TTX-sensitive Sodium Channels Research Articles

Prostaglandin E2 sensitizes the cough reflex centrally via EP3 receptor-dependent activation of NaV 1.8 channels

BackgroundCough hypersensitivity is a major characteristic feature associated with several types of cough, including chronic cough, but its underlying mechanisms remain to be fully understood. Inflammatory mediators, such as prostaglandin E2 (PGE2), have been implicated in both peripheral induction and sensitization of the cough reflex. In this study, using a conscious guinea pig model of cough, we investigated whether PGE2 can sensitize the cough reflex via central actions and, if so, via which mechanisms.MethodsAll drugs were administered by intracerebroventricular (i.c.v.) route and whole-body plethysmograph set-up was used for both induction, using aerosolized citric acid (0.2 M), and recording of cough. Immunohistochemistry was performed to confirm the expression of NaV 1.8 channels in the nucleus tractus solitarius (nTS).ResultsWe show that both PGE2 and the non-selective EP1/EP3 agonist, sulprostone, dose-dependently enhanced the citric acid-induced cough (P ≤ 0.001, P ≤ 0.01, respectively). Pretreatment with the EP1 antagonist, ONO-8130, did not affect the sulprostone-induced cough sensitization, whilst the EP3 antagonist, L-798,106, dose-dependently inhibited this effect (P ≤ 0.05). Furthermore, treatment with either the EP2 agonist, butaprost or the EP4 agonist, L-902,688, had no effect on cough sensitization. Additionally, pretreatment with either the TRPV1 antagonist, JNJ-17203212 or the TRPA1 antagonist, HC-030031, alone or in combination, nor with the NaV 1.1, 1.2, 1.3, 1.4, 1.6 and 1.7 channel blocker, tetrodotoxin, had any effect on the cough. In contrast, pretreatment with the NaV 1.8 antagonist, A-803467, dose-dependently inhibited this effect (P ≤ 0.05). Furthermore, NaV 1.8 channels were shown to be expressed in the nTS.ConclusionCollectively, our findings show that PGE2 sensitizes the cough reflex centrally via EP3 receptor-dependent activation of NaV 1.8 but independently of TRPV1,TRPA1 and TTX-sensitive sodium channel activation. These results indicate that PGE2 plays an important role in central sensitization of the cough reflex and suggest that central EP3 receptors and/or NaVv 1.8 channels may represent novel antitussive molecular targets.Graphical

Analysis of tetrodotoxin-sensitive sodium and low voltage-activated calcium channels in developing mouse retinal horizontal cells

Expression patterns of voltage-gated ion channels determine the spatio-temporal dynamics of ion currents that supply excitable neurons in developing tissue with proper electrophysiological properties. The purpose of the study was to identify fast cationic inward currents in mouse retinal horizontal cells (HCs) and describe their biophysical properties at different developmental stages. We also aimed to reveal their physiological role in shaping light responses (LRs) in adult HCs. HCs were recorded in horizontal slices of wild-type mouse retina at postnatal stages ranging from p8 through p60. Voltage-dependent inward currents were isolated with appropriate voltage protocols and blockers specific for sodium and T-type calcium channels. LRs were evoked with full-field flashes (130 μW/cm2). Transient and steady inward currents were identified at all developmental stages. Transient currents were mediated by T-type calcium and TTX-sensitive sodium channels, whereas steady currents were blocked by cadmium, indicating the presence of high voltage-activated calcium channels. Activation and steady-state inactivation kinetics of T-type calcium channels revealed a contribution to the resting membrane potential during postnatal development. Additionally, both sodium and T-type calcium channels had an impact on HC LRs at light offset in adult animals. Our results showed that the voltage-dependent inward currents of postnatally developing mouse HCs consist of T-type calcium, TTX-sensitive sodium, and high voltage-activated calcium channels, and that transient ionic currents contributed to light-evoked responses of adult HCs, suggesting a role in HC information processing.

CC chemokine ligand 2 (CCL2) enhances TTX-sensitive sodium channel activity of primary afferent neurons in the complete Freud adjuvant-induced inflammatory pain model.

