Voltage-gated sodium channels (Nav) mediate neuronal action potentials. Tetrodotoxin inhibits all Nav isoforms, but Nav1.8 and Nav1.9 are relatively tetrodotoxin-resistant (TTX-r) compared to other isoforms. Nav1.8 is highly expressed in dorsal root ganglion neurons and is functionally linked to nociception, but the sensitivity of TTX-r isoforms to inhaled anesthetics is unclear.
The sensitivities of heterologously expressed rat TTX-r Nav1.8 and endogenous tetrodotoxin-sensitive (TTX-s) Nav to the prototypic inhaled anesthetic isoflurane were tested in mammalian ND7/23 cells using patch-clamp electrophysiology.
From a holding potential of −70 mV, isoflurane (0.53±0.06 mM, ~1.8 MAC at 24°C) reduced normalized peak Na+ current (INa) of Nav1.8 to 0.55±0.03 and of endogenous TTX-s Nav to 0.56±0.06. Isoflurane minimally inhibited INa from a holding potential of −140 mV. Isoflurane did not affect voltage-dependence of activation, but significantly shifted voltage-dependence of steady-state inactivation by −6 mV for Nav1.8 and by −7 mV for TTX-s Nav. IC50 values for inhibition of peak INa were 0.67±0.06 mM for Nav1.8 and 0.66±0.09 mM for TTX-s Nav; significant inhibition occurred at clinically relevant concentrations as low as 0.58 MAC. Isoflurane produced use-dependent block of Nav1.8; at a stimulation frequency of 10 Hz, 0.56±0.08 mM isoflurane reduced INa to 0.64±0.01 vs. 0.78±0.01 for control.
Isoflurane inhibited the tetrodotoxin-resistant isoform Nav1.8 with potency comparable to that for endogenous tetrodotoxin-sensitive Nav isoforms, indicating that sensitivity to inhaled anesthetics is conserved across diverse Nav family members. Block of Nav1.8 in dorsal root ganglion neurons could contribute to the effects of inhaled anesthetics on peripheral nociceptive mechanisms.
The (−)-gallocatechin-3-gallate (GCG) concentration in some tea beverages can account for as much as 50% of the total catechins. It has been shown that catechins have analgesic properties. Voltage-gated sodium channels (Nav) mediate neuronal action potentials. Tetrodotoxin inhibits all Nav isoforms, but Nav1.8 and Nav1.9 are relatively tetrodotoxin-resistant compared to other isoforms and functionally linked to nociception. In this study, the effects of GCG on tetrodotoxin-resistant Na+ currents were investigated in rat primary cultures of dorsal root ganglion neurons via the whole-cell patch-clamp technique. We found that 1 μM GCG reduced the amplitudes of peak current density of tetrodotoxin-resistant Na+ currents significantly. Furthermore, the inhibition was accompanied by a depolarizing shift of the activation voltage and a hyperpolarizing shift of steady-state inactivation voltage. The percentage block of GCG (1 μM) on tetrodotoxin-resistant Na+ current was 45.1% ± 1.1% in 10 min. In addition, GCG did not produce frequency-dependent block of tetrodotoxin-resistant Na+ currents at stimulation frequencies of 1 Hz, 2 Hz and 5 Hz. On the basis of these findings, we propose that GCG may be a potential analgesic agent.
catechins; (−)-gallocatechin-3-gallate; Na+ channel; dorsal root ganglion; tetrodotoxin-resistant
Peripheral neuropathic pain is a disabling condition resulting from nerve injury. It is characterized by the dysregulation of voltage-gated sodium channels (Navs) expressed in dorsal root ganglion (DRG) sensory neurons. The mechanisms underlying the altered expression of Navs remain unknown. This study investigated the role of the E3 ubiquitin ligase NEDD4-2, which is known to ubiquitylate Navs, in the pathogenesis of neuropathic pain in mice. The spared nerve injury (SNI) model of traumatic nerve injury–induced neuropathic pain was used, and an Nav1.7-specific inhibitor, ProTxII, allowed the isolation of Nav1.7-mediated currents. SNI decreased NEDD4-2 expression in DRG cells and increased the amplitude of Nav1.7 and Nav1.8 currents. The redistribution of Nav1.7 channels toward peripheral axons was also observed. Similar changes were observed in the nociceptive DRG neurons of Nedd4L knockout mice (SNS-Nedd4L–/–). SNS-Nedd4L–/– mice exhibited thermal hypersensitivity and an enhanced second pain phase after formalin injection. Restoration of NEDD4-2 expression in DRG neurons using recombinant adenoassociated virus (rAAV2/6) not only reduced Nav1.7 and Nav1.8 current amplitudes, but also alleviated SNI-induced mechanical allodynia. These findings demonstrate that NEDD4-2 is a potent posttranslational regulator of Navs and that downregulation of NEDD4-2 leads to the hyperexcitability of DRG neurons and contributes to the genesis of pathological pain.
