NAV1 Research Articles

Tools for analysis and conditional deletion of subsets of sensory neurons

Background: Somatosensation depends on primary sensory neurons of the trigeminal and dorsal root ganglia (DRG). Transcriptional profiling of mouse DRG sensory neurons has defined at least 18 distinct neuronal cell types. Using an advillin promoter, we have generated a transgenic mouse line that only expresses diphtheria toxin A (DTA) in sensory neurons in the presence of Cre recombinase. This has allowed us to ablate specific neuronal subsets within the DRG using a range of established and novel Cre lines that encompass all sets of sensory neurons. Methods: A floxed-tdTomato-stop-DTA bacterial artificial chromosome (BAC) transgenic reporter line (AdvDTA) under the control of the mouse advillin DRG promoter was generated. The line was first validated using a Na v1.8 Cre and then crossed to CGRP CreER (Calca), Th CreERT2, Tmem45b Cre, Tmem233 Cre, Ntng1 Cre and TrkB CreER (Ntrk2) lines. Pain behavioural assays included Hargreaves’, hot plate, Randall-Selitto, cold plantar, partial sciatic nerve ligation and formalin tests. Results: Motor activity, as assessed by the rotarod test, was normal for all lines tested. Noxious mechanosensation was significantly reduced when either Na v1.8 positive neurons or Tmem45b positive neurons were ablated whilst acute heat pain was unaffected. In contrast, noxious mechanosensation was normal following ablation of CGRP-positive neurons but acute heat pain thresholds were significantly elevated and a reduction in nocifensive responses was observed in the second phase of the formalin test. Ablation of TrkB-positive neurons led to significant deficits in mechanical hypersensitivity in the partial sciatic nerve ligation neuropathic pain model. Conclusions: Ablation of specific DRG neuronal subsets using the AdvDTA line will be a useful resource for further functional characterization of somatosensory processing, neuro-immune interactions and chronic pain disorders.

Tools for analysis and conditional deletion of subsets of sensory neurons.

Background: Somatosensation depends on primary sensory neurons of the trigeminal and dorsal root ganglia (DRG). Transcriptional profiling of mouse DRG sensory neurons has defined at least 18 distinct neuronal cell types. Using an advillin promoter, we have generated a transgenic mouse line that only expresses diphtheria toxin A (DTA) in sensory neurons in the presence of Cre recombinase. This has allowed us to ablate specific neuronal subsets within the DRG using a range of established and novel Cre lines that encompass all sets of sensory neurons. Methods: A floxed-tdTomato-stop-DTA bacterial artificial chromosome (BAC) transgenic reporter line (AdvDTA) under the control of the mouse advillin DRG promoter was generated. The line was first validated using a Na v1.8 Cre and then crossed to CGRP CreER (Calca), Th CreERT2, Tmem45b Cre, Tmem233 Cre, Ntng1 Cre and TrkB CreER (Ntrk2) lines. Pain behavioural assays included Hargreaves', hot plate, Randall-Selitto, cold plantar, partial sciatic nerve ligation and formalin tests. Results: Motor activity, as assessed by the rotarod test, was normal for all lines tested. Noxious mechanosensation was significantly reduced when either Na v1.8 positive neurons or Tmem45b positive neurons were ablated whilst acute heat pain was unaffected. In contrast, noxious mechanosensation was normal following ablation of CGRP-positive neurons but acute heat pain thresholds were significantly elevated and a reduction in nocifensive responses was observed in the second phase of the formalin test. Ablation of TrkB-positive neurons led to significant deficits in mechanical hypersensitivity in the partial sciatic nerve ligation neuropathic pain model. Conclusions: Ablation of specific DRG neuronal subsets using the AdvDTA line will be a useful resource for further functional characterization of somatosensory processing, neuro-immune interactions and chronic pain disorders.

