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Diversity of composition and function of sodium channels in peripheral sensory neurons
... 8 Voltage gated sodium channels (VGSCs) underlie the transduction and propagation of nociceptive signals and VGSC subtypes are selectively expressed in dorsal root ganglia (DRG) neurons. 9 The expression and properties of VGSCs are dramatically altered by nerve injury, implying that the modulation of sodium currents critically contributes to the pathophysiological hyperexcitability that is associated with neuropathic pain states. Nav1.3, ...
... Their importance in pain signaling is demonstrated by animal models of pain and human pain disorders. 9 It is desirable but challenging to develop subtype-specific VGSC blockers which can minimize side effects outside the pain axis. ...
... These have distinct electrophysiological properties and characteristic patterns of tissue distribution. 9 Conserved transmembrane segments of the -subunit are organized into four homologous domains (I-IV), each with six transmembrane -helices (S1-S6). The -subunit makes up the voltage sensor and channel pore which includes the selectivity filter. ...
Being maladaptive and frequently unresponsive to pharmacotherapy, chronic pain presents a major unmet clinical need. While an intact central nervous system is required for conscious pain perception, nociceptor hyperexcitability induced by nerve injury in the peripheral nervous system (PNS) is sufficient and necessary to initiate and maintain neuropathic pain. The genesis and propagation of action potentials is dependent on voltage-gated sodium channels, in particular, Nav1.7, Nav1.8 and Nav1.9. However, nerve injury triggers changes in their distribution, expression and/or biophysical properties, leading to aberrant excitability. Most existing treatment for pain relief acts through non-selective, state-dependent sodium channel blockage and have narrow therapeutic windows. Natural toxins and developing subtype-specific and molecular-specific sodium channel blockers show promise for treatment of neuropathic pain with minimal side effects. New approaches to analgesia include combination therapy and gene therapy. Here, we review how individual sodium channel subtypes contribute to pain, and the attempts made to develop more effective analgesics for the treatment of chronic pain.
... antibody in the intracellular solution. (3) Similar to the Nav1.9 channel currents in neurons of the dorsal root ganglia (DRG; Coste et al., 2004;Dib-Hajj and Waxman, 2015) and mPFC pyramidal neurons ( Gawlak et al., 2017), RD was resistant to TTX. (4) The voltage threshold of RD was close to the resting membrane potential, similar to Nav1.9 channel currents in DRG neurons ( Coste et al., 2004;Dib-Hajj and Waxman, 2015) and mPFC pyramidal neurons ( Gawlak et al., 2017). (5) The RD threshold was reduced in the presence of F − , similar to the activation threshold of Nav1.9 channels in DRG neurons ( Coste et al., 2004). ...
... antibody in the intracellular solution. (3) Similar to the Nav1.9 channel currents in neurons of the dorsal root ganglia (DRG; Coste et al., 2004;Dib-Hajj and Waxman, 2015) and mPFC pyramidal neurons ( Gawlak et al., 2017), RD was resistant to TTX. (4) The voltage threshold of RD was close to the resting membrane potential, similar to Nav1.9 channel currents in DRG neurons ( Coste et al., 2004;Dib-Hajj and Waxman, 2015) and mPFC pyramidal neurons ( Gawlak et al., 2017). (5) The RD threshold was reduced in the presence of F − , similar to the activation threshold of Nav1.9 channels in DRG neurons ( Coste et al., 2004). ...
... (5) The RD threshold was reduced in the presence of F − , similar to the activation threshold of Nav1.9 channels in DRG neurons ( Coste et al., 2004). (6) Steady-state inactivation of RD was removed by membrane hyperpolarization to −80 mV, similar to the behavior of Nav1.9 currents, which were de-inactivated at −80 mV in DRG neurons ( Coste et al., 2004;Dib-Hajj and Waxman, 2015) and in mPFC pyramidal neurons ( Gawlak et al., 2017). (7) It was recently demonstrated that Nav1.9 channels are present in the mPFC ( Gawlak et al., 2017;Radzicki et al., 2017), including in layer V pyramidal neurons ( Kurowski et al., 2015). ...
Rebound depolarization (RD) occurs after membrane hyperpolarization and converts an arriving inhibitory signal into cell excitation. The purpose of our study was to clarify the ionic mechanism of RD in synaptically isolated layer V medial prefrontal cortex (mPFC) pyramidal neurons in slices obtained from 58-to 62-day-old male rats. The RD was evoked after a step hyperpolarization below −80 mV, longer than 150 ms in 192 of 211 (91%) tested neurons. The amplitude of RD was 30.6 ± 1.2 mV above the resting membrane potential (−67.9 ± 0.95 mV), and it lasted a few 100 ms (n = 192). RD could be observed only after preventing BK channel activation, which was attained either by using paxilline, by removal of Ca ++ from the extra-or intracellular solution, by blockade of Ca ++ channels or during protein kinase C (PKC) activation. RD was resistant to tetrodotoxin (TTX) and was abolished after the removal of Na + from the extracellular solution or application of an anti-Nav1.9 antibody to the cell interior. We conclude that two membrane currents are concomitantly activated after the step hyperpolarization in the tested neurons: a. a low-threshold, TTX-resistant, Na + current that evokes RD; and b. an outward K + current through BK channels that opposes Na +-dependent depolarization. The obtained results also suggest that a. low-level Ca ++ in the external medium attained upon intense neuronal activity may facilitate the formation of RD and seizures; and b. RD can be evoked during the activation of PKC, which is an effector of a number of transduction pathways.
... The TTX-S channel Na v 1.3 is predominantly expressed in embryonic sensory neurons, 116 but is up-regulated after traumatic or metabolic nerve injury in rodent dorsal root ganglion (DRG) neurons. 35,116 Functional studies of these channels within neurons in which they are normally expressed 38 as well as studies in knock-out mice have yielded important information about the contribution of individual sodium channels to electrogenesis within these neurons 42,47,97 (Fig. 1). ...