Sensory neurons of the dorsal root ganglia (DRG) express multiple voltage-gated sodium (Na) channels that substantially differ in gating kinetics and pharmacology. Small-diameter (<25 µm) neurons isolated from the rat DRG express a combination of fast tetrodotoxin-sensitive (TTX-S) and slow TTX-resistant (TTX-R) Na currents while large-diameter neurons (>30 µm) predominately express fast TTX-S Na current. Na channel expression was further investigated using single-cell RT-PCR to measure the transcripts present in individually harvested DRG neurons. Consistent with cellular electrophysiology, the small neurons expressed transcripts encoding for both TTX-S (Nav1.1, Nav1.2, Nav1.6, Nav1.7) and TTX-R (Nav1.8, Nav1.9) Na channels. Nav1.7, Nav1.8 and Nav1.9 were the predominant Na channels expressed in the small neurons. The large neurons highly expressed TTX-S isoforms (Nav1.1, Nav1.6, Nav1.7) while TTX-R channels were present at comparatively low levels. A unique subpopulation of the large neurons was identified that expressed TTX-R Na current and high levels of Nav1.8 transcript. DRG neurons also displayed substantial differences in the expression of neurofilaments (NF200, peripherin) and Necl-1, a neuronal adhesion molecule involved in myelination. The preferential expression of NF200 and Necl-1 suggests that large-diameter neurons give rise to thick myelinated axons. Small-diameter neurons expressed peripherin, but reduced levels of NF200 and Necl-1, a pattern more consistent with thin unmyelinated axons. Single-cell analysis of Na channel transcripts indicates that TTX-S and TTX-R Na channels are differentially expressed in large myelinated (Nav1.1, Nav1.6, Nav1.7) and small unmyelinated (Nav1.7, Nav1.8, Nav1.9) sensory neurons.
Sodium channel; dorsal root ganglia; single-cell RT-PCR; Necl-1; NF200; peripherin
Because of their prominent role in electro-excitability, voltage-gated sodium (NaV) channels have become the foremost important target of animal toxins. These toxins have developed the ability to discriminate between closely related NaV subtypes, making them powerful tools to study NaV channel function and structure. CgNa is a 47-amino acid residue type I toxin isolated from the venom of the Giant Caribbean Sea Anemone Condylactis gigantea. Previous studies showed that this toxin slows the fast inactivation of tetrodotoxin-sensitive NaV currents in rat dorsal root ganglion neurons. To illuminate the underlying NaV subtype-selectivity pattern, we have assayed the effects of CgNa on a broad range of mammalian isoforms (NaV1.2–NaV1.8) expressed in Xenopus oocytes. This study demonstrates that CgNa selectively slows the fast inactivation of rNaV1.3/β1, mNaV1.6/β1 and, to a lesser extent, hNaV1.5/β1, while the other mammalian isoforms remain unaffected. Importantly, CgNa was also examined on the insect sodium channel DmNaV1/tipE, revealing a clear phyla-selectivity in the efficacious actions of the toxin. CgNa strongly inhibits the inactivation of the insect NaV channel, resulting in a dramatic increase in peak current amplitude and complete removal of fast and steady-state inactivation. Together with the previously determined solution structure, the subtype-selective effects revealed in this study make of CgNa an interesting pharmacological probe to investigate the functional role of specific NaV channel subtypes. Moreover, further structural studies could provide important information on the molecular mechanism of NaV channel inactivation.