S11.1 - Navigating through discovery and development painlessly: Snapshots within the NAV1.7 Program

S11.1 - Navigating through discovery and development painlessly: Snapshots within the NAV1.7 Program

Mechanism of dexmedetomidine-induced reduction of acute visceral pain in rats: relationship between alpha2 adrenergic receptors and expression of Nav1.8 in dorsal root ganglion neurons

Objective To evaluate the relationship between alpha2 adrenergic receptors and expression of Nav1.8 in dorsal root ganglion(DRG)neurons, and to illuminate the mechanism of dexmedetomidine-induced reduction of acute visceral pain in rats. Methods Thirty-two clean-grade healthy adult male Sprague-Dawley rats, aged 6-8 weeks, weighing 180-220 g, were divided into 4 groups (n=8 each) using a random number table method: control group (group C), acute visceral pain group (group VP), dexmedetomidine group (group D), and dexmedetomidine plus atipamezole group (group DA). In VP, D and DA groups, 10-3 mmol/L capsaicin 1.3 ml was injected into the rectum at a dose of 10-3 mmol/L to establish the acute visceral pain model, while the equal volume of normal saline was given instead in group C. Atipamezole 1 mg/kg was subcutaneously injected through the back of the neck at 20 min before establishing the model in group DA.Dexmedetomidine 10 μg/kg was injected through the tail vein at 15 min before establishing the model in D and DA groups, while the equal volume of normal saline was given instead at the correspording time points in C and VP groups.The visceral pain behavior score was recorded at 1 h after establishing the model.The animals were then sacrificed, and DRGs of the lumbar segment (L3-6) were removed for determination of the number of Nav1.8 positive DRG neurons (by immunohistochemistry) and expression of Nav1.8 mRNA (by quantitative real-time polymerase chain reaction). Results Compared with group C, the visceral behavior scores and the number of Nav1.8 positive DRG neurons were significantly increased, and the expression of Nav1.8 mRNA was up-regulated in VP, D and DA groups (P<0.05). Compared with group VP, the visceral behavior score and the number of Nav1.8 positive DRG neurons were significantly decreased, and the expression of Nav1.8 mRNA was down-regulated in D and DA groups (P<0.05). Compared with group D, the visceral behavior scores and the number of Nav1.8 positive DRG neurons were significantly increased, and the expression of Nav1.8 mRNA was up-regulated in group DA (P<0.05). Conclusion The mechanism by which dexmedetomidine reduces acute visceral pain is related to activating alpha2 adrenergic receptors and to down-regulating the expression of Nav1.8 in DRG neurons of rats. Key words: Dexmedetomidine; Visceral pain; Receptors, adrenergic, alpha-2; Ganglia, spinal; NAV1.8 voltage-gated sodium channel

Possible mechanism of bursting suppression in nociceptive neurons.

The use of the mathematical model of rat nociceptive neuron membrane allowed us to predict a new mechanism of suppression of ectopic bursting discharges, which arise in neurons of dorsal root ganglia and are one of the causes of neuropathic pain. The treatment with comenic acid leads to switching off the ectopic bursting discharges due to a decrease in the effective charge transferring via the activation gating structure of the slow sodium channels (Na V1.8a). Comenic acid is a drug substance of a new non-opioid analgesic [1] Thus, this analgesic not only reduces the frequency of rhythmic discharges of nociceptive neuron membrane [2] but also it suppresses its ectopic bursting discharges.

Evaluation of recombinant monoclonal antibody SVmab1 binding to Na V1.7 target sequences and block of human Na V1.7 currents

Identification of small and large molecule pain therapeutics that target the genetically validated voltage-gated sodium channel Na V1.7 is a challenging endeavor under vigorous pursuit. The monoclonal antibody SVmab1 was recently published to bind the Na V1.7 DII voltage sensor domain and block human Na V1.7 sodium currents in heterologous cells. We produced purified SVmab1 protein based on publically available sequence information, and evaluated its activity in a battery of binding and functional assays. Herein, we report that our recombinant SVmAb1 does not bind peptide immunogen or purified Na V1.7 DII voltage sensor domain via ELISA, and does not bind Na V1.7 in live HEK293, U-2 OS, and CHO-K1 cells via FACS. Whole cell manual patch clamp electrophysiology protocols interrogating diverse Na V1.7 gating states in HEK293 cells, revealed that recombinant SVmab1 does not block Na V1.7 currents to an extent greater than observed with an isotype matched control antibody. Collectively, our results show that recombinant SVmab1 monoclonal antibody does not bind Na V1.7 target sequences or specifically inhibit Na V1.7 current.