... TTX-S channels Na v 1.3, Na v 1.6 (especially at nodes of Ranvier) and TTX-R channel Na v 1.8 (in the neurons in which it is expressed) contribute most of the current for the action potential. Adapted with permission from Ref. 42. Adaptations are themselves works protected by copyright. ...
... 33,63 This ability of Na v 1.7 to boost subthreshold stimuli increases the probability of neurons reaching their threshold for action potential firing. 42,47,97 The implementation of dynamic-clamp recordings in native rodent DRG neurons using physiologically relevant levels of Na v 1.7 Figure 2. Species-specific differences in Na v 1.8 and action potential properties. (A and B) Representative Na v 1.8 current family traces recorded from rat (A) or human DRG neurons (B). ...
... Navs are composed of one α subunit and one or more β subunits (Figure 2). In mammals, Nav channels have nine known alpha members, Nav1.1-Nav1.9, which are selectively expressed in dorsal root ganglia (DRG) neurons [59]. Pharmacologically, Navs may be classified by their sensitivity to the neurotoxin tetrodotoxin (TTX). ...
... Navs are composed of one α subunit and one or more β subunits ( Figure 2). In mammals, Nav channels have nine known alpha members, Nav1.1-Nav1.9, which are selectively expressed in dorsal root ganglia (DRG) neurons [59]. Pharmacologically, Navs may be classified by their sensitivity to the neurotoxin tetrodotoxin (TTX). ...
... Navs are composed of one α subunit and one or more β subunits (Figure 2). In mammals, Nav channels have nine known alpha members, Nav1.1-Nav1.9, which are selectively expressed in dorsal root ganglia (DRG) neurons [59]. Pharmacologically, Navs may be classified by their sensitivity to the neurotoxin tetrodotoxin (TTX). ...
... Navs are composed of one α subunit and one or more β subunits ( Figure 2). In mammals, Nav channels have nine known alpha members, Nav1.1-Nav1.9, which are selectively expressed in dorsal root ganglia (DRG) neurons [59]. Pharmacologically, Navs may be classified by their sensitivity to the neurotoxin tetrodotoxin (TTX). ...
Centipedes are among the oldest venomous arthropods that use their venom to subdue the prey. The major components of centipede venom are a variety of low-molecular-weight peptide toxins that have evolved to target voltage-gated ion channels to interfere with the central system of prey and produce pain or paralysis for efficient hunting. Peptide toxins usually contain several intramolecular disulfide bonds, which confer chemical, thermal and biological stability. In addition, centipede peptides generally have novel structures and high potency and specificity and therefore hold great promise both as diagnostic tools and in the treatment of human disease. Here, we review the centipede peptide toxins with reported effects on ion channels, including Nav, Kv, Cav and the nonselective cation channel polymodal transient receptor potential vanilloid 1 (TRPV1).
... 97 Nerve injury can modify sodium channel properties and expression, and alterations in sodium currents significantly contribute to the hyperexcitability observed in neuropathic pain conditions. 98 The deactivation of sodium channels appears to be one of the mechanisms by which BoNT/A can modulate pain behavior. Min-Chul Shin et al. demonstrated that BoNT/A blocked sodium channel activity in central and peripheral neuronal cells, reducing sodium current in cultured hippocampal and DRG cells. ...
Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, have been used for the treatment of various central and peripheral neurological conditions. Recent studies have suggested that BoNTs may also have a beneficial effect on pain conditions. It has been hypothesized that one of the mechanisms underlying BoNTs' analgesic effects is the inhibition of pain-related receptors' transmission to the neuronal cell membrane. BoNT application disrupts the integration of synaptic vesicles with the cellular membrane, which is responsible for transporting various receptors, including pain receptors such as TRP channels, calcium channels, sodium channels, purinergic receptors, neurokinin-1 receptors, and glutamate receptors. BoNT also modulates the opioidergic system and the GABAergic system, both of which are involved in the pain process. Understanding the cellular and molecular mechanisms underlying these effects can provide valuable insights for the development of novel therapeutic approaches for pain management. This review aims to summarize the experimental evidence of the analgesic functions of BoNTs and discuss the cellular and molecular mechanisms by which they can act on pain conditions by inhibiting the transmission of pain-related receptors.
... Nav1.8, and Nav1.9 are associated with various pain states. 17 They are useful analgesic drug targets for various types of pain, including neuropathic pain. Sodium channel blockers include carbamazepine, lidocaine, and bulleyaconitine A (BLA). ...
Ion channel drugs have been increasing used for chronic pain management with progress in the development of selective calcium channel modulators. Although ion channel drugs have been proven safe and effective in clinical practice, uncertainty remains regarding its use to treat chronic pain. To standardize the clinical practice of ion channel drug for the treatment of chronic pain, the National Health Commission Capacity Building and Continuing Education Center for Pain Diagnosis and Treatment Special Ability Training Project established an expert group to form an expert consensus on the use of ion channel drugs for the treatment of chronic pain after repeated discussions on existing medical evidence combined with the well clinical experience of experts. The consensus provided information on the mechanism of action of ion channel drugs and their recommendations, caution use, contraindications, and precautions for their use in special populations to support doctors in their clinical decision-making.
... TTX-S channels SCN3A, SCN8A (especially at nodes of Ranvier), and TTX-R channel SCN10A (in the neurons in which it is expressed), contribute most of the current for the action potential. Adapted from Dib-Hajj, S.D. et al., 2015[134]. ...
The SCN10A sodium channel is expressed in peripheral sensory neurons. More precisely, it is present in primary nociceptive neurons and is involved in the pain signal transmission. Gain-of-function mutations of SCN10A have been found in patients with small fiber neuropathy (SFN). Three patients carried the c.4984G> A, p.G1662S mutation in SCN10A gene. We have created a mouse model carrying the Scn10aG1663S mutation, corresponding to the human G1662S mutation. Scn10aG1663S mice have increased sensitivity to touch in the von Frey test, cold in the acetone test, and warm in the Hargreaves and hot plate tests. The phenotype found is similar to the symptoms of SFN patients, suggesting that this mutation contributes to their pain symptoms. Our results provide a better understanding of the role of SCN10A in pain mechanisms and opens the way for future pharmacological studies.