sea anemone; toxin; inactivation; sodium channel; subtype; selectivity
Tetrodotoxin-resistant (TTX-R) sodium channels NaV1.8 and NaV1.9 in sensory neurons were known as key pain modulators. Comparing with the widely reported NaV1.8, roles of NaV1.9 on inflammatory pain are poorly studied by antisense-induced specific gene knockdown. Here, we used molecular, electrophysiological and behavioral methods to examine the effects of antisense oligodeoxynucleotide (AS ODN) targeting NaV1.8 and NaV1.9 on inflammatory pain. Following complete Freund's adjuvant (CFA) inflammation treatment, NaV1.8 and NaV1.9 in rat dorsal root ganglion (DRG) up-regulated mRNA and protein expressions and increased sodium current densities. Immunohistochemical data demonstrated that NaV1.8 mainly localized in medium and small-sized DRG neurons, whereas NaV1.9 only expressed in small-sized DRG neurons. Intrathecal (i.t.) delivery of AS ODN was used to down-regulate NaV1.8 or NaV1.9 expressions confirmed by immunohistochemistry and western blot. Unexpectedly, behavioral tests showed that only NaV1.8 AS ODN, but not NaV1.9 AS ODN could reverse CFA-induced heat and mechanical hypersensitivity. Our data indicated that TTX-R sodium channels NaV1.8 and NaV1.9 in primary sensory neurons played distinct roles in CFA-induced inflammatory pain and suggested that antisense oligodeoxynucleotide-mediated blocking of key pain modulator might point toward a potential treatment strategy against certain types of inflammatory pain.
Tetrodotoxin (TTX)-resistant voltage-gated Na (NaV) channels have been implicated in nociception. In particular, NaV1.9 contributes to expression of persistent Na current in small diameter, nociceptive sensory neurons in dorsal root ganglia and is required for inflammatory pain sensation. Using ND7/23 cells stably expressing human NaV1.9, we elucidated the biophysical mechanisms responsible for potentiation of channel activity by G-protein signaling to better understand the response to inflammatory mediators. Heterologous NaV1.9 expression evoked TTX-resistant Na current with peak activation at −40 mV with extensive overlap in voltage dependence of activation and inactivation. Inactivation kinetics were slow and incomplete, giving rise to large persistent Na currents. Single-channel recording demonstrated long openings and correspondingly high open probability (Po) accounting for the large persistent current amplitude. Channels exposed to intracellular GTPγS, a proxy for G-protein signaling, exhibited twofold greater current density, slowing of inactivation, and a depolarizing shift in voltage dependence of inactivation but no change in activation voltage dependence. At the single-channel level, intracellular GTPγS had no effect on single-channel amplitude but caused an increased mean open time and greater Po compared with recordings made in the absence of GTPγS. We conclude that G-protein activation potentiates human NaV1.9 activity by increasing channel open probability and mean open time, causing the larger peak and persistent current, respectively. Our results advance our understanding about the mechanism of NaV1.9 potentiation by G-protein signaling during inflammation and provide a cellular platform useful for the discovery of NaV1.9 modulators with potential utility in treating inflammatory pain.
The expression of voltage-gated sodium channels is regulated at multiple levels, and in this study we addressed the potential for alternative splicing of the Nav1.2, Nav1.3, Nav1.6 and Nav1.7 mRNAs. We isolated novel mRNA isoforms of Nav1.2 and Nav1.3 from adult mouse and rat dorsal root ganglia (DRG), Nav1.3 and Nav1.7 from adult mouse brain, and Nav1.7 from neonatal rat brain. These alternatively spliced isoforms introduce an additional exon (Nav1.2 exon 17A and topologically equivalent Nav1.7 exon 16A) or exon pair (Nav1.3 exons 17A and 17B) that contain an in-frame stop codon and result in predicted two-domain, truncated proteins. The mouse and rat orthologous exon sequences are highly conserved (94-100% identities), as are the paralogous Nav1.2 and Nav1.3 exons (93% identity in mouse) to which the Nav1.7 exon has only 60% identity. Previously, Nav1.3 mRNA has been shown to be upregulated in rat DRG following peripheral nerve injury, unlike the downregulation of all other sodium channel transcripts. Here we show that the expression of Nav1.3 mRNA containing exons 17A and 17B is unchanged in mouse following peripheral nerve injury (axotomy), whereas total Nav1.3 mRNA expression is upregulated by 33% (P=0.003), suggesting differential regulation of the alternatively spliced transcripts. The alternatively spliced rodent exon sequences are highly conserved in both the human and chicken genomes, with 77-89% and 72-76% identities to mouse, respectively. The widespread conservation of these sequences strongly suggests an additional level of regulation in the expression of these channels, that is also tissue-specific.
