutation, K345Q, in the NaV1.3 DI/S5-6 linker has recently been identified in a patient with cryptogenic partial epilepsy. Pathogenicity of the NaV1.3/K354Q mutation has been inferred from the conservation of this residue in all sodium channels and its absence from control alleles, but functional analysis has been limited to the corresponding substitution in the cardiac muscle sodium channel NaV1.5. Since identical mutations may produce different effects within different sodium channel isoforms, we assessed the K354Q mutation within its native NaV1.3 channel and studied the effect of the mutant NaV1.3/K354Q channels on hippocampal neuron excitability. We show here that the K354Q mutation enhances the persistent and ramp currents of NaV1.3, reduces current threshold and produces spontaneous firing and paroxysmal depolarizing shift-like complexes in hippocampal neurons. Our data provide a pathophysiological basis for the pathogenicity of the first epilepsy-linked mutation within NaV1.3 channels and hippocampal neurons.
Jan Tytgat - One of the best experts on this subject based on the ideXlab platform.
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Differential effects of the recombinant toxin PnTx4(5-5) from the spider Phoneutria nigriventer on mammalian and insect sodium channels
Biochimie, 2015Co-Authors: Ana Luiza B. Paiva, Alessandra Matavel, Marta N. Cordeiro, Marcelo R.v. Diniz, Steve Peigneur, Jan Tytgat, Maria Elena De LimaAbstract:Abstract The toxin PnTx4(5-5) from the spider Phoneutria nigriventer is extremely toxic/lethal to insects but has no macroscopic behavioral effects observed in mice after intracerebral injection. Nevertheless, it was demonstrated that it inhibits the N-methyl- d -aspartate (NMDA) - subtype of glutamate receptors of cultured rat hippocampal neurons. PnTx4(5-5) has 63% identity to PnTx4(6-1), another insecticidal toxin from P. nigriventer, which can slow down the sodium current inactivation in insect central nervous system, but has no effect on Nav1.2 and Nav1.4 rat sodium channels. Here, we have cloned and heterologous expressed the toxin PnTx4(5-5) in Escherichia coli. The recombinant toxin rPnTx4(5-5) was tested on the sodium channel NavBg from the cockroach Blatella germanica and on mammalian sodium channels Nav1.2-1.6, all expressed in Xenopus leavis oocytes. We showed that the toxin has different affinity and mode of action on insect and mammalian sodium channels. The most remarkable effect was on NavBg, where rPnTx4(5-5) strongly slowed down channel inactivation (EC50 = 212.5 nM), and at 1 μM caused an increase on current peak amplitude of 105.2 ± 3.1%. Interestingly, the toxin also inhibited sodium current on all the mammalian channels tested, with the higher current inhibition on Nav1.3 (38.43 ± 8.04%, IC50 = 1.5 μM). Analysis of activation curves on Nav1.3 and Nav1.5 showed that the toxin shifts channel activation to more depolarized potentials, which can explain the sodium current inhibition. Furthermore, the toxin also slightly slowed down sodium inactivation on Nav1.3 and Nav1.6 channels. As far as we know, this is the first araneomorph toxin described which can shift the sodium channel activation to more depolarized potentials and also slows down channel inactivation.