CC chemokine ligand 2 (CCL2) has been implicated in pathological pain, but the mechanism underlying the pronociceptive effect of CCL2 is not fully understood. Voltage-gated sodium (Nav) channels are important determinants of the excitability of sensory neurons. Hence we tested the hypothesis that CCL2 contributes to inflammatory pain via modulating Nav channel activity of primary afferent neurons. Chronic inflammatory pain was induced in rats by intraplantar injection of the complete Freud adjuvant (CFA) to one of the hind paws. Control rats received intraplantar injection of equal volume of saline. A significant increase of CCL2 mRNA and CCL2 receptor (CCR2) protein expression was detected in the ipsilateral dorsal root ganglion (DRG) in CFA-treated rats. Intraplantar injection of CCL2 protein in the control rats had minimal effect on the paw withdrawal threshold (PWT) in response to mechanical stimulation. However, in CFA-treated rats, intraplantar CCL2 led to an increase in pain responses. Patch-clamp recording of acutely dissociated DRG neurons revealed that CCL2 had minimum effect on the excitability of sensory neurons from control rats. However, CCL2 directly depolarized a large proportion of small to medium-sized sensory neurons from CFA-treated rats. In addition, CCL2 was found to enhance whole-cell TTX-sensitive sodium currents without significantly affecting the TTX-resistant sodium currents and the potassium currents. These results are in agreement with previous reports concerning the involvement of CCL2-CCR2 signaling in inflammatory hyperalgesia and further indicate that enhanced TTX-sensitive channel activity may partly underlie the pronociceptive effects of CCL2.

A novel \u03b1-conopeptide Eu1.6 inhibits N-type (CaV2.2) calcium channels and exhibits potent analgesic activity

We here describe a novel α-conopeptide, Eu1.6 from Conus eburneus, which exhibits strong anti-nociceptive activity by an unexpected mechanism of action. Unlike other α-conopeptides that largely target nicotinic acetylcholine receptors (nAChRs), Eu1.6 displayed only weak inhibitory activity at the α3β4 and α7 nAChR subtypes and TTX-resistant sodium channels, and no activity at TTX-sensitive sodium channels in rat dorsal root ganglion (DRG) neurons, or opiate receptors, VR1, KCNQ1, L- and T-type calcium channels expressed in HEK293 cells. However, Eu1.6 inhibited high voltage-activated N-type calcium channel currents in isolated mouse DRG neurons which was independent of GABAB receptor activation. In HEK293 cells expressing CaV2.2 channels alone, Eu1.6 reversibly inhibited depolarization-activated Ba2+ currents in a voltage- and state-dependent manner. Inhibition of CaV2.2 by Eu1.6 was concentration-dependent (IC50 ~1 nM). Significantly, systemic administration of Eu1.6 at doses of 2.5–5.0 μg/kg exhibited potent analgesic activities in rat partial sciatic nerve injury and chronic constriction injury pain models. Furthermore, Eu1.6 had no significant side-effect on spontaneous locomotor activity, cardiac and respiratory function, and drug dependence in mice. These findings suggest α-conopeptide Eu1.6 is a potent analgesic for the treatment of neuropathic and chronic pain and opens a novel option for future analgesic drug design.

Effects of Tetrodotoxin in Mouse Models of Visceral Pain.

Visceral pain is very common and represents a major unmet clinical need for which current pharmacological treatments are often insufficient. Tetrodotoxin (TTX) is a potent neurotoxin that exerts analgesic actions in both humans and rodents under different somatic pain conditions, but its effect has been unexplored in visceral pain. Therefore, we tested the effects of systemic TTX in viscero-specific mouse models of chemical stimulation of the colon (intracolonic instillation of capsaicin and mustard oil) and intraperitoneal cyclophosphamide-induced cystitis. The subcutaneous administration of TTX dose-dependently inhibited the number of pain-related behaviors in all evaluated pain models and reversed the referred mechanical hyperalgesia (examined by stimulation of the abdomen with von Frey filaments) induced by capsaicin and cyclophosphamide, but not that induced by mustard oil. Morphine inhibited both pain responses and the referred mechanical hyperalgesia in all tests. Conditional nociceptor‑specific Nav1.7 knockout mice treated with TTX showed the same responses as littermate controls after the administration of the algogens. No motor incoordination after the administration of TTX was observed. These results suggest that blockade of TTX-sensitive sodium channels, but not Nav1.7 subtype alone, by systemic administration of TTX might be a potential therapeutic strategy for the treatment of visceral pain.