... Voltage-gated sodium channels (NaV channels) are of paramount importance for nociception, since they generate and propagate action potentials [16,17]. Moreover, HCN channels have recently been associated with inflammatory, neuropathic and postoperative pain [18]. ...
Introduction
Neuropathic pain (NP) is caused by a lesion or disease of the somatosensory system, which can severely impact patients’ quality of life. The current-approved treatments for NP comprise of both centrally acting agents and topical drugs, including capsaicin 8% dermal patches, which is approved for the treatment of peripheral NP.
Areas covered
The authors summarize literature data regarding capsaicin use in patients who suffer from NP and discuss the clinical applications of this topical approach.
Expert opinion
Overall, the capsaicin 8% dermal patch is as effective in reducing pain intensity as other centrally active agents (i.e. pregabalin). Some studies have also reported fewer systemic side effects, a faster onset of action and superior treatment satisfaction compared with systemic agents. In our opinion, capsaicin 8% dermal patches also present additional advantages, such as a good systemic tolerability, the scarcity of adverse events, the possibility to combine it with other agents, and a good cost-effective profile. It is important to note that, as the mechanism of action of capsaicin 8% is the ‘defunctionalization’ of small afferent fibers through interaction with TRPV1 receptors, the peripheral expression of this receptor on nociceptor fibers, is crucial to predict patient’s response to treatment.
... According to our findings and previous data, the three channels may have different roles in the regulation of membrane excitability and AP firing in DRG neurons. AP generation is the process of neuron membrane depolarization, during which the membrane potential can be described to reach three levels: the RMP, threshold, and upstroke 47,54 . Na v 1.7, Na v 1.8, and Na v 1.9 play distinct roles in these three levels, and their collaboration is required for the generation of a complete AP and repeated firing 47 . ...
The sodium channels Nav1.7, Nav1.8 and Nav1.9 are critical for pain perception in peripheral nociceptors. Loss of function of Nav1.7 leads to congenital insensitivity to pain in humans. Here we show that the spider peptide toxin called HpTx1, first identified as an inhibitor of Kv4.2, restores nociception in Nav1.7 knockout (Nav1.7-KO) mice by enhancing the excitability of dorsal root ganglion neurons. HpTx1 inhibits Nav1.7 and activates Nav1.9 but does not affect Nav1.8. This toxin produces pain in wild-type (WT) and Nav1.7-KO mice, and attenuates nociception in Nav1.9-KO mice, but has no effect in Nav1.8-KO mice. These data indicate that HpTx1-induced hypersensitivity is mediated by Nav1.9 activation and offers pharmacological insight into the relationship of the three Nav channels in pain signalling. Loss of function of Nav1.7 leads to congenital insensitivity to pain in humans. Here the authors found that activation of Nav1.9 can restore nociception in Nav1.7 knockout mice, revealed by a venom-derived peptide as a probe.
... Ion channels play a major role in the process of maintaining central sensitization. In particular, voltage-gated sodium (NaV) channels are responsible for the generation of pathogenic action potentials in nociceptors [39,40]. Targeting these peripheral ion channels may thus prevent painful stimuli from reaching the CNS. ...
The ‘Science of Relief’ event, held in Milan on 10–11 May 2019, was aimed at promoting dialog between different stakeholders among scientific associations, pharma industry, healthcare services and related institutions. The goal was to renew interest and attention on the management of pain, sharing new solutions in order to bring the patients and their quality of life to the center of attention. An international group of scientists and clinicians presented and discussed new and known evidence in the field of chronic pain, from physiopathology and diagnosis to the choice of appropriate and timely pharmacological treatments. This paper reports the highlights of those presentations.
... In our present study, only one peak of the ramp current is observed and occurs at −30 mV, ruling out a role for Na v 1.3. In addition, Na v 1.3 channels are expressed only in DRG neurons after nerve injury 47 . Na v 1.6 is highly expressed in large DRG neurons while small DRG neurons express low mRNA levels of this sodium channel 53 . ...
17β-Estradiol mediates the sensitivity to pain and is involved in sex differences in nociception. The widespread environmental disrupting chemical bisphenol A (BPA) has estrogenic activity, but its implications in pain are mostly unknown. Here we show that treatment of male mice with BPA (50 µg/kg/day) during 8 days, decreases the latency to pain behavior in response to heat, suggesting increased pain sensitivity. We demonstrate that incubation of dissociated dorsal root ganglia (DRG) nociceptors with 1 nM BPA increases the frequency of action potential firing. SCN9A encodes the voltage-gated sodium channel Nav1.7, which is present in DRG nociceptors and is essential in pain signaling. Nav1.7 and other voltage-gated sodium channels in mouse DRG are considered threshold channels because they produce ramp currents, amplifying small depolarizations and enhancing electrical activity. BPA increased Nav-mediated ramp currents elicited with slow depolarizations. Experiments using pharmacological tools as well as DRG from ERβ−/− mice indicate that this BPA effect involves ERα and phosphoinositide 3-kinase. The mRNA expression and biophysical properties other than ramp currents of Nav channels, were unchanged by BPA. Our data suggest that BPA at environmentally relevant doses affects the ability to detect noxious stimuli and therefore should be considered when studying the etiology of pain conditions.
... It has previously been hypothesized that Na V 1.7 is an amplifier of subthreshold stimuli, responsible for depolarizing the membrane potential to a point when Na V 1.8 and 1.6 activate, which would then carry the upstroke of the action potential. 2,13,14,34 Our results argue that Na V 1.7 is unlikely to be a subthreshold channel because the slope of the subthreshold depolarization of the action potential is not changed in IEM nociceptors. In addition, blocking Na V 1.7 by ProTx-II displayed a hyperpolarized voltage dependence of activation, suggesting that the remaining Na V isoforms open before Na V 1.7 and are therefore likely to carry subthreshold depolarizations. ...