Immunotherapy Branches Out: A Novel Approach to Itch and Pain Control

Sodium channels are central mediators of action potentials and, therefore, critical to neural and cardiac function. The NAV1.7 sodium channel is important in nociceptive neurons that sense pain — humans missing function of this channel are insensitive to pain and have anosmia; those with hyperactive mutant channels …

The Voltage-Gated Sodium Channel Nav1.8 Is Expressed in Human Sperm

The role of Na+ fluxes through voltage-gated sodium channels in the regulation of sperm cell function remains poorly understood. Previously, we reported that several genes encoding voltage-gated Na+ channels were expressed in human testis and mature spermatozoa. In this study, we analyzed the presence and function of the TTX-resistant VGSC α subunit Nav1.8 in human capacitated sperm cells. Using an RT-PCR assay, we found that the mRNA of the gene SCN10A, that encode Na v1.8, was abundantly and specifically expressed in human testis and ejaculated spermatozoa. The Na v1.8 protein was detected in capacitated sperm cells using three different specific antibodies against this channel. Positive immunoreactivity was mainly located in the neck and the principal piece of the flagellum. The presence of Na v1.8 in sperm cells was confirmed by Western blot. Functional studies demonstrated that the increases in progressive motility produced by veratridine, a voltage-gated sodium channel activator, were reduced in sperm cells preincubated with TTX (10 μM), the Na v1.8 antagonist A-803467, or a specific Na v1.8 antibody. Veratridine elicited similar percentage increases in progressive motility in sperm cells maintained in Ca2+-containing or Ca2+-free solution and did not induce hyperactivation or the acrosome reaction. Veratridine caused a rise in sperm intracellular Na+, [Na+]i, and the sustained phase of the response was inhibited in the presence of A-803467. These results verify that the Na+ channel Na v1.8 is present in human sperm cells and demonstrate that this channel participates in the regulation of sperm function.

Electrophysiological properties of mouse and epitope-tagged human cardiac sodium channel Na v1.5 expressed in HEK293 cells

Background: The pore-forming subunit of the cardiac sodium channel, Na v1.5, has been previously found to be mutated in genetically determined arrhythmias. Na v1.5 associates with many proteins that regulate its function and cellular localisation. In order to identify more in situ Na v1.5 interacting proteins, genetically-modified mice with a high-affinity epitope in the sequence of Na v1.5 can be generated. Methods: In this short study, we (1) compared the biophysical properties of the sodium current (I Na) generated by the mouse Na v1.5 (mNa v1.5) and human Na v1.5 (hNa v1.5) constructs that were expressed in HEK293 cells, and (2) investigated the possible alterations of the biophysical properties of the human Na v1.5 construct that was modified with specific epitopes. Results: The biophysical properties of mNa v1.5 were similar to the human homolog. Addition of epitopes either up-stream of the N-terminus of hNa v1.5 or in the extracellular loop between the S5 and S6 transmembrane segments of domain 1, significantly decreased the amount of I Na and slightly altered its biophysical properties. Adding green fluorescent protein (GFP) to the N-terminus did not modify any of the measured biophysical properties of hNa v1.5. Conclusions: These findings have to be taken into account when planning to generate genetically-modified mouse models that harbour specific epitopes in the gene encoding mNa v1.5.

Treatment of Na v1.7-mediated pain in inherited erythromelalgia using a novel sodium channel blocker

Mutations in the SCN9A gene leading to deficiency of its protein product, Na v1.7, cause congenital indifference to pain (CIP). CIP is characterized by the absence of the ability to sense pain associated with noxious stimuli. In contrast, the opposite phenotype to CIP, inherited erythromelalgia (IEM), is a disorder of spontaneous pain caused by missense mutations resulting in gain-of-function in Na v1.7 that promote neuronal hyperexcitability. The primary aim of this study was to demonstrate that Na v1.7 antagonism could alleviate the pain of IEM, thereby demonstrating the utility of this opposite phenotype model as a tool for rapid proof-of-concept for novel analgesics. An exploratory, randomized, double-blind, 2-period crossover study was conducted in 4 SCN9A mutation-proven IEM patients. In each treatment period (2 days), separated by a 2-day washout period, patients were orally administered XEN402 (400 mg twice daily) or matching placebo. In 3 patients, pain was induced by heat or exercise during each treatment arm. A fourth patient, in constant severe pain, required no induction. Patient-reported outcomes of pain intensity and/or relief were recorded, and the time taken to induce pain was measured. The ability to induce pain in IEM patients was significantly attenuated by XEN402 compared with placebo. XEN402 increased the time to maximal pain induction and significantly reduced the amount of pain (42% less) after induction ( P = .014). This pilot study showed that XEN402 blocks Na v1.7-mediated pain associated with IEM, thereby demonstrating target engagement in humans and underscoring the use of rare genetic disorders with mutant target channels as a novel approach to rapid proof-of-concept.