A Novel Toxin from Haplopelma lividum Selectively Inhibits the NaV1.8 Channel and Possesses Potent Analgesic Efficacy.

Spider venoms are a complex mixture of peptides with a large number of neurotoxins targeting ion channels. Although thousands of peptide toxins have been identified from venoms of numerous species of spiders, many unknown species urgently need to be investigated. In this study, a novel sodium channel inhibitor, µ-TRTX-Hl1a, was identified from the venom of Haplopelma lividum. It contained eight cysteines and formed a conserved cysteine pattern of ICK motif. µ-TRTX-Hl1a inhibited the TTX-resistant (TTX-r) sodium channel current rather than the TTX-sensitive (TTX-s) sodium channel current. Meanwhile, µ-TRTX-Hl1a selectively inhibited NaV1.8 with an IC50 value of 2.19 μM. Intraperitoneal injection of µ-TRTX-Hl1a dose-dependently reduced inflammatory and neuropathic pain in rodent models of formalin-induced paw licking, tail-flicking, acetic acid-induced writhing, and hot plate test. It showed a better analgesic effect than morphine in inflammatory pain and equipotent effect to morphine in neuropathic pain. These findings demonstrate that µ-TRTX-Hl1a might be a valuable tool for physiology studies on NaV1.8 and a promising lead molecule for pain therapeutics.

Characterization of ion channels on subesophageal ganglion neurons from Chinese tarantula Ornithoctonus huwena: Exploring the myth of the spider insensitive to its venom

Chinese tarantula Ornithoctonus huwena is one of the most venomous spiders distributing in the hilly areas of southern China. In this study, using whole-cell patch-clamp technique we investigated electrophysiological and pharmacological properties of ion channels from tarantula subesophageal ganglion neurons. It was found that the neurons express multiple kinds of ion channels at least including voltage-gated calcium channels, TTX-sensitive sodium channels and two types of potassium channels. They exhibit pharmacological properties similar to mammalian subtypes. Spider calcium channels were sensitive to ω-conotoxin GVIA and diltiazem, two well-known inhibitors of mammalian neuronal high-voltage-activated (HVA) subtypes. 4-Aminopyridine and tetraethylammonium could inhibit spider outward transient and delayed-rectifier potassium channels, respectively. Huwentoxin-I and huwentoxin-IV are two abundant toxic components in the venom of Ornithoctonus huwena. Interestingly, although in our previous work they inhibit HVA calcium channels and TTX-sensitive sodium channels from mammalian sensory neurons, respectively, they fail to affect the subtypes from spider neurons. Moreover, the crude venom has no effect on delayed-rectifier potassium channels and only slightly reduces transient outward potassium channels with an IC50 value of ∼51.3 mg/L. Therefore, our findings provide important evidence for ion channels from spiders having an evolution as self-defense and prey mechanism.

Tetrodotoxin suppresses thermal hyperalgesia and mechanical allodynia in a rat full thickness thermal injury pain model

Burn injuries have been identified as the primary cause of injury in 5% of U.S. military personnel evacuated from Operations Iraqi Freedom and Enduring Freedom. Severe burn-associated pain is typically treated with opioids such as fentanyl, morphine, and methadone. Side effects of opioids include respiratory depression, cardiac depression, decrease in motor and cognitive function, as well as the development of hyperalgesia, tolerance and dependence. These effects have led us to search for novel analgesics for the treatment of burn-associated pain in wounded combat service members. Tetrodotoxin (TTX) is a selective voltage-gated sodium channel blocker currently in clinical trials as an analgesic. A phase 3 clinical trial for cancer-related pain has been completed and phase 3 clinical trials on chemotherapy-induced neuropathic pain are planned. It has also been shown in mice to inhibit the development of chemotherapy-induced neuropathic pain. TTX was originally identified as a neurotoxin in marine animals but has now been shown to be safe in humans at therapeutic doses. The antinociceptive effects of TTX are thought to be due to inhibition of Na+ ion influx required for initiation and conduction of nociceptive impulses. One TTX sensitive sodium channel, Nav1.7, has been shown to be essential in lowering the heat pain threshold after burn injuries. To date, the analgesic effect of TTX has not been tested in burn-associated pain. Male Sprague-Dawley rats were subjected to a full thickness thermal injury on the right hind paw. TTX (8μg/kg) was administered once a day systemically by subcutaneous injection beginning 3 days post thermal injury and continued through 7 days post thermal injury. Thermal hyperalgesia and mechanical allodynia were assessed 60 and 120min post injection on each day of TTX treatment. TTX significantly reduced thermal hyperalgesia at all days tested and had a less robust, but statistically significant suppressive effect on mechanical allodynia. These results suggest that systemic TTX may be an effective, rapidly acting analgesic for battlefield burn injuries and has the potential for replacing or reducing the need for opioid analgesics.