The chronic pain syndrome inherited erythromelalgia (IEM) is attributed to mutations in the voltage-gated sodium channel (Nav) 1.7. Still, recent studies targeting Nav1.7 in clinical trials have provided conflicting results. Here, we differentiated induced pluripotent stem cells (iPS cells) from IEM patients with the Nav1.7/I848T mutation into sensory nociceptors. Action potentials in these IEM nociceptors displayed a decreased firing threshold, an enhanced upstroke and afterhyperpolarization, all of which may explain the increased pain experienced by patients. Subsequently, we investigated the voltage dependence of the tetrodotoxin-sensitive Nav activation in these human sensory neurons using a specific pre-pulse voltage protocol. The IEM mutation induced a hyperpolarizing shift of Nav activation which leads to activation of Nav1.7 at more negative potentials. Our results indicate that Nav1.7 is not active during subthreshold depolarizations, but that its activity defines the action potential threshold and contributes significantly to the action potential upstroke. Thus, our model system with iPS cell-derived sensory neurons provides a new rationale for Nav1.7 function and promises to be valuable as a translational tool to profile and develop more efficacious clinical analgesics.
... While potassium and other ions generally function to repolarize and/or stabilize the cell membrane, voltagegated sodium channels (VGSCs) are critical elements determining the initiation and propagation of action potentials in dorsal root ganglion (DRG) neurons responsible for pain transmission. 1 VGSCs also help to set the resting membrane potential. 2,3 However, the influx of sodium ions through these channels causes depolarization of the cell membrane, which is critical for amplification or propagation of receptor potentials generated by the initial nociceptive stimuli. ...
Background
Abdominal pain is a frequent and persistent problem in the most common gastrointestinal disorders, including irritable bowel syndrome and inflammatory bowel disease. Pain adversely impacts quality of life, incurs significant healthcare expenditures, and remains a challenging issue to manage with few safe therapeutic options currently available. It is imperative that new methods are developed for identifying and treating this symptom. A variety of peripherally active neuroendocrine signaling elements have the capability to influence gastrointestinal pain perception. A large and growing body of evidence suggests that voltage‐gated sodium channels (VGSCs) play a critical role in the development and modulation of nociceptive signaling associated with the gut. Several VGSC isoforms demonstrate significant promise as potential targets for improved diagnosis and treatment of gut‐based disorders associated with hyper‐ and hyposensitivity to abdominal pain.
Purpose
In this article, we critically review key investigations that have evaluated the potential role that VGSCs play in visceral nociception and discuss recent advances related to this topic. Specifically, we discuss the following: (a) what is known about the structure and basic function of VGSCs, (b) the role that each VGSC plays in gut nociception, particularly as it relates to human physiology, and (c) potential diagnostic and therapeutic uses of VGSCs to manage disorders associated with chronic abdominal pain.
... Resistance to local anesthetic effects has rarely been reported, with only one report of decreased efficacy and duration of topical and intradermal local anesthetic administration in patients with Ehler-Danlos syndrome (it was postulated that this was due to the laxity and density of abnormal connective tissues [4], which is clearly not related to muscular dystrophy). There is also research on specific sodium channel variants that may be linked to resistance to local anesthetics [5]. We acknowledge that there are many points to confirm before declaring that our patient clearly demonstrated resistance to local anesthetic. ...
Emery–Dreifuss muscular dystrophy (EDMD) presenting for bilateral Achilles lengthening and equinoplanovalgus deformity correction. EDMD is a rare X-linked condition that primarily affects skeletal muscle, leading to joint contractures with progressive weakness, and cardiac muscle, leading to malignant arrhythmias and cardiomyopathy. EDMD presents unique challenges to the anesthetist, including arrhythmias (i.e., atrioventricular heart block), cardiomyopathy, possible compromised airway mechanics leading to difficult intubation (i.e., cervical paraspinal contractures and hypoplasia of the third to fifth cervical vertebral bodies that can influence neck flexion and extension), potential for difficult neuraxial access due to paraspinal muscle contractures, and concern for adverse medication reactions (i.e., succinylcholine exaggerated hyperkalemic response or risk of rhabdomyolysis and negative volatile anesthetic reactions). Considering the challenges of anesthetic management in this patient, it seemed reasonable that bilateral sciatic and femoral nerve blockade could avoid airway instrumentation and general anesthesia. Notably, however, the patient commented preoperatively that “...every time I received a local anesthetic injection at the dentist's office, I required multiple injections in order to feel any effect.”
In preclinical rodent models, spinal cord injury (SCI) manifests in gastric vagal afferent dysfunction both acutely and chronically. However, the mechanism that underlies this dysfunction remains unknown. In the current study, we examined the effect of SCI on gastric nodose ganglia (NG) neuron excitability and voltage-gated Na⁺ (NaV) channels expression and function in rats after an acute (i.e. 3-days) and chronic period (i.e. 3-weeks). Rats randomly received either T3-SCI or sham control surgery 3-days or 3-weeks prior to experimentation as well as injections of 3% DiI solution into the stomach to identify gastric NG neurons. Single cell qRT-PCR was performed on acutely dissociated DiI-labeled NG neurons to measure NaV1.7, NaV1.8 and NaV1.9 expression levels. The results indicate that all 3 channel subtypes decreased. Current- and voltage-clamp whole-cell patch-clamp recordings were performed on acutely dissociated DiI-labeled NG neurons to measure active and passive properties of C- and A-fibers as well as the biophysical characteristics of NaV1.8 channels in gastric NG neurons. Acute and chronic SCI did not demonstrate deleterious effects on either passive properties of dissociated gastric NG neurons or biophysical properties of NaV1.8. These findings suggest that although NaV gene expression levels change following SCI, NaV1.8 function is not altered. The disruption throughout the entirety of the vagal afferent neuron has yet to be investigated.
An 18-month-old patient with hereditary sensory and autonomic neuropathy, type VII undergoing general anesthesia for Nissen fundoplication and gastrostomy tube is presented. This is the first reported case of a patient with this particular genetic mutation receiving general anesthesia. We presented the major intraoperative events during the procedure. The anesthetic considerations and implications of caring for a patient with this particular mutation and patients with other variations of hereditary sensory and autonomic neuropathy are also discussed. We show that a patient with de novo hereditary sensory and autonomic neuropathy, type VII without anhidrosis did not require intraoperative narcotics and did not experience bradycardia, asystole, or hemodynamic compromise.