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Electrophysiological characterization of the first Tityus serrulatus alpha-like toxin, Ts5: Evidence of a pro-inflammatory toxin on macrophages
Biochimie, 2015Co-Authors: Manuela Berto Pucca, Felipe A. Cerni, Karina Furlan Zoccal, Karla De Castro Figueiredo Bordon, Camila Takeno Cologna, Steve Peigneur, Lúcia Helena Faccioli, Jan Tytgat, Eliane Candiani ArantesAbstract:Abstract Tityus serrulatus (Ts) venom is composed of mainly neurotoxins specific for voltage-gated K+ and Na+ channels, which are expressed in many cells such as macrophages. Macrophages are the first line of defense invasion and they participate in the inflammatory response of Ts envenoming. However, little is known about the effect of Ts toxins on macrophage activation. This study investigated the effect of Ts5 toxin on different sodium channels as well as its role on the macrophage immunomodulation. The electrophysiological assays showed that Ts5 inhibits the rapid inactivation of the mammalian sodium channels Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6 and Nav1.7. Interestingly, Ts5 also inhibits the inactivation of the insect Drosophila melanogaster sodium channel (DmNav1), and it is therefore classified as the first Ts α-like toxin. The immunological experiments on macrophages reveal that Ts5 is a pro-inflammatory toxin inducing the cytokine production of tumor necrosis factor (TNF)-α and interleukin (IL)-6. On the basis of recent literature, our study also stresses a possible mechanism responsible for venom-associated molecular patterns (VAMPs) internalization and macrophage activation and moreover we suggest two main pathways of VAMPs signaling: direct and indirect. This work provides useful insights for a better understanding of the involvement of VAMPs in macrophage modulation.
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investigation of the relationship between the structure and function of ts2 a neurotoxin from tityus serrulatus venom
FEBS Journal, 2012Co-Authors: Camila Takeno Cologna, Steve Peigneur, Jan Tytgat, Joane K Rustiguel, Cristina M Nonato, Eliane Candiani ArantesAbstract:Scorpion toxins targeting voltage-gated sodium (NaV) channels are peptides that comprise 60–76 amino acid residues cross-linked by four disulfide bridges. These toxins can be divided in two groups (α and β toxins), according to their binding properties and mode of action. The scorpion α-toxin Ts2, previously described as a β-toxin, was purified from the venom of Tityus serrulatus, the most dangerous Brazilian scorpion. In this study, seven mammalian NaV channel isoforms (rNaV1.2, rNaV1.3, rNaV1.4, hNaV1.5, mNaV1.6, rNaV1.7 and rNav1.8) and one insect NaV channel isoform (DmNaV1) were used to investigate the subtype specificity and selectivity of Ts2. The electrophysiology assays showed that Ts2 inhibits rapid inactivation of NaV1.2, NaV1.3, NaV1.5, NaV1.6 and NaV1.7, but does not affect NaV1.4, Nav1.8 or DmNaV1. Interestingly, Ts2 significantly shifts the voltage dependence of activation of NaV1.3 channels. The 3D structure of this toxin was modeled based on the high sequence identity (72%) shared with Ts1, another T. serrulatus toxin. The overall fold of the Ts2 model consists of three β-strands and one α-helix, and is arranged in a triangular shape forming a cysteine-stabilized α-helix/β-sheet (CSαβ) motif. Database Model data are available in the PMDB under accession number PM0077533.
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potent modulation of the voltage gated sodium channel nav1 7 by od1 a toxin from the scorpion odonthobuthus doriae
Molecular Pharmacology, 2006Co-Authors: Chantal Maertens, Eva Cuypers, Mehriar Amininasab, Amir Jalali, Hossein Vatanpour, Jan TytgatAbstract:Voltage-gated sodium channels are essential for the propagation of action potentials in nociceptive neurons. Nav1.7 is found in peripheral sensory and sympathetic neurons and involved in short-term and inflammatory pain. Nav1.8 and Nav1.3 are major players in nociception and neuropathic pain, respectively. In our effort to identify isoform-specific and high-affinity ligands for these channels, we investigated the effects of OD1, a scorpion toxin isolated from the venom of the scorpion Odonthobuthus doriae. Nav1.3, Nav1.7, and Nav1.8 channels were coexpressed with beta1-subunits in Xenopus laevis oocytes. Na+ currents were recorded with the two-electrode voltage-clamp technique. OD1 modulates Nav1.7 at low nanomolar concentrations: 1) fast inactivation is dramatically impaired, with an EC50 value of 4.5 nM; 2) OD1 substantially increases the peak current at all voltages; and 3) OD1 induces a substantial persistent current. Nav1.8 was not affected by concentrations up to 2 microM, whereas Nav1.3 was sensitive only to concentrations higher than 100 nM. OD1 impairs the inactivation process of Nav1.3 with an EC50 value of 1127 nM. Finally, the effects of OD1 were compared with a classic alpha-toxin, AahII from Androctonus australis Hector and a classic alpha-like toxin, BmK M1 from Buthus martensii Karsch. At a concentration of 50 nM, both toxins affected Nav1.7. Nav1.3 was sensitive to AahII but not to BmK M1, whereas Nav1.8 was affected by neither toxin. In conclusion, the present study shows that the scorpion toxin OD1 is a potent modulator of Nav1.7, with a unique selectivity pattern.