Hunting for origins of migraine pain: cluster analysis of spontaneous and capsaicin-induced firing in meningeal trigeminal nerve fibers.

Trigeminal nerves in meninges are implicated in generation of nociceptive firing underlying migraine pain. However, the neurochemical mechanisms of nociceptive firing in meningeal trigeminal nerves are little understood. In this study, using suction electrode recordings from peripheral branches of the trigeminal nerve in isolated rat meninges, we analyzed spontaneous and capsaicin-induced orthodromic spiking activity. In control, biphasic single spikes with variable amplitude and shapes were observed. Application of the transient receptor potential vanilloid 1 (TRPV1) agonist capsaicin to meninges dramatically increased firing whereas the amplitudes and shapes of spikes remained essentially unchanged. This effect was antagonized by the specific TRPV1 antagonist capsazepine. Using the clustering approach, several groups of uniform spikes (clusters) were identified. The clustering approach combined with capsaicin application allowed us to detect and to distinguish “responder” (65%) from “non-responder” clusters (35%). Notably, responders fired spikes at frequencies exceeding 10 Hz, high enough to provide postsynaptic temporal summation of excitation at brainstem and spinal cord level. Almost all spikes were suppressed by tetrodotoxin (TTX) suggesting an involvement of the TTX-sensitive sodium channels in nociceptive signaling at the peripheral branches of trigeminal neurons. Our analysis also identified transient (desensitizing) and long-lasting (slowly desensitizing) responses to the continuous application of capsaicin. Thus, the persistent activation of nociceptors in capsaicin-sensitive nerve fibers shown here may be involved in trigeminal pain signaling and plasticity along with the release of migraine-related neuropeptides from TRPV1 positive neurons. Furthermore, cluster analysis could be widely used to characterize the temporal and neurochemical profiles of other pain transducers likely implicated in migraine.

Characterization of Specific Roles of Sodium Channel Subtypes in Regional Anesthesia.

Commonly used local anesthetics (eg, lidocaine) are nonselective in blocking sodium channel subtypes, potentially resulting in adverse events, such as prolonged muscle paralysis and unstable hemodynamics. Subtype-selective sodium channel block might avoid these unwanted adverse effects while preserving desirable anesthetic effects. The contributions of sodium channel subtypes in different components of regional anesthesia are unclear and this study assumed that selective sodium channel subtype block might produce selective nerve block. Sciatic nerve block was performed in mice with lidocaine (nonselective sodium channel blocker), tetrodotoxin (TTX, TTX-sensitive sodium channel blocker), and A-803467 (selective Nav1.8 subtype blocker). Tactile sensory, pinprick, and thermal sensory block as well as motor block were evaluated after injection of study drugs. Median effective concentration (EC50) of lidocaine, TTX, and A-803467 as well as their blocking durations were determined. Lidocaine produced regional anesthetic effects including tactile, pinprick, and thermal sensory block as well as motor block, with EC50 [mean, 95% confidence intervals (CIs)] of 4.4 (3.7-5.2), 9.4 (8.0-10.9), 5.2 (4.3-6.2), and 3.7 (3.3-4.2) mmol/L, respectively. Tetrodotoxin produced tactile sensory block and motor block with EC50 (mean, 95% CIs) of 7.7 (6.0-11.0) and 8.3 (7.4-9.8) μmol/L, respectively; whereas A-803467 produced tactile sensory block only, with EC50 (mean, 95% CIs) of 12.6 (11.7-15.6) μmol/L. Sodium channel subtype selective blockers could induce selective nerve blocks. Tetrodotoxin-sensitive sodium channel subtypes contribute to low-threshold sensory block (eg, tactile) and motor block. Unexpectedly, selective Nav1.8 subtype block induced low-threshold sensory block rather than nociceptive or motor block.