The importance of a neuroinflammatory response to the development and maintenance of inflammatory and neuropathic pain have been highlighted in recent years. Inflammatory cells contributing to this response include circulating immune cells such as monocytes, T and B lymphocytes, and neutrophils, as well as microglia in the central nervous system. Pain signals are transmitted via sensory neurons in the peripheral nervous system, which express various receptors and channels that respond to mediators secreted from these inflammatory cells. Chronobiological rhythms, which include the 24-hr circadian cycle, have recently been shown to regulate both nervous and immune cell activity and function. This review examines the current literature on chronobiological control of neuroinflammatory processes, with a focus on inflammatory and neuropathic pain states. While the majority of this work has stemmed from observational studies in humans, recent advances in using animal models have highlighted distinct mechanisms underlying these interactions. Better understanding interactions between the circadian and neuroimmune systems can help guide the development of new treatments and provide improved care for patients suffering from acute and chronic pain.
Although species-specific differences in ion channel properties are well-documented, little has been known about the properties of the human Nav1.8 channel, an important contributor to pain signaling. Here we show, using techniques that include voltage-clamp, current-clamp and dynamic clamp in dorsal root ganglion (DRG) neurons, that human Nav1.8 channels display slower inactivation kinetics, and produce larger persistent current and ramp current than previously reported in other species. DRG neurons expressing human Nav1.8 channels unexpectedly produce significantly longer-lasting action potentials including action potentials, with half-widths in some cells >10 msec, and increased firing frequency, compared to the narrower and usually single action potentials generated by DRG neurons expressing rat Nav1.8 channels. We also show that native human DRG neurons recapitulate these properties of Nav1.8 current and the long-lasting action potentials. Together, our results demonstrate strikingly distinct properties of human Nav1.8, which contribute to the firing properties of human DRG neurons.
Copyright © 2015, Journal of Neurophysiology.
Objectives
We evaluated the contribution of sensory neurons in ankle joints and adjacent tissue to the development of pain in collagen-induced arthritis (CIA), and the relationship between pain and the appearance of clinical signs.Methods
Mechanical and heat hypersensitivity and hindpaw swelling were assessed in Lewis rats before and until 18 days following collagen immunization. The effect of intrathecal administration of a CGRP antagonist (CGRP8-37; days 11-18) was examined on CIA-induced hypersensitivity. During CIA development CGRP and pERK immunoreactivity was quantified in lumbar dorsal root ganglia (DRG) in which sensory neurons innervating the ankle joint were identified by Fluorogold back-labelling. In the lumbar dorsal horn microgliosis was assessed by immunohistochemistry and activity-evoked CGRP release measured from the dorsal horn-with dorsal roots attached preparation.ResultsCIA was associated with mechanical hypersensitivity, evident before hindpaw swelling, and exacerbated with the development of swelling. Heat hyperalgesia developed alongside swelling. Concomitant with the development of mechanical hypersensitivity, joint innervating neurons exhibited enhanced CGRP expression and an activated phenotype (pERK), and significant microgliosis became evident in the dorsal horn; these peripheral and central changes augmented further with disease progression. CGRP release evoked by dorsal root stimulation was higher in day18-CIA dorsal horn compared to controls. Prolonged intrathecal administration of CGRP8-37 attenuated established mechanical hypersensitivity and reduced spinal microgliosis.Conclusions
Sensory neuron derived CGRP sustains mechanical hypersensitivity and spinal microglial reactivity in CIA suggesting that central mechanisms play critical roles in chronic inflammatory pain. Blockade of these central events may provide pain relief in RA patients. This article is protected by copyright. All rights reserved.
Sodium channel Nav1.9 is expressed in peripheral nociceptive neurons, as well as visceral afferents, and has been shown to act as a threshold channel. Painful peripheral neuropathy represents a significant public health challenge and may involve gain-of-function variants in sodium channels that are preferentially expressed in peripheral sensory neurons. Although gain-of-function variants of peripheral sodium channels Nav1.7 and Nav1.8 have recently been found in painful small fibre neuropathy, the aetiology of peripheral neuropathy in many cases remains unknown. We evaluated 459 patients who were referred for possible painful peripheral neuropathy, and confirmed the diagnosis of small fibre neuropathy in a cohort of 393 patients (369 patients with pure small fibre neuropathy, and small fibre neuropathy together with large fibre involvement in an additional 24 patients). From this cohort of 393 patients with peripheral neuropathy, we sequenced SCN11A in 345 patients without mutations in SCN9A and SCN10A, and found eight variants in 12 patients. Functional profiling by electrophysiological recordings showed that these Nav1.9 mutations confer gain-of-function attributes to the channel, depolarize resting membrane potential of dorsal root ganglion neurons, enhance spontaneous firing, and increase evoked firing of these neurons. Our data show, for the first time, missense mutations of Nav1.9 in individuals with painful peripheral neuropathy. These genetic and functional observations identify missense mutations of Nav1.9 as a cause of painful peripheral neuropathy.
The sensation of pain protects the body from serious injury. Using exome sequencing, we identified a specific de novo missense mutation in SCN11A in individuals with the congenital inability to experience pain who suffer from recurrent tissue damage and severe mutilations. Heterozygous knock-in mice carrying the orthologous mutation showed reduced sensitivity to pain and self-inflicted tissue lesions, recapitulating aspects of the human phenotype. SCN11A encodes Nav1.9, a voltage-gated sodium ion channel that is primarily expressed in nociceptors, which function as key relay stations for the electrical transmission of pain signals from the periphery to the central nervous system. Mutant Nav1.9 channels displayed excessive activity at resting voltages, causing sustained depolarization of nociceptors, impaired generation of action potentials and aberrant synaptic transmission. The gain-of-function mechanism that underlies this channelopathy suggests an alternative way to modulate pain perception.