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four novel tarantula toxins as selective modulators of voltage gated sodium channel subtypes
Molecular Pharmacology, 2006Co-Authors: Frank Bosmans, Lachlan D Rash, Shunyi Zhu, Sylvie Diochot, Michel Lazdunski, Pierre Escoubas, Jan TytgatAbstract:Four novel peptide toxins that act on voltage-gated sodium channels have been isolated from tarantula venoms: ceratotoxins 1, 2, and 3 (CcoTx1, CcoTx2, and CcoTx3) from Ceratogyrus cornuatus and phrixotoxin 3 (PaurTx3) from Phrixotrichus auratus. The pharmacological profiles of these new toxins were characterized by electrophysiological measurements on six cloned voltage-gated sodium channel subtypes expressed in Xenopus laevis oocytes (Nav1.1/beta1, Nav1.2/beta1, Nav1.3/beta1, Nav1.4/beta1, Nav1.5/beta1, and Nav1.8/beta1). These novel toxins modulate voltage-gated sodium channels with properties similar to those of typical gating-modifier toxins, both by causing a depolarizing shift in gating kinetics and by blocking the inward component of the sodium current. PaurTx3 is one of the most potent peptide modulators of voltage-gated sodium channels described thus far from spider venom, modulating Nav1.2 with an IC50 value of 0.6 ± 0.1 nM. CcoTx1 and CcoTx2, differing by only one amino acid, are potent modulators of different voltage-gated sodium channel subtypes from the central nervous system, except for Nav1.3, which is only affected by CcoTx2. The potency of CcoTx3 is lower, although this toxin seems to be more selective for the tetrodotoxin-resistant channel subtype Nav1.5/beta1 (IC50 = 447 ± 32 nM). In addition to these results, molecular modeling indicates that subtle differences in toxin surfaces may relate to their different pharmacological profiles. Furthermore, an evolutionary trace analysis of these toxins and other structurally related three-disulfide spider toxins provides clues for the exploration of toxin-channel interaction and future structure-function research.
Koichi Noguchi - One of the best experts on this subject based on the ideXlab platform.
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de novo expression of nav1 7 in injured putative proprioceptive afferents multiple tetrodotoxin sensitive sodium channels are retained in the rat dorsal root after spinal nerve ligation
Neuroscience, 2015Co-Authors: Tetsuo Fukuoka, Kan Miyoshi, Koichi NoguchiAbstract:Tetrodotoxin-sensitive (TTX-s) spontaneous activity is recorded from the dorsal roots after peripheral nerve injury. Primary sensory neurons in the dorsal root ganglion (DRG) express multiple TTX-s voltage-gated sodium channel α-subunits (Navs). Since Nav1.3 increases, whereas all other Navs decrease, in the DRG neurons after peripheral nerve lesion, Nav1.3 is proposed to be critical for the generation of these spontaneous discharges and the contributions of other Navs have been ignored. Here, we re-evaluate the changes in expression of three other TTX-s Navs, Nav1.1, Nav1.6 and Nav1.7, in the injured 5th lumbar (L5) primary afferent components following L5 spinal nerve ligation (SNL) using in situ hybridization histochemistry and immunohistochemistry. While the overall signal intensities for these Nav mRNAs decreased, many injured DRG neurons still expressed these transcripts at clearly detectable levels. All these Nav proteins accumulated at the proximal stump of the ligated L5 spinal nerve. The immunostaining patterns of Nav1.6 and Nav1.7 associated with the nodes of Ranvier were maintained in the ipsilateral L5 dorsal root. Interestingly, putative proprioceptive neurons characterized by α3 Na+/K+ ATPase-immunostaining specifically lacked Nav1.7 mRNA in naive DRG but displayed de novo expression of this transcript following SNL. Nav1.7-immunoreactive fibers were significantly increased in the ipsilateral gracile nucleus where central axonal branches of the injured A-fiber afferents terminated. These data indicate that multiple TTX-s channel subunits could contribute to the generation and propagation of the spontaneous discharges in the injured primary afferents. Specifically, Nav1.7 may cause some functional changes in sensory processing in the gracile nucleus after peripheral nerve injury.