Role of sodium channels in the spontaneous excitability of early embryonic cardiomyocytes.

Sodium channels play an important role in action potentials. Moreover, some evidences recently suggested that sodium channels were responsible for murine sinoatrial node pacemaking. The aim of this study was to investigate the role of sodium channels in pacemaking in embryonic cardiomyocytes in early development stage (EDS). Whole-cell patch-clamp technique was employed to record sodium current of murine early embryonic cardiomyocytes. Current clamp technique was used to record the effect of 0.1, 1 and 10 μM tetrodotoxin (TTX) on embryonic cardiomyocytes pacemaking. Electro- physiology properties of sodium channels in embryonic cardiomyocytes corresponded to Nav1.5, and the IC₅₀ of TTX was 5.24 μM. TTX at 0.1 μM concentration had no effects on the pacemaking. TTX at 1 μM concentration, however, dramatically slowed the spontaneous beating rate from 73.975 ± 10.478 to 50.268 ± 10.476 cycle/min (P < 0.05), and the maximum upstroke velocity (dV/dtmax) of phase 4 from 0.074 ± 0.006 to 0.046 ± 0.007 V/s (P < 0.01). Furthermore, 1 μM TTX reduced the dV/dtmax of phase 0 from 16.405 ± 0.056 to 12.801 ± 0.084 V/s (P < 0.01), and increased the period of phase 4 from 710.342 ± 110.983 to 1320.618 ± 250.483 ms (P < 0.05). TTX at 1 μM also had some effects on the peak of phase 0 decreasing it from 40.621 ± 3.012 to 37.407 ± 2.749 mV (P < 0.05). But TTX at 1 μM had no effects on the period of phase 0. In some cells (9/13), TTX at 10 μM caused complete cessation of spontaneous action potentials. Our results suggested that the main expression subtype of sodium channels was Nav1.5 of early embryonic cardiomyocytes. And TTX-resistant sodium channels contributed to the initiation of action potentials of early embryonic cardiomyocytes, while TTX-sensitive sodium channels were not involved in initiation of action potentials.

Contribution of sodium channels to lamellipodial protrusion and Rac1 and ERK1/2 activation in ATP-stimulated microglia.

Microglia are motile resident immune cells of the central nervous system (CNS) that continuously explore their territories for threats to tissue homeostasis. Following CNS insult (e.g., cellular injury, infection, or ischemia), microglia respond to signals such as ATP, transform into an activated state, and migrate towards the threat. Directed migration is a complex and highly-coordinated process involving multiple intersecting cellular pathways, including signal transduction, membrane adhesion and retraction, cellular polarization, and rearrangement of cytoskeletal elements. We previously demonstrated that the activity of sodium channels contributes to ATP-induced migration of microglia. Here we show that TTX-sensitive sodium channels, specifically NaV 1.6, participate in an initial event in the migratory process, i.e., the formation in ATP-stimulated microglia of polymerized actin-rich membrane protrusions, lamellipodia, containing accumulations of Rac1 and phosphorylated ERK1/2. We also examined Ca(2+) transients in microglia and found that blockade of sodium channels with TTX produced a downward shift in the level of [Ca(2+) ]i during the delayed, slower recovery of [Ca(2+) ]i following ATP stimulation. These observations demonstrate a modulatory role of sodium channels on Ca(2+) transients in microglia that are likely to affect down-stream signaling cascades. Consistent with these observations, we demonstrate that ATP-induced microglial migration is mediated via Rac1 and ERK1/2, but not p38α/β and JNK, dependent pathways, and that activation of both Rac1 and ERK1/2 is modulated by sodium channel activity. Our results provide evidence for a direct link between sodium channel activity and modulation of Rac1 and ERK1/2 activation in ATP-stimulated microglia, possibly by regulating Ca(2+) transients.