Coexpression with auxiliary β subunits modulates the action of tefluthrin on rat Na v1.6 and Na v1.3 sodium channels

We expressed the rat Na v1.3 and Na v1.6 sodium channel α subunit isoforms in Xenopus oocytes either alone or with the rat β1 and β2 auxiliary subunits in various combinations and assessed the sensitivity of the expressed channels to resting and use-dependent modification by the pyrethroid insecticide tefluthrin using the two-electrode voltage clamp technique. Coexpression with the β1 and β2 subunits, either individually or in combination, did not affecting the resting sensitivity of Na v1.6 channels to tefluthrin. Modification by tefluthrin of Na v1.6 channels in the absence of β subunits was not altered by the application of trains of high-frequency depolarizing prepulses. By contrast, coexpression of the Na v1.6 channel with the β1 subunit enhanced the extent of channel modification 2-fold following repeated depolarization. Coexpression of Na v1.6 with the β2 subunit also slightly enhanced modification following repeated depolarization, but coexpression of Na v1.6 with both β subunits caused enhanced modification following repeated depolarization that was indistinguishable from that found with Na v1.6 + β1 channels. In contrast to Na v1.6, the resting modification by tefluthrin of Na v1.3 channels expressed in the absence of β subunits was reduced by repeated depolarization. However, tefluthrin modification of the Na v1.3 α subunit expressed with both β subunits was enhanced 1.7-fold by repeated depolarization, thereby confirming that β subunit modulation of use-dependent effects was not confined to the Na v1.6 isoform. These results show that the actions of pyrethroids on mammalian sodium channels in the Xenopus oocyte expression system are determined in part by the interactions of the sodium channel α subunit with the auxiliary β subunits that are part of the heteromultimeric sodium channel complexes found in neurons and other excitable cells.

Utility of frozen cell lines in medium throughput electrophysiology screening of hERG and Na V1.5 blockade

Introduction The development of drug candidates must take into account that many compounds have off-target activity against voltage-gated ion channels (VGIC) which may prevent their progression to market. Of particular concern are hERG and hNa V1.5. Screening against these ion channels is necessary but expensive, partially due to maintenance of constantly cultured cell lines. Here, we show that frozen HEK-293 cells can be maintained indefinitely, reducing variability in cell performance, time and expense of cell culture. Methods Cells, constantly cultured or frozen, were assayed on the PatchXpress 7000A using tool compounds. Results Amitriptyline, quinidine, compound A, fluoxetine and imipramine inhibited hERG with IC 50s (paired values denote constantly cultured and frozen, respectively) of 4.8 ± 0.4 and 5.1 ± 0.4, 1.4 ± 0.1 and 1.1 ± 0.1, 24.4 ± 2.4 and 21.9 ± 1.8, 2.1 ± 0.4 and 2.1 ± 0.1, 5.2 ± 0.4 and 4.0 ± 0.2 μM. Quinidine, flecainide, mexiletine and amitriptyline inhibited hNa V1.5 with IC 50s of 46.6 ± 4.3 and 28.0 ± 2.3, 7.6 ± 0.7 and 6.2 ± 0.5, 153.5 ± 13.0 and 106.0 ± 4.7, 5.5 ± 0.5 and 4.8 ± 0.2 μM. Voltage dependences of activation (V 1/2) for hERG were statistically identical, 0.4 ± 0.8 mV and 2.5 ± 0.5 mV. In hNa V1.5, the V 1/2 of inactivation and activation were statistically identical, −82.7 ± 0.1 mV versus − 84.9 ± 0.3 mV, −47.5 ± 0.3 mV versus − 45.0 ± 0.6 mV. Current density in both conditions in hERG experiments was similar, 47.0 ± 4.1 pA versus 42.3 ± 6.0 pA/pF. Discussion hERG and hNa V1.5 screens run using frozen cells have statistically identical IC 50s, voltage dependence of activation, IV relationships and current density to screens using continuously cultured cells. Frozen cells have more constant performance and allow rapid switching between experiments on several cell lines without sacrificing data quality.