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re evaluation of the phenotypic changes in l4 dorsal root ganglion neurons after l5 spinal nerve ligation
Pain, 2012Co-Authors: Tetsuo Fukuoka, Kimiko Kobayashi, Hiroki Yamanaka, Yi Dai, Kan Miyoshi, Masamichi Okubo, Koichi NoguchiAbstract:The L5 spinal nerve ligation (SNL) is a widely used animal neuropathic pain model. There are conflicting reports regarding the extent of injury to the L4 dorsal root ganglion (DRG) neurons in this model. If a significant number of these neurons were injured, the previously reported phenotypic and electrophysiological changes at this level are in need of re-evaluation by separating the injured neurons and the frankly spared ones. So, we immunostained activating transcription factor 3 (ATF3) and examined the change in expression of transcripts for neuropeptide Y (NPY), brain-derived neurotrophic factor (BDNF) and several voltage-gated sodium channel α-subunits (Nav1.1, Nav1.3, Nav1.6, Nav1.7, Nav1.8, and Nav1.9) in the L4 DRG by comparing signal intensities of individual neurons using in situ hybridization histochemistry. ATF3-immunoreactivity was similarly observed in 4-6% of neuronal nuclei of the SNL and sham-operated ipsilateral L4 DRGs. Comparison between ATF3+ and ATF3- neurons in the SNL L4 DRG revealed that (1) whereas NPY induction occurred in ATF3+ cells, BDNF increased mainly in ATF3- neurons; (2) although ATF3+ neurons had higher Nav1.3 signals than ATF3- neurons, these signals were much lower than those of the L5 DRG neurons; and (3) ATF3+/N52- neurons selectively lost Nav1.8 and Nav1.9 mRNAs. Comparison of the total neuronal populations among naive, SNL, and sham-operated rats revealed no significant differences for all examined Nav mRNAs. Because neuropathic pain behaviors were developed by rats with SNL but not the sham-operation, the small number of injured L4 neurons likely do not contribute to the pathomechanisms of neuropathic pain.
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comparative study of the distribution of the α subunits of voltage gated sodium channels in normal and axotomized rat dorsal root ganglion neurons
The Journal of Comparative Neurology, 2008Co-Authors: Tetsuo Fukuoka, Kimiko Kobayashi, Hiroki Yamanaka, Koichi Obata, Yi Dai, Koichi NoguchiAbstract:We compared the distribution of the alpha-subunit mRNAs of voltage-gated sodium channels Nav1.1-1.3 and Nav1.6-1.9 and a related channel, Nax, in histochemically identified neuronal subpopulations of the rat dorsal root ganglia (DRG). In the naive DRG, the expression of Nav1.1 and Nav1.6 was restricted to A-fiber neurons, and they were preferentially expressed by TrkC neurons, suggesting that proprioceptive neurons possess these channels. Nav1.7, -1.8, and -1.9 mRNAs were more abundant in C-fiber neurons compared with A-fiber ones. Nax was evenly expressed in both populations. Although Nav1.8 and -1.9 were preferentially expressed by TrkA neurons, other alpha-subunits were expressed independently of TrkA expression. Actually, all IB4(+) neurons expressed both Nav1.8 and -1.9, and relatively limited subpopulations of IB4(+) neurons (3% and 12%, respectively) expressed Nav1.1 and/or Nav1.6. These findings provide useful information in interpreting the electrophysiological characteristics of some neuronal subpopulations of naive DRG. After L5 spinal nerve ligation, Nav1.3 mRNA was up-regulated mainly in A-fiber neurons in the ipsilateral L5 DRG. Although previous studies demonstrated that nerve growth factor (NGF) and glial cell-derived neurotrophic factor (GDNF) reversed this up-regulation, the Nav1.3 induction was independent of either TrkA or GFRalpha1 expression, suggesting that the induction of Nav1.3 may be one of the common responses of axotomized DRG neurons without a direct relationship to NGF/GDNF supply.