Impact of tetrodotoxin application and lidocaine supplementation on equine jejunal smooth muscle contractility and activity of the enteric nervous system in vitro

By blocking the enteric nervous system (ENS) using tetrodotoxin (TTX), previous studies have documented the contractility-enhancing (CE) effects of lidocaine in equine intestinal smooth muscle (SM) at the level of SM cells and/or interstitial cells of Cajal (ICC). The present study examined the impact of ENS deactivation on CE lidocaine effects, and investigated the effects of lidocaine on ENS activity. TTX application did not affect the CE effects of lidocaine, indicating that these were not mediated by TTX-sensitive sodium channels. Application of TTX or ≥100 mg/L lidocaine reduced ENS activity. Although such concentrations of lidocaine exceed therapeutic blood concentrations, tissue concentrations may be higher with the potential to reduce ENS activity and impair intestinal motility in vivo. Improved understanding of underlying mechanisms is relevant for therapeutic use of lidocaine in horses with postoperative ileus.

The Trpa1 Agonist Cinnamaldehyde Acts as a Local Anesthetic Inhibiting Voltage-Gated Sodium Channels in Sensory Neurons

Cinnamaldehyde (CA) is a highly reactive compound that has been used in experimental models of neurogenic inflammation as specific agonist of the broadly tuned chemosensor TRPA1. In several models of chemonociception in mice and humans, we have found that CA only produces very weak avoidance, pain or visceral irritation responses. Furthermore, CA induces a dramatic decrease in the velocity of conduction and the amplitude of compound action potentials in the mouse tail. Noting the structural similarities between CA and classical local anesthetics, we investigated whether CA inhibits voltage-gated sodium channels (VGSC) expressed in sensory neurons. We found that CA inhibits TTX-sensitive voltage-dependent sodium currents in a concentration dependent manner in mouse trigeminal neurons with an IC50 of 1.5 mM. TTX-resistant sodium currents were inhibited with an IC50 of 0.8 mM. TTX-sensitive VGSC recorded in the immortalized DRG neuron-derived F11 cells were inhibited with an IC50 of 1.3 mM. CA modified the gating of TTX-sensitive sodium channels, inducing concentration-dependent shifts of the activation curve to depolarized voltages and shifts of the availability curve to more hyperpolarized voltages. In the presence of CA, the corresponding slope factors became significantly larger. CA-induced inhibition was enhanced at depolarized holding potentials. Inhibition of TTX-sensitive sodium channels was not frequency dependent and can occur when the channels are in resting/closed state(s). We conclude that CA inhibits VGSC in sensory neurons at concentrations that can be pharmacologically relevant, and that the mechanism of inhibition shares similarities with the action of local anesthetics.

Two tarantula venom peptides as potent and differential NaV channels blockers

Voltage dependent sodium (NaV) channels are large membrane spanning proteins which lie in the basis of action potential generation and propagation in excitable cells and hence are essential mediators of neuronal signaling. Inhibition of NaV channel activity is one of the core mechanisms to treat conditions related to neuronal hyperexcitability, such as epilepsy in the clinic. NaV channel blockers are also extensively used to locally inhibit action potential generation and related pain perceptions in the form of local anesthetics. Here we describe the isolation, biochemical characterization, synthesis and in vitro characterization of two potent NaV channel blockers from the venom of the Paraphysa scrofa (Phrixotrichus auratus) tarantula spider. Both Voltage sensor toxin 3 (VSTx-3, κ-theraphotoxin-Gr4a) and GTx1-15 (Toxin Gtx1-15), were originally isolated from the venom of the related tarantula Grammostola rosea and described as KV and CaV channel blockers, respectively. In our hands, GTx1-15 was shown to be a potent inhibitor of tetrodotoxin (TTX)-sensitive channels (IC50 0.007 μM for hNaV1.7 and 0.12 μM for hNaV1.3 channels), with very little effect on TTX-resistant (NaV1.5 and NaV1.8) channels. VSTx-3 was demonstrated to be a potent, TTX-sensitive sodium channel blocker and especially, potent blocker of NaV1.8 channels (IC50 0.19 μM for hNaV1.3, 0.43 μM for hNaV1.7 and 0.77 μM for hNaV1.8 channels). Such potent inhibitors with differential selectivity among NaV channel isoforms may be used as tools to study the roles of the different channels in processes related to hyperexcitability and as lead compounds to treat pathological pain conditions.