Painful peripheral neuropathy often occurs without apparent underlying cause. Gain-of-function variants of sodium channel Na(v)1.7 have recently been found in ∼30% of cases of idiopathic painful small-fiber neuropathy. Here, we describe mutations in Na(v)1.8, another sodium channel that is specifically expressed in dorsal root ganglion (DRG) neurons and peripheral nerve axons, in patients with painful neuropathy. Seven Na(v)1.8 mutations were identified in 9 subjects within a series of 104 patients with painful predominantly small-fiber neuropathy. Three mutations met criteria for potential pathogenicity based on predictive algorithms and were assessed by voltage and current clamp. Functional profiling showed that two of these three Na(v)1.8 mutations enhance the channel's response to depolarization and produce hyperexcitability in DRG neurons. These observations suggest that mutations of Na(v)1.8 contribute to painful peripheral neuropathy.
Neuropathic pain is a chronic condition that is often refractory to treatment with available therapies and thus an unmet medical need. We have previously shown that the voltage-gated sodium channel Na(v)1.3 is upregulated in peripheral and central nervous system (CNS) of rats following nerve injury, and that it contributes to nociceptive neuron hyperexcitability in neuropathic conditions. To evaluate the therapeutic potential of peripheral Na(v)1.3 knockdown at a specific segmental level, we constructed adeno-associated viral (AAV) vector expressing small hairpin RNA against rat Na(v)1.3 and injected it into lumbar dorsal root ganglion (DRG) of rats with spared nerve injury (SNI). Our data show that direct DRG injection provides a model that can be used for proof-of-principle studies in chronic pain with respect to peripheral delivery route of gene transfer constructs, high transduction efficiency, flexibility in terms of segmental localization, and limited behavioral effects of the surgical procedure. We show that knockdown of Na(v)1.3 in lumbar 4 (L4) DRG results in an attenuation of nerve injury-induced mechanical allodynia in the SNI model. Taken together, our studies support the contribution of peripheral Na(v)1.3 to pain in adult rats with neuropathic pain, validate Na(v)1.3 as a target, and provide validation for this approach of AAV-mediated peripheral gene therapy.
Although microglia have been implicated in nerve injury-induced neuropathic pain, the manner by which injured sensory neurons engage microglia remains unclear. We found that peripheral nerve injury induced de novo expression of colony-stimulating factor 1 (CSF1) in injured sensory neurons. CSF1 was transported to the spinal cord, where it targeted the microglial CSF1 receptor (CSF1R). Cre-mediated sensory neuron deletion of Csf1 completely prevented nerve injury-induced mechanical hypersensitivity and reduced microglial activation and proliferation. In contrast, intrathecal injection of CSF1 induced mechanical hypersensitivity and microglial proliferation. Nerve injury also upregulated CSF1 in motoneurons, where it was required for ventral horn microglial activation and proliferation. Downstream of CSF1R, we found that the microglial membrane adaptor protein DAP12 was required for both nerve injury- and intrathecal CSF1-induced upregulation of pain-related microglial genes and the ensuing pain, but not for microglial proliferation. Thus, both CSF1 and DAP12 are potential targets for the pharmacotherapy of neuropathic pain.
Chronic pain, both inflammatory and neuropathic, is a debilitating condition in which the pain experience persists after the painful stimulus has resolved. The efficacy of current treatment strategies using opioids, NSAIDS and anticonvulsants is limited by the extensive side effects observed in patients, underlining the necessity for novel therapeutic targets. Preclinical models of chronic pain have recently provided evidence for a critical role played by glial cells in the mechanisms underlying the chronicity of pain, both at the site of damage in the periphery and in the dorsal horn of the spinal cord. Here microglia and astrocytes respond to the increased input from the periphery and change morphology, increase in number and release pro-nociceptive mediators such as ATP, cytokines and chemokines. These gliotransmitters can sensitise neurons by activation of their cognate receptors thereby contributing to central sensitization which is fundamental for the generation of allodynia, hyperalgesia and spontaneous pain.
The discovery of genetic variants that substantially alter an individual's perception of pain has led to a step-change in our understanding of molecular events underlying the detection and transmission of noxious stimuli by the peripheral nervous system. For example, the voltage-gated sodium ion channel Nav1.7 is expressed selectively in sensory and autonomic neurons; inactivating mutations in SCN9A, which encodes Nav1.7, result in congenital insensitivity to pain, whereas gain-of-function mutations in this gene produce distinct pain syndromes such as inherited erythromelalgia, paroxysmal extreme pain disorder, and small-fibre neuropathy. Heterozygous mutations in TRPA1, which encodes the transient receptor potential cation channel, can cause familial episodic pain syndromes, and variants of genes coding for the voltage-gated sodium channels Nav1.8 (SCN10A) and Nav1.9 (SCN11A) lead to small-fibre neuropathy and congenital insensitivity to pain, respectively. Furthermore, other genetic polymorphisms have been identified that contribute to risk or severity of more complex pain phenotypes. Novel models of sensory disorders are in development-eg, using human sensory neurons differentiated from human induced pluripotent stem cells. Understanding rare heritable pain disorders not only improves diagnosis and treatment of patients but may also reveal new targets for analgesic drug development.
Many ion channel genes have been associated with human genetic pain disorders. Here we report two large Chinese families with autosomal-dominant episodic pain. We performed a genome-wide linkage scan with microsatellite markers after excluding mutations in three known genes (SCN9A, SCN10A, and TRPA1) that cause similar pain syndrome to our findings, and we mapped the genetic locus to a 7.81 Mb region on chromosome 3p22.3-p21.32. By using whole-exome sequencing followed by conventional Sanger sequencing, we identified two missense mutations in the gene encoding voltage-gated sodium channel Nav1.9 (SCN11A): c.673C>T (p.Arg225Cys) and c.2423C>G (p.Ala808Gly) (one in each family). Each mutation showed a perfect cosegregation with the pain phenotype in the corresponding family, and neither of them was detected in 1,021 normal individuals. Both missense mutations were predicted to change a highly conserved amino acid residue of the human Nav1.9 channel. We expressed the two SCN11A mutants in mouse dorsal root ganglion (DRG) neurons and showed that both mutations enhanced the channel's electrical activities and induced hyperexcitablity of DRG neurons. Taken together, our results suggest that gain-of-function mutations in SCN11A can be causative of an autosomal-dominant episodic pain disorder.