William A. Catterall - One of the best experts on this subject based on the ideXlab platform.
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localization of sodium channel subtypes in mouse ventricular myocytes using quantitative immunocytochemistry
Journal of Molecular and Cellular Cardiology, 2013Co-Authors: Ruth E Westenbroek, Sebastian Bischoff, Sebastian K G Maier, Ying Fu, William A. Catterall, Todd ScheuerAbstract:Voltage-gated sodium channels are responsible for the rising phase of the action potential in cardiac muscle. Previously, both TTX-sensitive neuronal sodium channels (NaV1.1, NaV1.2, NaV1.3, NaV1.4 and NaV1.6) and the TTX-resistant cardiac sodium channel (NaV1.5) have been detected in cardiac myocytes, but relative levels of protein expression of the isoforms were not determined. Using a quantitative approach, we analyzed z-series of confocal microscopy images from individual mouse myocytes stained with either anti-NaV1.1, anti-NaV1.2, anti-NaV1.3, anti-NaV1.4, anti-NaV1.5, or anti-NaV1.6 antibodies and calculated the relative intensity of staining for these sodium channel isoforms. Our results indicate that the TTX-sensitive channels represented approximately 23% of the total channels, whereas the TTX-resistant NaV1.5 channel represented 77% of the total channel staining in mouse ventricular myocytes. These ratios are consistent with previous electrophysiological studies in mouse ventricular myocytes. NaV1.5 was located at the cell surface, with high density at the intercalated disc, but was absent from the transverse (t)-tubular system, suggesting that these channels support surface conduction and inter-myocyte transmission. Low-level cell surface staining of NaV1.4 and NaV1.6 channels suggest a minor role in surface excitation and conduction. Conversely, NaV1.1 and NaV1.3 channels are localized to the t-tubules and are likely to support t-tubular transmission of the action potential to the myocyte interior. This quantitative immunocytochemical approach for assessing sodium channel density and localization provides a more precise view of the relative importance and possible roles of these individual sodium channel protein isoforms in mouse ventricular myocytes and may be applicable to other species and cardiac tissue types.
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distribution and function of sodium channel subtypes in human atrial myocardium
Journal of Molecular and Cellular Cardiology, 2013Co-Authors: Susann G Kaufmann, Ruth E Westenbroek, Volkmar Lange, Andre Renner, Andreas Bonz, Alexander H Maass, Erhard Wischmeyer, Jenny Muck, Georg Ertl, William A. CatterallAbstract:Voltage-gated sodium channels composed of a pore-forming α subunit and auxiliary β subunits are responsible for the upstroke of the action potential in cardiac muscle. However, their localization and expression patterns in human myocardium have not yet been clearly defined. We used immunohistochemical methods to define the level of expression and the subcellular localization of sodium channel α and β subunits in human atrial myocytes. Nav1.2 channels are located in highest density at intercalated disks where β1 and β3 subunits are also expressed. Nav1.4 and the predominant Nav1.5 channels are located in a striated pattern on the cell surface at the z-lines together with β2 subunits. Nav1.1, Nav1.3, and Nav1.6 channels are located in scattered puncta on the cell surface in a pattern similar to β3 and β4 subunits. Nav1.5 comprised approximately 88% of the total sodium channel staining, as assessed by quantitative immunohistochemistry. Functional studies using whole cell patch-clamp recording and measurements of contractility in human atrial cells and tissue showed that TTX-sensitive (non-Nav1.5) α subunit isoforms account for up to 27% of total sodium current in human atrium and are required for maximal contractility. Overall, our results show that multiple sodium channel α and β subunits are differentially localized in subcellular compartments in human atrial myocytes, suggesting that they play distinct roles in initiation and conduction of the action potential and in excitation–contraction coupling. TTX-sensitive sodium channel isoforms, even though expressed at low levels relative to TTX-sensitive Nav1.5, contribute substantially to total cardiac sodium current and are required for normal contractility. This article is part of a Special Issue entitled “Na+ Regulation in Cardiac Myocytes”.