DRG; brain; alternative splicing; Scn2a; Scn3a; Scn9a
In rats expression of the Nav1.7 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 Nav1.7 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 Nav1.7 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 two-fold by the application of a train of up to 100 5-ms depolarizing prepulses. These effects of tefluthrin on Nav1.7 channels were qualitatively similar to its effects on rat Nav1.2, Nav1.3 and Nav1.6 channels assayed previously under identical conditions. However, Nav1.7 sodium channels were distinguished by their low sensitivity to modification by tefluthrin, especially compared to Nav1.3 and Nav1.6 channels. It is likely that Nav1.7 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 Nav1.3, Nav1.6 and Nav1.8) and two pyrethroid-resistant isoforms (rat Nav1.2 and Nav1.7) 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.
voltage-gated sodium channel; Nav1.7 isoform; pyrethroid; tefluthrin; peripheral nervous system; dorsal root ganglion
Voltage-gated sodium channels (Nav) are essential for the generation and conduction of action potentials. Peripheral inflammation increases the expression of Nav1.7 and Nav1.8 in dorsal root ganglion (DRG) neurons, suggesting that they participate in the induction and maintenance of chronic inflammatory pain. However, how Nav1.7 and Nav1.8 are regulated in the DRG under inflammatory pain conditions remains unclear. Using a complete Freund’s adjuvant (CFA)-induced chronic inflammatory pain model and Western blot analysis, we found that phosphorylated Akt (p-Akt) was significantly increased in the ipsilateral L4/5 DRGs of rats on days 3 and 7 after intraplantar CFA injection. Immunohistochemistry showed that the percentage of p-Akt-positive neurons in the DRG was also significantly increased in the ipsilateral L4/5 DRGs at these times. Moreover, CFA injection increased the colocalization of p-Akt with Nav1.7 and Nav1.8 in L4/5 DRG neurons. Pretreatment of rats with an intrathecal injection of Akt inhibitor IV blocked CFA-induced thermal hyperalgesia and CFA-induced increases in Nav1.7 and Nav1.8 in the L4/5 DRGs on day 7 after CFA injection. Our findings suggest that the Akt pathway participates in inflammation-induced upregulation of Nav1.7 and Nav1.8 expression in DRG neurons. This participation might contribute to the maintenance of chronic inflammatory pain.
This article presents that inhibition of Akt blocks CFA-induced thermal hyperalgesia and CFA-induced increases in dorsal root ganglion Nav1.7 and Nav1.8. These findings have potential implications for use of Akt inhibitors to prevent and/or treat persistent inflammatory pain.
Nav1.7; Nav1.8; Akt; Dorsal root ganglion; Inflammatory pain
The NaV1.7 tetrodotoxin-sensitive voltage-gated sodium channel isoform plays a critical role in nociception. In rodent models of diabetic neuropathy, increased NaV1.7 in dorsal root ganglion (DRG) neurons correlates with the emergence of pain-related behaviors characteristic of painful diabetic neuropathy (PDN). We examined the effect of transgene-mediated expression of enkephalin on pain-related behaviors and their biochemical correlates in DRG neurons. Transfection of DRG neurons by subcutaneous inoculation of a herpes simplex virus (HSV)-based vector expressing proenkephalin (PE) reversed nocisponsive behavioral responses to heat, cold, and mechanical pressure characteristic of PDN. Vector-mediated enkephalin production in vivo prevented the increase in DRG NaV1.7 observed in PDN, an effect that correlated with inhibition of phosphorylation of p38 MAP kinase and protein kinase C (PKC). Primary DRG neurons in vitro exposed to 45 mM glucose for 18 hrs also demonstrated an increase in NaV1.7 and increased phosphorylation of p38 and PKC; these changes were prevented by transfection in vitro with the enkephalin-expressing vector. The effect of hyperglycemia on NaV1.7 production in vitro was mimicked by exposure to PMA, and blocked by the myristolated PKC inhibitor 20–28 or the p38 inhibitor SB202190; the effect of vector-mediated enkephalin on NaV1.7 levels was prevented by naltrindole. The results of these studies suggest that activation of the presynaptic delta opioid receptor by enkephalin prevents the increase in neuronal NaV1.7 in DRG through inhibition of PKC and p38. These results establish a novel interaction between the delta opioid receptor and voltage gated sodium channels.