The voltage-gated sodium channel Na(V)1.7 is preferentially expressed in peripheral somatic and visceral sensory neurons, olfactory sensory neurons and sympathetic ganglion neurons. Na(V)1.7 accumulates at nerve fibre endings and amplifies small subthreshold depolarizations, poising it to act as a threshold channel that regulates excitability. Genetic and functional studies have added to the evidence that Na(V)1.7 is a major contributor to pain signalling in humans, and homology modelling based on crystal structures of ion channels suggests an atomic-level structural basis for the altered gating of mutant Na(V)1.7 that causes pain.
The induction of rheumatoid arthritis (RA) by active and passive immunization of mice results in the development of pain at the same time as the swelling and inflammation, with both peripheral and central sensitization contributing to joint pain. The purpose of this study was to examine the development of pain in the rat model of collagen-induced arthritis (CIA) and to evaluate the contribution of neuroimmune interactions to established arthritis pain.
Mechanical hypersensitivity was assessed in female Lewis rats before and up to 18 days after induction of CIA by immunization with type II collagen. The effect of selective inhibitors of microglia were then evaluated by prolonged intrathecal delivery of a cathepsin S (CatS) inhibitor and a fractalkine (FKN) neutralizing antibody, from day 11 to day 18 following immunization.
Rats with CIA developed significant mechanical hypersensitivity, which started on day 9, before the onset of clinical signs of arthritis. Mechanical hypersensitivity peaked with the severity of the disease, when significant microglial and astrocytic responses, alongside T cell infiltration, were observed in the spinal cord. Intrathecal delivery of microglial inhibitors, a CatS inhibitor, or an FKN neutralizing antibody attenuated mechanical hypersensitivity and spinal microglial response in rats with CIA.
The inhibition of microglial targets by centrally penetrant CatS inhibitors and CX(3) CR1 receptor antagonists represents a potential therapeutic avenue for the treatment of pain in RA.
Small nerve fiber neuropathy (SFN) often occurs without apparent cause, but no systematic genetic studies have been performed in patients with idiopathic SFN (I-SFN). We sought to identify a genetic basis for I-SFN by screening patients with biopsy-confirmed idiopathic SFN for mutations in the SCN9A gene, encoding voltage-gated sodium channel Na(V)1.7, which is preferentially expressed in small diameter peripheral axons.
Patients referred with possible I-SFN, who met the criteria of ≥2 SFN-related symptoms, normal strength, tendon reflexes, vibration sense, and nerve conduction studies, and reduced intraepidermal nerve fiber density (IENFD) plus abnormal quantitative sensory testing (QST) and no underlying etiology for SFN, were assessed clinically and by screening of SCN9A for mutations and functional analyses.
Twenty-eight patients who met stringent criteria for I-SFN including abnormal IENFD and QST underwent SCN9A gene analyses. Of these 28 patients with biopsy-confirmed I-SFN, 8 were found to carry novel mutations in SCN9A. Functional analysis revealed multiple gain of function changes in the mutant channels; each of the mutations rendered dorsal root ganglion neurons hyperexcitable.
We show for the first time that gain of function mutations in sodium channel Na(V)1.7, which render dorsal root ganglion neurons hyperexcitable, are present in a substantial proportion (28.6%; 8 of 28) of patients meeting strict criteria for I-SFN. These results point to a broader role of Na(V)1.7 mutations in neurological disease than previously considered from studies on rare genetic syndromes, and suggest an etiological basis for I-SFN, whereby expression of gain of function mutant sodium channels in small diameter peripheral axons may cause these fibers to degenerate.
Microglia respond rapidly to injury, increasing their synthesis and release of inflammatory mediators, many of which contribute to the maintenance of persistent pain following CNS or PNS injury. We have recently shown that the lysosomal cysteine protease Cathepsin S (CatS) expressed by spinal microglia is vital for the full expression of neuropathic pain. Here we evaluated the mechanisms by which CatS release occurs from primary microglia in culture. Stimulation of microglia with lipopolysaccharide (LPS) or adenosine tri-phosphate (ATP) alone was insufficient to induce release of enzymatically active CatS in extracellular media. However, following priming with LPS, ATP at 1 mM but not 50 μM resulted in significant release of CatS in the media and maturation of CatS protein in cell extracts. The enzymatic activity measured in media at neutral pH was specific for CatS as it was completely prevented by the CatS inhibitor LHVS. ATP-induced release of CatS required potassium efflux and both extracellular calcium influx and mobilization of intracellular calcium. Pharmacological modulation of ATP-induced release of CatS enzymatic activity revealed that this was dependent on activation of the P2X7 receptor and intracellular phospholipase C and phospholipase A(2). In addition, ATP-induced CatS release involved p38 mitogen activated protein kinase (MAPK) phosphorylation, but not ERK and PI3K signalling pathways. Thus, as high concentration of extracellular ATP promotes release of active CatS from microglia via P2X7 receptor activation, we suggest that the inhibition of CatS release is one of the mechanisms responsible for P2X7 antagonist efficacy in neuropathic pain.
Nociception is essential for survival whereas pathological pain is maladaptive and often unresponsive to pharmacotherapy. Voltage-gated sodium channels, Na(v)1.1-Na(v)1.9, are essential for generation and conduction of electrical impulses in excitable cells. Human and animal studies have identified several channels as pivotal for signal transmission along the pain axis, including Na(v)1.3, Na(v)1.7, Na(v)1.8, and Na(v)1.9, with the latter three preferentially expressed in peripheral sensory neurons and Na(v)1.3 being upregulated along pain-signaling pathways after nervous system injuries. Na(v)1.7 is of special interest because it has been linked to a spectrum of inherited human pain disorders. Here we review the contribution of these sodium channel isoforms to pain.