Impairment of Ubiquitin–Proteasome System by E334K cMyBPC Modifies Channel Proteins, Leading to Electrophysiological Dysfunction

Cardiac arrhythmogenesis is regulated by channel proteins whose protein levels are in turn regulated by the ubiquitin–proteasome system (UPS). We have previously reported on UPS impairment induced by E334K cardiac myosin-binding protein C (cMyBPC), which causes hypertrophic cardiomyopathy (HCM) accompanied by arrhythmia. We hypothesized that UPS impairment induced by E334K cMyBPC causes accumulation of cardiac channel proteins, leading to electrophysiological dysfunction. Wild-type or E334K cMyBPC was overexpressed in HL-1 cells and primary cultured neonatal rat cardiac myocytes. The expression of E334K cMyBPC suppressed cellular proteasome activities. The protein levels of K v1.5, Na v1.5, Hcn4, Ca v3.2, Ca v1.2, Serca, RyR2, and Ncx1 were significantly higher in cells expressing E334K cMyBPC than in wild type. They further increased in cells pretreated with MG132 and had longer protein decays. The channel proteins retained the correct localization. Cells expressing E334K cMyBPC exhibited higher Ca 2+ transients and longer action potential durations (APDs), accompanied by afterdepolarizations and higher apoptosis. Those augments of APD and Ca 2+ transients were recapitulated by a simulation model. Although a Ca 2+ antagonist, azelnidipine, neither protected E334K cMyBPC from degradation nor affected E334K cMyBPC incorporation into the sarcomere, it normalized the APD and Ca 2+ transients and partially reversed the levels of those proteins regulating apoptosis, thereby attenuating apoptosis. In conclusion, UPS impairment caused by E334K cMyBPC may modify the levels of channel proteins, leading to electrophysiological dysfunction. Therefore, UPS impairment due to a mutant cMyBPC may partly contribute to the observed clinical arrhythmias in HCM patients.

Actions of tefluthrin on rat Na v1.7 voltage-gated sodium channels expressed in Xenopus oocytes

In rats expression of the Na v1.7 voltage-gated sodium channel isoform is restricted to the peripheral nervous system and is abundant in the sensory neurons of the dorsal root ganglion. We expressed the rat Na v1.7 sodium channel α subunit together with the rat auxiliary β1 and β2 subunits in Xenopus laevis oocytes and assessed the effects of the pyrethroid insecticide tefluthrin on the expressed currents using the two-electrode voltage clamp method. Tefluthrin at 100 μM modified of Na v1.7 channels to prolong inactivation of the peak current during a depolarizing pulse, resulting in a marked “late current” at the end of a 40 ms depolarization, and induced a sodium tail current following repolarization. Tefluthrin modification was enhanced up to twofold by the application of a train of up to 100 5 ms depolarizing prepulses. These effects of tefluthrin on Na v1.7 channels were qualitatively similar to its effects on rat Na v1.2, Na v1.3 and Na v1.6 channels assayed previously under identical conditions. However, Na v1.7 sodium channels were distinguished by their low sensitivity to modification by tefluthrin, especially compared to Na v1.3 and Na v1.6 channels. It is likely that Na v1.7 channels contribute significantly to the tetrodotoxin-sensitive, pyrethroid-resistant current found in cultured dorsal root ganglion neurons. We aligned the complete amino acid sequences of four pyrethroid-sensitive isoforms (house fly Vssc1; rat Na v1.3, Na v1.6 and Na v1.8) and two pyrethroid-resistant isoforms (rat Na v1.2 and Na v1.7) and found only a single site, located in transmembrane segment 6 of homology domain I, at which the amino acid sequence was conserved among all four sensitive isoform sequences but differed in the two resistant isoform sequences. This position, corresponding to Val410 of the house fly Vssc1 sequence, also aligns with sites of multiple amino acid substitutions identified in the sodium channel sequences of pyrethroid-resistant insect populations. These results implicate this single amino acid polymorphism in transmembrane segment 6 of sodium channel homology domain I as a determinant of the differential pyrethroid sensitivity of rat sodium channel isoforms.