Native Pyroglutamation of Huwentoxin-IV: A Post-Translational Modification that Increases the Trapping Ability to the Sodium Channel

Huwentoxin-IV (HWTX-IV), a tetrodotoxin-sensitive (TTX-s) sodium channel antagonist, is found in the venom of the Chinese spider Ornithoctonus huwena. A naturally modified HWTX-IV (mHWTX-IV), having a molecular mass 18 Da lower than HWTX-IV, has also been isolated from the venom of the same spider. By a combination of enzymatic fragmentation and MS/MS de novo sequencing, mHWTX-IV has been shown to have the same amino acid sequence as that of HWTX-IV, except that the N-terminal glutamic acid replaced by pyroglutamic acid. mHWTX-IV inhibited tetrodotoxin-sensitive voltage-gated sodium channels of dorsal root ganglion neurons with an IC50 nearly equal to native HWTX-IV. mHWTX-IV showed the same activation and inactivation kinetics seen for native HWTX-IV. In contrast with HWTX-IV, which dissociates at moderate voltage depolarization voltages (+50 mV, 180000 ms), mHWTX-IV inhibition of TTX-sensitive sodium channels is not reversed by strong depolarization voltages (+200 mV, 500 ms). Recovery of Nav1.7current was voltage-dependent and was induced by extreme depolarization in the presence of HWTX-IV, but no obvious current was elicited after application of mHWTX-IV. Our data indicate that the N-terminal modification of HWTX-IV gives the peptide toxin a greater ability to trap the voltage sensor in the sodium channel. Loss of a negative charge, caused by cyclization at the N-terminus, is a possible reason why the modified toxin binds much stronger. To our knowledge, this is the first report of a pyroglutamic acid residue in a spider toxin; this modification seems to increase the trapping ability of the voltage sensor in the sodium channel.

Distribution and function of sodium channel subtypes in human atrial myocardium

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”.

Whither Art Thou, SCN10A, and What Art Thou Doing?

Since its initial cloning in 1992, SCN5A (Nav1.5) has become known as “the” cardiac sodium channel.1 SCN5A encodes a tetrodotoxin (TTX)-resistant channel that is responsible for the inward sodium current (INa), which initiates the cardiac action potential. Its RNA and protein expression dominate the sodium channel landscape in the ventricle, atrium, and specialized conduction system. Mice heterozygous for a targeted Scn5a deletion (Scn5a+/− mice) have slowed conduction along with atrial and ventricular arrhythmias, whereas homozygous targeted deletion in mice is embryonic lethal.2 In humans, pharmacological agents that block SCN5A such as flecainide, developed as antiarrhythmics, are proarrhythmic and increase mortality in patients with structural heart disease.3 In addition, SCN5A mutations cause a potpourri of diseases that lead to sudden cardiac death including long-QT syndrome type 3 (LQT3, gain-of-function mutations that slow inactivation and increase late sodium current, INa,L), Brugada syndrome (BRS1, loss of function mutations that decrease peak INa), conduction disease, atrial fibrillation, and dilated cardiomyopathy.4 More recently, changes in SCN5A (expression, splicing, posttranslational modifications including CaMKII phosphorylation) and/or its β-subunits that decrease peak INa or increase INa,L have been implicated as potential contributing factors for more common types of sudden death from acquired heart diseases such as heart failure.5–8 Articles, see p 322 and 333 A number of independent lines of evidence have suggested that other sodium channels might play important roles in the heart. In the mouse, Scn5a is preferentially localized at intercalated disks, whereas Nav1.1, Nav1.3, and Nav1.6 are localized in t-tubules.9 Consistent with this, TTX-sensitive sodium channels were shown to be responsible for 16% of the sodium channel mRNA and 8% of the peak INa in isolated adult cardiac myocytes and were upregulated in cardiac myocytes isolated from rats with thoracic …