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abstract 171 localization and function of sodium channel subtypes in human heart
Circulation, 2007Co-Authors: Ruth E Westenbroek, Todd Scheuer, William A. Catterall, Susann G Kaufmann, Volkmar Lange, Andre Renner, Andreas Bonz, Sebastian K G MaierAbstract:Introduction: Voltage-gated sodium channels are responsible for the rising phase of the action potential in cardiac muscle. They are composed of pore-forming α- and auxiliary β-subunits. Different α-subunit isoforms have distinct pharmacological properties and subcellular localization in the heart. The puffer fish toxin, tetrodotoxin (TTX), is a specific sodium channel blocker with different affinity for α-subunit isoforms. Nav1.5 is blocked by micromolar concentrations whereas CNS and skeletal muscle α-subunit isoforms are blocked by nanomolar concentrations of TTX. Methods: We studied acutely isolated human atrial cells with the patch-clamp technique and studied expression and localization by immunoblotting and immunocytochemistry. Results: We show that both TTX-resistant NaV1.5 channels and TTX-sensitive isoforms are expressed in human atrial cells, as assessed by immunoblotting with specific antibodies. At intercalated disks, we find NaV1.2 in association with β1 and β3 subunits by immunocytochemistry. In the cell surface at z-lines, we find NaV1.2, NaV1.4, and NaV1.5 channels in association with β2 subunits. In addition, NaV1.1, NaV1.3, and NaV1.6 channels are localized in scattered punctate clusters on the cell surface in association with β3 and β4 subunits. NaV1.5 immunostaining comprises approximately 88% of total. Our patch-clamp studies suggest that TTX-sensitive isoforms conduct approximately 15% of total sodium current whereas TTX-resistant isoforms, primarily NaV1.5, are responsible for approximately 85%. Specific blockade of TTX-sensitive isoforms with low concentrations of TTX leads to a reduction of myocardial contractility. Conclusions: We show that both TTX-sensitive and cardiac, TTX-resistant sodium channels are functionally expressed and are differentially localized in subcellular compartments in atrial myocytes in human heart. TTX-sensitive sodium channels are required for normal contractility.
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molecular determinants for modulation of persistent sodium current by g protein βγ subunits
The Journal of Neuroscience, 2005Co-Authors: Massimo Mantegazza, William A. Catterall, Frank H Yu, Andrew J Powell, Jeffrey J Clare, Todd ScheuerAbstract:Voltage-gated sodium channels are responsible for the upstroke of the action potential in most excitable cells, and their fast inactivation is essential for controlling electrical signaling. In addition, a noninactivating, persistent component of sodium current, I NaP, has been implicated in integrative functions of neurons including threshold for firing, neuronal bursting, and signal integration. G-protein βγ subunits increase I NaP, but the sodium channel subtypes that conduct I NaP and the target site(s) on the sodium channel molecule required for modulation by Gβγ are poorly defined. Here, we show that I NaP conducted by Nav1.1 and Nav1.2 channels (Nav1.1 > Nav1.2) is modulated by Gβγ; Nav1.4 and Nav1.5 channels produce smaller I NaP that is not regulated by Gβγ. These qualitative differences in modulation by Gβγ are determined by the transmembrane body of the sodium channels rather than their cytoplasmic C-terminal domains, which have been implicated previously in modulation by Gβγ. However, the C-terminal domains determine the quantitative extent of modulation of Nav1.2 channels by Gβγ. Studies of chimeric and truncated Nav1.2 channels identify molecular determinants that affect modulation of I NaP located between amino acid residue 1890 and the C terminus at residue 2005. The last 28 amino acid residues of the C terminus are sufficient to support modulation by Gβγ when attached to the proximal C-terminal domain. Our results further define the sodium channel subtypes that generate I NaP and identify crucial molecular determinants in the C-terminal domain required for modulation by Gβγ when attached to the transmembrane body of a responsive sodium channel.