pain; diabetic neuropathy; sodium channel; gene therapy; herpes simplex; enkephalins
The mechanisms underlying neuropathic pain induction are very complex but might involve abnormal spontaneous activity in the sensory dorsal root ganglion (DRG). Voltage-gated sodium channels in the DRG are essential for the genesis of abnormal spontaneous neuronal activity. In the present study, we examined the changes in expression of the voltage-gated sodium channel Nav1.1 in the DRG after peripheral nerve injury. Western blot analysis showed that the level of Nav1.1 protein in the ipsilateral L5 DRG was significantly increased on days 3 and 7 after fifth lumbar spinal nerve ligation. Immunohistochemical study further confirmed a marked increase in the percentage of Nav1.1-positive cells in the ipsilateral DRG on day 3 after fifth lumbar spinal nerve ligation. Similarly, on day 7 after sciatic nerve axotomy, the amount of Nav1.1 protein and the percentage of Nav1.1-positive cells in the ipsilateral L5 DRG were also significantly increased. Our results suggest that an early increase in DRG Nav1.1 expression after peripheral nerve injury might be involved in the induction of neuropathic pain.
Nav1.1; sodium channel; neuropathic pain; dorsal root ganglion; rat
Voltage-gated Nav channels are required for normal electrical activity in neurons, skeletal muscle, and cardiomyocytes. In the heart, Nav1.5 is the predominant Nav channel, and Nav1.5-dependent activity regulates rapid upstroke of the cardiac action potential. Nav1.5 activity requires precise localization at specialized cardiomyocyte membrane domains. However, the molecular mechanisms underlying Nav channel trafficking in the heart are unknown. In this paper, we demonstrate that ankyrin-G is required for Nav1.5 targeting in the heart. Cardiomyocytes with reduced ankyrin-G display reduced Nav1.5 expression, abnormal Nav1.5 membrane targeting, and reduced Na+ channel current density. We define the structural requirements on ankyrin-G for Nav1.5 interactions and demonstrate that loss of Nav1.5 targeting is caused by the loss of direct Nav1.5–ankyrin-G interaction. These data are the first report of a cellular pathway required for Nav channel trafficking in the heart and suggest that ankyrin-G is critical for cardiac depolarization and Nav channel organization in multiple excitable tissues.
Dysregulation of voltage-gated sodium channels (Navs) is believed to play a major role in nerve fiber hyperexcitability associated with neuropathic pain. A complete transcriptional characterization of the different isoforms of Navs under normal and pathological conditions had never been performed on mice, despite their widespread use in pain research. Navs mRNA levels in mouse dorsal root ganglia (DRG) were studied in the spared nerve injury (SNI) and spinal nerve ligation (SNL) models of neuropathic pain. In the SNI model, injured and non-injured neurons were intermingled in lumbar DRG, which were pooled to increase the tissue available for experiments.
A strong downregulation was observed for every Navs isoform expressed except for Nav1.2; even Nav1.3, known to be upregulated in rat neuropathic pain models, was lower in the SNI mouse model. This suggests differences between these two species. In the SNL model, where the cell bodies of injured and non-injured fibers are anatomically separated between different DRG, most Navs were observed to be downregulated in the L5 DRG receiving axotomized fibers. Transcription was then investigated independently in the L3, L4 and L5 DRG in the SNI model, and an important downregulation of many Navs isoforms was observed in the L3 DRG, suggesting the presence of numerous injured neurons there after SNI. Consequently, the proportion of axotomized neurons in the L3, L4 and L5 DRG after SNI was characterized by studying the expression of activating transcription factor 3 (ATF3). Using this marker of nerve injury confirmed that most injured fibers find their cell bodies in the L3 and L4 DRG after SNI in C57BL/6 J mice; this contrasts with their L4 and L5 DRG localization in rats. The spared sural nerve, through which pain hypersensitivity is measured in behavioral studies, mostly projects into the L4 and L5 DRG.