Modulation of spinal cord synaptic activity by tumor necrosis factor α in a model of peripheral neuropathy

BackgroundThe cytokine tumor necrosis factor α (TNFα) is an established pain modulator in both the peripheral and central nervous systems. Modulation of nociceptive synaptic transmission in the spinal cord dorsal horn (DH) is thought to be involved in the development and maintenance of several pathological pain states. Increased levels of TNFα and its receptors (TNFR) in dorsal root ganglion (DRG) cells and in the spinal cord DH have been shown to play an essential role in neuropathic pain processing. In the present experiments the effect of TNFα incubation on modulation of primary afferent synaptic activity was investigated in a model of peripheral neuropathy.MethodsSpontaneous and miniature excitatory postsynaptic currents (sEPSC and mEPSCs) were recorded in superficial DH neurons in acute spinal cord slices prepared from animals 5 days after sciatic nerve transection and in controls.ResultsIn slices after axotomy the sEPSC frequency was 2.8 ± 0.8 Hz, while neurons recorded from slices after TNFα incubation had significantly higher sEPSC frequency (7.9 ± 2.2 Hz). The effect of TNFα treatment was smaller in the slices from the control animals, where sEPSC frequency was 1.2 ± 0.2 Hz in slices without and 2.0 ± 0.5 Hz with TNFα incubation. Tetrodotoxin (TTX) application in slices from axotomized animals and after TNFα incubation decreased the mEPSC frequency to only 37.4 ± 6.9% of the sEPSC frequency. This decrease was significantly higher than in the slices without the TNFα treatment (64.4 ± 6.4%). TTX application in the control slices reduced the sEPSC frequency to about 80% in both TNFα untreated and treated slices. Application of low concentration TRPV1 receptors endogenous agonist N-oleoyldopamine (OLDA, 0.2 μM) in slices after axotomy induced a significant increase in mEPSC frequency (175.9 ± 17.3%), similar to the group with TNFα pretreatment (158.1 ± 19.5%).ConclusionsOur results indicate that TNFα may enhance spontaneous transmitter release from primary afferent fibres in the spinal cord DH by modulation of TTX-sensitive sodium channels following sciatic nerve transection. This nerve injury also leads to enhanced sensitivity of presynaptic TRPV1 receptors to endogenous agonist. Modulation of presynaptic receptor activity on primary sensory terminals by TNFα may play an important role in neuropathic pain development.

Light-Evoked Lateral GABAergic Inhibition at Single Bipolar Cell Synaptic Terminals Is Driven by Distinct Retinal Microcircuits

Inhibitory amacrine cells (ACs) filter visual signals crossing the retina by modulating the excitatory, glutamatergic output of bipolar cells (BCs) on multiple temporal and spatial scales. Reciprocal feedback from ACs provides focal inhibition that is temporally locked to the activity of presynaptic BC activity, whereas lateral feedback originates from ACs excited by distant BCs. These distinct feedback mechanisms permit temporal and spatial computation at BC terminals. Here, we used a unique preparation to study light-evoked IPSCs recorded from axotomized terminals of ON-type mixed rod/cone BCs (Mb) in goldfish retinal slices. In this preparation, light-evoked IPSCs could only reach axotomized BC terminals via the lateral feedback pathway, allowing us to study lateral feedback in the absence of overlapping reciprocal feedback components. We found that light evokes ON and OFF lateral IPSCs (L-IPSCs) in Mb terminals having different temporal patterns and conveyed via distinct retinal pathways. The relative contribution of rods versus cones to ON and OFF L-IPSCs was light intensity dependent. ACs presynaptic to Mb BC terminals received inputs via AMPA/KA- and NMDA-type receptors in both the ON and OFF pathways, and used TTX-sensitive sodium channels to boost signal transfer along their processes. ON and OFF L-IPSCs, like reciprocal feedback IPSCs, were mediated by both GABA(A) and GABA(C) receptors. However, our results suggest that lateral and reciprocal feedback do not cross-depress each other, and are therefore mediated by distinct populations of ACs. These findings demonstrate that retinal inhibitory circuits are highly specialized to modulate BC output at different light intensities.