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an unexpected role for brain type sodium channels in coupling of cell surface depolarization to contraction in the heart
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Sebastian K G Maier, Ruth E Westenbroek, Kenneth A Schenkman, Eric O Feigl, Todd Scheuer, William A. CatterallAbstract:Voltage-gated sodium channels composed of pore-forming α and auxiliary β subunits are responsible for the rising phase of the action potential in cardiac muscle, but the functional roles of distinct sodium channel subtypes have not been clearly defined. Immunocytochemical studies show that the principal cardiac pore-forming α subunit isoform Nav1.5 is preferentially localized in intercalated disks, whereas the brain α subunit isoforms Nav1.1, Nav1.3, and Nav1.6 are localized in the transverse tubules. Sodium currents due to the highly tetrodotoxin (TTX)-sensitive brain isoforms in the transverse tubules are small and are detectable only after activation with β scorpion toxin. Nevertheless, they play an important role in coupling depolarization of the cell surface membrane to contraction, because low TTX concentrations reduce left ventricular function. Our results suggest that the principal cardiac isoform in the intercalated disks is primarily responsible for action potential conduction between cells and reveal an unexpected role for brain sodium channel isoforms in the transverse tubules in coupling electrical excitation to contraction in cardiac muscle.
Tetsuo Fukuoka - One of the best experts on this subject based on the ideXlab platform.
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de novo expression of nav1 7 in injured putative proprioceptive afferents multiple tetrodotoxin sensitive sodium channels are retained in the rat dorsal root after spinal nerve ligation
Neuroscience, 2015Co-Authors: Tetsuo Fukuoka, Kan Miyoshi, Koichi NoguchiAbstract:Tetrodotoxin-sensitive (TTX-s) spontaneous activity is recorded from the dorsal roots after peripheral nerve injury. Primary sensory neurons in the dorsal root ganglion (DRG) express multiple TTX-s voltage-gated sodium channel α-subunits (Navs). Since Nav1.3 increases, whereas all other Navs decrease, in the DRG neurons after peripheral nerve lesion, Nav1.3 is proposed to be critical for the generation of these spontaneous discharges and the contributions of other Navs have been ignored. Here, we re-evaluate the changes in expression of three other TTX-s Navs, Nav1.1, Nav1.6 and Nav1.7, in the injured 5th lumbar (L5) primary afferent components following L5 spinal nerve ligation (SNL) using in situ hybridization histochemistry and immunohistochemistry. While the overall signal intensities for these Nav mRNAs decreased, many injured DRG neurons still expressed these transcripts at clearly detectable levels. All these Nav proteins accumulated at the proximal stump of the ligated L5 spinal nerve. The immunostaining patterns of Nav1.6 and Nav1.7 associated with the nodes of Ranvier were maintained in the ipsilateral L5 dorsal root. Interestingly, putative proprioceptive neurons characterized by α3 Na+/K+ ATPase-immunostaining specifically lacked Nav1.7 mRNA in naive DRG but displayed de novo expression of this transcript following SNL. Nav1.7-immunoreactive fibers were significantly increased in the ipsilateral gracile nucleus where central axonal branches of the injured A-fiber afferents terminated. These data indicate that multiple TTX-s channel subunits could contribute to the generation and propagation of the spontaneous discharges in the injured primary afferents. Specifically, Nav1.7 may cause some functional changes in sensory processing in the gracile nucleus after peripheral nerve injury.