The complex regulation of Navs, together with the anatomical rostral shift of the DRG harboring injured fibers in C57BL/6 J mice, emphasize that caution is necessary and preliminary anatomical experiments should be carried out for gene and protein expression studies after SNI in mouse strains.
Activating transcription factor 3 (ATF3); Dorsal root ganglia (DRG); Nerve injury; Neuropathic pain; Quantitative real time polymerase chain reaction (qRT-PCR); Sciatic nerve; Spared nerve injury (SNI); Spinal nerve ligation (SNL); Voltage-gated sodium channels (Navs)
▶ The β3 subunit masks the ER retention signal of NaV1.8 and release the channel from the ER. ▶ p11 directly binds to NaV1.8 and help its translocation to the plasma membrane. ▶ PDZD2 is responsible for the functional expression of NaV1.8 on the plasma membrane. ▶ Contactin KO mice exhibit a reduction of NaV1.8 along unmyelinated axons in the sciatic nerve. ▶ PKA activation increases the NaV1.8 density on the membrane through direct phosphorylation.
The α-subunit of tetrodotoxin-resistant voltage-gated sodium channel NaV1.8 is selectively expressed in sensory neurons. It has been reported that NaV1.8 is involved in the transmission of nociceptive information from sensory neurons to the central nervous system in nociceptive  and neuropathic  pain conditions. Thus NaV1.8 has been a promising target to treat chronic pain. Here we discuss the recent advances in the study of trafficking mechanism of NaV1.8. These pieces of information are particularly important as such trafficking machinery could be new targets for painkillers.
Sodium Channel; Sensory Neuron; Pain; Trafficking
Voltage-gated sodium channel Nav1.7 is preferentially expressed in dorsal root ganglion (DRG) and sympathetic neurons within the peripheral nervous system. Homozygous or compound heterozygous loss-of-function mutations in SCN9A, the gene which encodes Nav1.7, cause congenital insensitivity to pain (CIP) accompanied by anosmia. Global knock-out of Nav1.7 in mice is neonatal lethal reportedly from starvation, suggesting anosmia. These findings led us to hypothesize that Nav1.7 is the main sodium channel in the peripheral olfactory sensory neurons (OSN, also known as olfactory receptor neurons).
We used multiplex PCR-restriction enzyme polymorphism, in situ hybridization and immunohistochemistry to determine the identity of sodium channels in rodent OSNs.
We show here that Nav1.7 is the predominant sodium channel transcript, with low abundance of other sodium channel transcripts, in olfactory epithelium from rat and mouse. Our in situ hybridization data show that Nav1.7 transcripts are present in rat OSNs. Immunostaining of Nav1.7 and Nav1.6 channels in rat shows a complementary accumulation pattern with Nav1.7 in peripheral presynaptic OSN axons, and Nav1.6 primarily in postsynaptic cells and their dendrites in the glomeruli of the olfactory bulb within the central nervous system.
Our data show that Nav1.7 is the dominant sodium channel in rat and mouse OSN, and may explain anosmia in Nav1.7 null mouse and patients with Nav1.7-related CIP.