Differential gene expression of neonatal and adult DRG neurons correlates with the differential sensitization of TRPV1 responses to nerve growth factor

Cultures of neonatal and adult dorsal root ganglion (DRG) neurons are commonly used in in vitro models to study the ion channels and signaling events associated with peripheral sensation under various conditions. Differential responsiveness between neonatal and adult DRG neurons to physiological or pathological stimuli suggests potential differences in their gene expression profiles. We performed a microarray analysis of cultured adult and neonatal rat DRG neurons, which revealed distinct gene expression profiles especially of ion channels and signaling molecules at the genomic level. For example, Ca 2+-stimulated adenylyl cyclase (AC) isoforms AC3 and AC8, PKCδ and CaMKIIα, the voltage-gated sodium channel β1 and β4, and potassium channels K v1.1, K v3.2, K v4.1, K v9.1, K v9.3, K ir3.4, K ir7.1, K 2P1.1/TWIK-1 had significantly higher mRNA expression in adult rat DRG neurons, while Ca 2+-inhibited AC5 and AC6, sodium channel Na v1.3 α subunit, potassium channels K ir6.1, K 2P10.1/TREK-2, calcium channel Ca v2.2 α1 subunit, and its auxiliary subunits β1 and β3 were conversely down regulated in adult neurons. Importantly, higher adult neuron expression of ERK1/2, PI3K/P110α, but not of TRPV1 and TrkA, was found and confirmed by PCR and western blot. These latter findings are consistent with the key role of ERK and PI3K signaling in sensitization of TRPV1 by NGF and may explain our previously published observation that adult, but not neonatal, rat DRG neurons are sensitized by NGF.

Functional Expression of Spider Neurotoxic Peptide Huwentoxin-I in E. coli

The coding sequence of huwentoxin-I, a neurotoxic peptide isolated from the venom of the Chinese spider Ornithoctonus huwena, was amplified by PCR using the cDNA library constructed from the spider venom glands. The cloned fragment was inserted into the expression vector pET-40b and transformed into the E. coli strain BL21 (DE3). The expression of a soluble fusion protein, disulfide interchange protein (DsbC)-huwentoxin-I, was auto-induced in the periplasm of E. coli in the absence of IPTG. After partial purification using a Ni-NTA column, the expressed fusion protein was digested using enterokinase to release heteroexpressed huwentoxin-I and was further purified using RP-HPLC. The resulting peptide was subjected to gel electrophoresis and mass spectrometry analysis. The molecular weight of the heteroexpressed huwentoxin-I was 3750.69, which is identical to that of the natural form of the peptide isolated from spider venom. The physiological properties of the heteroexpressed huwentoxin-I were further analyzed using a whole-cell patch clamp assay. The heteroexpressed huwentoxin-I was able to block currents generated by human Nav1.7 at an IC50 of 640 nmole/L, similar to that of the natural huwentoxin-I, which is 630 nmole/L.

Inhibition of human Na v1.5 sodium channels by strychnine and its analogs

Strychnine and brucine from the seeds of the plant Strychnos nux vomica have been shown to have interesting pharmacological effects on several neurotransmitter receptors. In this study, we have characterized the pharmacological properties of strychnine and its analogs on human Na v1.5 channels to assess their potential therapeutic advantage in certain arrhythmias. Among the eight alkaloids, only strychnine and icajine exhibited inhibition potency on the Na v1.5 channel with the half-maximum inhibition (IC 50) values of 83.1 μM and 104.6 μM, respectively. Structure–function analysis indicated that the increased bulky methoxy groups on the phenyl ring or the negatively charged oxygen atom may account for this lack of inhibition on the Na v1.5 channel. Strychnine and icajine may bind to the channel by cation–π interactions. The substitution with a large side chain on the phenyl ring or the increased molecular volume may alter the optimized position for the compound close to the binding sites of the channel. Strychnine and icajine bind to the Na v1.5 channel with a new mechanism that is different from TTX and local anesthetics. They bind to the outer vestibule of the channel pore with fast association and dissociation rates at resting state. Strychnine and icajine had little effect on steady-state fast inactivation but markedly shifted the slow inactivation of Na v1.5 currents toward more hyperpolarized potentials. The property of icajine influencing slow-inactivated state of Na v1.5 channel would be potential therapeutic advantages in certain arrhythmias.