The nervous system detects and interprets a wide range of thermal and mechanical stimuli, as well as environmental and endogenous chemical irritants. When intense, these stimuli generate acute pain, and in the setting of persistent injury, both peripheral and central nervous system components of the pain transmission pathway exhibit tremendous plasticity, enhancing pain signals and producing hypersensitivity. When plasticity facilitates protective reflexes, it can be beneficial, but when the changes persist, a chronic pain condition may result. Genetic, electrophysiological, and pharmacological studies are elucidating the molecular mechanisms that underlie detection, coding, and modulation of noxious stimuli that generate pain.
Two tetrodotoxin-resistant (TTX-R) voltage-gated sodium channels, SNS and NaN, are preferentially expressed in small dorsal root ganglia (DRG) and trigeminal ganglia neurons, most of which are nociceptive, of rat and mouse. We report here the sequence of NaN from human DRG, and demonstrate the presence of two TTX-R currents in human DRG neurons. One current has physiological properties similar to those reported for SNS, while the other displays hyperpolarized voltage-dependence and persistent kinetics; a similar TTX-R current was recently identified in DRG neurons of sns-null mouse. Thus SNS and NaN channels appear to produce different currents in human DRG neurons.
In the CNS, immune-like competent cells (microglia and astrocytes) were first described as potential sites of chemokine synthesis, but more recent evidence has indicated that neurones might also express chemokines and their receptors. The aim of the present work was to investigate further, both in vivo and in vitro, CC Chemokine Family Receptor 2 (CCR2) expression and functionality in rat spinal cord neurones. First, we demonstrated by RT-PCR and western blot analysis that CCR2 mRNA and protein were present in spinal extracts. Furthermore, we showed by immunolabelling that CCR2 was exclusively expressed by neurones in spinal sections of healthy rat. Finally, to test the functionality of CCR2, we used primary cultures of rat spinal neurones. In this model, similar to what was observed in vivo, CCR2 mRNA and protein were expressed by neurones. Cultured neurones stimulated with Monocyte Chemoattractant Protein-1 (MCP-1)/CCL2, the best characterized CCR2 agonist, showed activation of the Akt pathway. Finally, patch-clamp recording of cultured spinal neurones was used to investigate whether MCP-1/CCL2 could modulate their electrophysiological properties. MCP-1 alone did not affect the electrical properties of spinal neurones, but potently and efficiently inhibited GABA(A)-mediated GABAergic responses in these neurones. These data constitute the first demonstration of a modulatory role of MCP-1 on GABAergic neurotransmission and contribute to our understanding of the roles of CCR2 and MCP-1/CCL2 in spinal cord physiology, in particular with respect to nociceptive transmission, as well as the implication of this chemokine in neuronal adaptation or dysfunction during neuropathy.
Nociceptive dorsal root ganglion (DRG) neurons can be classified into nonpeptidergic IB(4)(+) and peptidergic IB(4)(-) subtypes, which terminate in different layers in dorsal horn and transmit pain along different ascending pathways, and display different firing properties. Voltage-gated, tetrodotoxin-resistant (TTX-R) Na(v)1.8 channels are expressed in both IB(4)(+) and IB(4)(-) cells and produce most of the current underlying the depolarizing phase of action potential (AP). Slow inactivation of TTX-R channels has been shown to regulate repetitive DRG neuron firing behavior. We show in this study that use-dependent reduction of Na(v)1.8 current in IB(4)(+) neurons is significantly stronger than that in IB(4)(-) neurons, although voltage dependency of activation and steady-state inactivation are not different. The time constant for entry of Na(v)1.8 into slow inactivation in IB(4)(+) neurons is significantly faster and more Na(v)1.8 enter the slow inactivation state than in IB(4)(-) neurons. In addition, recovery from slow inactivation of Na(v)1.8 in IB(4)(+) neurons is slower than that in IB(4)(-) neurons. Using current-clamp recording, we demonstrate a significantly higher current threshold for generation of APs and a longer latency to onset of firing in IB(4)(+), compared with those of IB(4)(-) neurons. In response to a ramp stimulus, IB(4)(+) neurons produce fewer APs and display stronger adaptation, with a faster decline of AP peak than IB(4)(-) neurons. Our data suggest that differential use-dependent reduction of Na(v)1.8 current in these two DRG subpopulations, which results from their different rate of entry into and recovery from the slow inactivation state, contributes to functional differences between these two neuronal populations.
Dorsal root ganglion neurons express an array of sodium channel isoforms allowing precise control of excitability. An increasing body of literature indicates that regulation of firing behaviour in these cells is linked to their patterns of expression of specific sodium channel isoforms, which have been discovered to possess distinct biophysical characteristics. The pattern of expression of sodium channels differs in different subclasses of DRG neurons and is not fixed but, on the contrary, changes in response to a variety of disease insults. Moreover, modulation of channels by their environment has been found to play an important role in the response of these neurons to stimuli. In this review we illustrate how excitability can be finely tuned to provide contrasting firing templates in different subclasses of DRG neurons by selective deployment of various sodium channel isoforms, by plasticity of expression of these proteins, and by interactions of these sodium channel isoforms with each other and with other modulatory molecules.
In order to deal effectively with danger, it is imperative to know about it. This is what nociceptors do--these primary sensory neurons are specialized to detect intense stimuli and represent, therefore, the first line of defense against any potentially threatening or damaging environmental inputs. By sensing noxious stimuli and contributing to the necessary reactions to avoid them--rapid withdrawal and the experience of an intensely unpleasant or painful sensation, nociceptors are essential for the maintenance of the body's integrity. Although nociceptive pain is clearly an adaptive alarm system, persistent pain is maladaptive, essentially an ongoing false alarm. Here, we highlight the genesis of nociceptors during development and the intrinsic properties of nociceptors that enable them to transduce, conduct, and transmit nociceptive information and also discuss how their phenotypic plasticity contributes to clinical pain.