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re evaluation of the phenotypic changes in l4 dorsal root ganglion neurons after l5 spinal nerve ligation
Pain, 2012Co-Authors: Tetsuo Fukuoka, Kimiko Kobayashi, Hiroki Yamanaka, Yi Dai, Kan Miyoshi, Masamichi Okubo, Koichi NoguchiAbstract:The L5 spinal nerve ligation (SNL) is a widely used animal neuropathic pain model. There are conflicting reports regarding the extent of injury to the L4 dorsal root ganglion (DRG) neurons in this model. If a significant number of these neurons were injured, the previously reported phenotypic and electrophysiological changes at this level are in need of re-evaluation by separating the injured neurons and the frankly spared ones. So, we immunostained activating transcription factor 3 (ATF3) and examined the change in expression of transcripts for neuropeptide Y (NPY), brain-derived neurotrophic factor (BDNF) and several voltage-gated sodium channel α-subunits (Nav1.1, Nav1.3, Nav1.6, Nav1.7, Nav1.8, and Nav1.9) in the L4 DRG by comparing signal intensities of individual neurons using in situ hybridization histochemistry. ATF3-immunoreactivity was similarly observed in 4-6% of neuronal nuclei of the SNL and sham-operated ipsilateral L4 DRGs. Comparison between ATF3+ and ATF3- neurons in the SNL L4 DRG revealed that (1) whereas NPY induction occurred in ATF3+ cells, BDNF increased mainly in ATF3- neurons; (2) although ATF3+ neurons had higher Nav1.3 signals than ATF3- neurons, these signals were much lower than those of the L5 DRG neurons; and (3) ATF3+/N52- neurons selectively lost Nav1.8 and Nav1.9 mRNAs. Comparison of the total neuronal populations among naive, SNL, and sham-operated rats revealed no significant differences for all examined Nav mRNAs. Because neuropathic pain behaviors were developed by rats with SNL but not the sham-operation, the small number of injured L4 neurons likely do not contribute to the pathomechanisms of neuropathic pain.
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comparative study of the distribution of the α subunits of voltage gated sodium channels in normal and axotomized rat dorsal root ganglion neurons
The Journal of Comparative Neurology, 2008Co-Authors: Tetsuo Fukuoka, Kimiko Kobayashi, Hiroki Yamanaka, Koichi Obata, Yi Dai, Koichi NoguchiAbstract:We compared the distribution of the alpha-subunit mRNAs of voltage-gated sodium channels Nav1.1-1.3 and Nav1.6-1.9 and a related channel, Nax, in histochemically identified neuronal subpopulations of the rat dorsal root ganglia (DRG). In the naive DRG, the expression of Nav1.1 and Nav1.6 was restricted to A-fiber neurons, and they were preferentially expressed by TrkC neurons, suggesting that proprioceptive neurons possess these channels. Nav1.7, -1.8, and -1.9 mRNAs were more abundant in C-fiber neurons compared with A-fiber ones. Nax was evenly expressed in both populations. Although Nav1.8 and -1.9 were preferentially expressed by TrkA neurons, other alpha-subunits were expressed independently of TrkA expression. Actually, all IB4(+) neurons expressed both Nav1.8 and -1.9, and relatively limited subpopulations of IB4(+) neurons (3% and 12%, respectively) expressed Nav1.1 and/or Nav1.6. These findings provide useful information in interpreting the electrophysiological characteristics of some neuronal subpopulations of naive DRG. After L5 spinal nerve ligation, Nav1.3 mRNA was up-regulated mainly in A-fiber neurons in the ipsilateral L5 DRG. Although previous studies demonstrated that nerve growth factor (NGF) and glial cell-derived neurotrophic factor (GDNF) reversed this up-regulation, the Nav1.3 induction was independent of either TrkA or GFRalpha1 expression, suggesting that the induction of Nav1.3 may be one of the common responses of axotomized DRG neurons without a direct relationship to NGF/GDNF supply.