CCL2 [chemokine (C–C motif) ligand 2] contributes to the inflammation-induced neuropathic pain through activating VGSC (voltage-gated sodium channel)-mediated nerve impulse conduction, but the underlying mechanism is currently unknown. Our study aimed to investigate whether PKC (protein kinase C)–NF-κB (nuclear factor κB) is involved in CCL2-induced regulation of voltage-gated sodium Nav1.8 currents and expression. DRG (dorsal root ganglion) neurons were prepared from adult male Sprague–Dawley rats and incubated with various concentration of CCL2 for 24 h. Whole-cell patch-clamps were performed to record the Nav1.8 currents in response to the induction by CCL2. After being pretreated with 5 and10 nM CCL2 for 16 h, CCR2 [chemokine (C–C motif) receptor 2] and Nav1.8 expression significantly increased and the peak currents of Nav1.8 elevated from the baseline 46.53±4.53 pA/pF to 64.28±3.12 pA/pF following 10 nM CCL2 (P<0.05). Compared with the control, significant change in Nav1.8 current density was observed when the CCR2 inhibitor INCB3344 (10 nM) was applied. Furthermore, inhibition of PKC by AEB071 significantly eliminated CCL2-induced elevated Nav1.8 currents. In vitro PKC kinase assays and autoradiograms suggested that Nav1.8 within DRG neurons was a substrate of PKC and direct phosphorylation of the Nav1.8 channel by PKC regulates its function in these neurons. Moreover, p65 expression was significantly higher in CCL2-induced neurons (P<0.05), and was reversed by treatment with INCB3344 and AEB071. PKC–NF-κB are involved in CCL2-induced elevation of Nav1.8 current density by promoting the phosphorylation of Nav1.8 and its expression.
Cytokine CCL2 is responsible for promoting voltage-gated sodium Nav1.8 current density and expression, which mediates nerve impulse conduction and induces inflammatory nociception. PKC phosphorylates Nav1.8 to increase its current density and PKC–NF-κB are involved in inducing the up-regulation of Nav1.8.
CCL2; CCR2; dorsal root ganglion (DRG); Nav1.8; nociception; PKC; CCL2, chemokine (C–C motif) ligand 2; CCR2, chemokine (C–C motif) receptor 2; DRG, dorsal root ganglion; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NF-κB, nuclear factor κB; PKC, protein kinase C; TEA-Cl, tetraethylammonium-Cl; TRPV1, transient receptor potential vanilloid 1; TTX-R, tetrodotoxin-resistant; VGSC, voltage-gated sodium channel
Nav1.5 is the principal voltage-gated sodium channel expressed in heart, and is also expressed at lower abundance in embryonic dorsal root ganglia (DRG) with little or no expression reported postnatally. We report here the expression of Nav1.5 mRNA isoforms in adult mouse and rat DRG. The major isoform of mouse DRG is Nav1.5a, which encodes a protein with an IDII/III cytoplasmic loop reduced by 53 amino acids. Western blot analysis of adult mouse DRG membrane proteins confirmed the expression of Nav1.5 protein. The Na+ current produced by the Nav1.5a isoform has a voltage-dependent inactivation significantly shifted to more negative potentials (by ~5 mV) compared to the full-length Nav1.5 when expressed in the DRG neuroblastoma cell line ND7/23. These results imply that the alternatively spliced exon 18 of Nav1.5 plays a role in channel inactivation and that Nav1.5a is likely to make a significant contribution to adult DRG neuronal function.
Inflammation or nerve injury-induced upregulation and release of chemokine CC chemokine ligand 2 (CCL2) within the dorsal root ganglion (DRG) is believed to enhance the activity of DRG nociceptive neurons and cause hyperalgesia. Transient receptor potential vanilloid receptor 1 (TRPV1) and tetrodotoxin (TTX)-resistant Nav1.8 sodium channels play an essential role in regulating the excitability and pain transmission of DRG nociceptive neurons. We therefore tested the hypothesis that CCL2 causes peripheral sensitization of nociceptive DRG neurons by upregulating the function and expression of TRPV1 and Nav1.8 channels.
DRG neuronal culture was prepared from 3-week-old Sprague–Dawley rats and incubated with various concentrations of CCL2 for 24 to 36 hours. Whole-cell voltage-clamp recordings were performed to record TRPV1 agonist capsaicin-evoked inward currents or TTX-insensitive Na+ currents from control or CCL2-treated small DRG sensory neurons. The CCL2 effect on the mRNA expression of TRPV1 or Nav1.8 was measured by real-time quantitative RT-PCR assay.