Na V1.7 accumulates and co-localizes with phosphorylated ERK1/2 within transected axons in early experimental neuromas

Peripheral nerve injury can result in formation of a neuroma, which is often associated with heightened sensitivity to normally innocuous stimuli as well as spontaneous dysesthesia and pain. The onset and persistence of neuropathic pain have been linked to spontaneous ectopic electrogenesis in axons within neuromas, suggesting an involvement of voltage-gated sodium channels. Sodium channel isoforms Na V1.3, Na V1.7 and Na V1.8 have been shown to accumulate in chronic painful human neuromas, while, to date, only Na V1.3 has been reported to accumulate within experimental neuromas. Although recent evidence strongly support a major contribution for Na V1.7 in nociception, the expression of Na V1.7 in injured axons within acute neuromas has not been studied. The current study examined whether Na V1.7 accumulates in experimental rat neuromas. We further investigated whether activated (phosphorylated) mitogen-activated protein (MAP) kinase ERK1/2, which is known to modulate Na V1.7 properties, is co-localized with Na V1.7 within axons in neuromas. We demonstrate increased levels of Na V1.7 in experimental rat sciatic nerve neuromas, 2 weeks after nerve ligation and transaction. We further show elevated levels of phosphorylated ERK1/2 within individual neuroma axons that exhibit Na V1.7 accumulation. These results extend previous descriptions of sodium channel and MAP kinase accumulation within experimental and human neuromas, and suggest that targeted blockade of Na V1.7 or ERK1/2 may provide a strategy for amelioration of chronic pain that often follows nerve injury and formation of neuromas.

Mutations in SCN5A From Patients With IBS Result in Abnormal Na V1.5 Function

Background: Central and peripheral serotonergic systems have been implicated in symptom pathogenesis in Irritable Bowel Syndrome (IBS) on the basis of serotonin's involvement in regulating gastrointestinal tract (GIT) homeostasis, in addition to its central role in the regulation of mood; accumulating evidence also points to the presence of low level immune activation in the condition. We previously showed that the activity of indoleamine-2,3dioxygenase (IDO), the immunoresponsive enzyme which is responsible for the degradation of tryptophan along the kynurenine pathway, is enhanced in IBS. The mechanism underpinning this phenomenon is not well understood. Recently, altered function in specific members of the toll-like receptor (TLR) family (TLR1-10) has also been demonstrated in IBS. However, the relationship between TLR activation and IDO activity in IBS is unknown. Aim: To investigate whether specific TLR activation elicits exaggerated kynurenine production in IBS patients compared to controls. Methods: 25 IBS patients and 37 healthy controls (HC) were recruited and venous blood was donated. Whole blood was cultured with a panel of nine different TLR agonists for 24 hours. Cell culture supernatants were then analysed for both trytophan and kynurenine by HPLC in a single isocratic run using sequential fluorescent and UV detection. Results: Kynurenine concentrations were elevated to similar levels in both IBS (240.9 ± 11.58 vs 322.4 ± 19.73ng/ml, p<0.001) and HC samples (224.2 ± 6.330 vs 316.5 ± 13.60ng/ml, p<0.001) following treatment with the TLR4 agonist lipopolysaccheride (LPS). A corresponding elevation in the kynurenine:tryptophan ratio, an indicator of IDO activity, was observed in both IBS patients (0.01774 ± 0.00089 vs 0.02367 ± 0.00137, p<0.001) and HCs (0.01681 ± 0.0006 vs 0.02354 ± 0.001117, p<0.001). In contrast, this ratio was elevated in IBS patients alone (0.01774 ± 0.00089 vs 0.02166 ± 0.00162, p<0.05) following treatment with the TLR8 ligand ssRNA40. Conclusion: These results demonstrate that TLR activation can drive elevations in IDO activity in a manner that corresponds to previously described functional TLR-agonist mediated responses in IBS. Moreover, it implicates TLR8 in the differential IDO activity observed in IBS patients compared to controls. This provides novel evidence for a putative TLR-dependant mechanism underpinning IDO activation in IBS and may point to new therapeutic avenues in this debilitating condition.

Sodium Hydrogen Sulfide is a Partly Redox-Independent Activator of Na V1.5, the Na + Channel in Human Jejunal Myocytes

Sodium Hydrogen Sulfide is a Partly Redox-Independent Activator of Na V1.5, the Na + Channel in Human Jejunal Myocytes