Pretreatment of CCL2 for 24 to 36 hours dose-dependently (EC50 value = 0.6 ± 0.05 nM) increased the density of capsaicin-induced currents in small putative DRG nociceptive neurons. TRPV1 mRNA expression was greatly upregulated in DRG neurons preincubated with 5 nM CCL2. Pretreating small DRG sensory neurons with CCL2 also increased the density of TTX-resistant Na+ currents with a concentration-dependent manner (EC50 value = 0.7 ± 0.06 nM). The Nav1.8 mRNA level was significantly increased in DRG neurons pretreated with CCL2. In contrast, CCL2 preincubation failed to affect the mRNA level of TTX-resistant Nav1.9. In the presence of the specific phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002 or Akt inhibitor IV, CCL2 pretreatment failed to increase the current density of capsaicin-evoked inward currents or TTX-insensitive Na+ currents and the mRNA level of TRPV1 or Nav1.8.
Our results showed that CCL2 increased the function and mRNA level of TRPV1 channels and Nav1.8 sodium channels in small DRG sensory neurons via activating the PI3K/Akt signaling pathway. These findings suggest that following tissue inflammation or peripheral nerve injury, upregulation and release of CCL2 within the DRG could facilitate pain transmission mediated by nociceptive DRG neurons and could induce hyperalgesia by upregulating the expression and function of TRPV1 and Nav1.8 channels in DRG nociceptive neurons.
CC chemokine ligand 2; Dorsal root ganglion neurons; Transient receptor potential vanilloid receptor 1; Tetrodotoxin-resistant Nav1.8 sodium channel
Amitriptyline (AMI) is tricyclic antidepressant that has been widely used to manage various chronic pains such as migraines. Its efficacy is attributed to its blockade of voltage-gated sodium channels (VGSCs). However, the effects of AMI on the tetrodotoxin-resistant (TTX-r) sodium channel Nav1.9 currents have been unclear to present.
Using a whole-cell patch clamp technique, this study showed that AMI efficiently inhibited Nav1.9 currents in a concentration-dependent manner and had an IC50 of 15.16 μM in acute isolated trigeminal ganglion (TG) neurons of the rats. 10 μM AMI significantly shifted the steady-state inactivation of Nav1.9 channels in the hyperpolarizing direction without affecting voltage-dependent activation. Surprisingly, neither 10 nor 50 μM AMI caused a use-dependent blockade of Nav1.9 currents elicited by 60 pulses at 1 Hz.
These data suggest that AMI is a state-selective blocker of Nav1.9 channels in rat nociceptive trigeminal neurons, which likely contributes to the efficacy of AMI in treating various pains, including migraines.
Amitriptyline; Nav1.9; Patch clamp; Trigeminal ganglion; Pain
Human voltage-activated sodium (Nav) channels are adept at rapidly transmitting electrical signals across long distances in various excitable tissues. As such, they are amongst the most widely targeted ion channels by drugs and animal toxins. Of the nine isoforms, Nav1.8 and Nav1.9 are preferentially expressed in DRG neurons where they are thought to play an important role in pain signaling. Although the functional properties of Nav1.8 have been relatively well characterized, difficulties with expressing Nav1.9 in established heterologous systems limit our understanding of the gating properties and toxin pharmacology of this particular isoform. This review summarizes our current knowledge of the role of Nav1.8 and Nav1.9 in pain perception and elaborates on the approaches used to identify molecules capable of influencing their function.
Nav1.8; Nav1.9; pain; animal toxins; voltage sensor; voltage-activated sodium channel
Functional alterations in the properties of Aβ afferent fibers may account for the increased pain sensitivity observed under peripheral chronic inflammation. Among the voltage-gated sodium channels involved in the pathophysiology of pain, Nav1.8 has been shown to participate in the peripheral sensitization of nociceptors. However, to date, there is no evidence for a role of Nav1.8 in controlling Aβ-fiber excitability following persistent inflammation.
Distribution and expression of Nav1.8 in dorsal root ganglia and sciatic nerves were qualitatively or quantitatively assessed by immunohistochemical staining and by real time-polymerase chain reaction at different time points following complete Freund’s adjuvant (CFA) administration. Using a whole-cell patch-clamp configuration, we further determined both total INa and TTX-R Nav1.8 currents in large-soma dorsal root ganglia (DRG) neurons isolated from sham or CFA-treated rats. Finally, we analyzed the effects of ambroxol, a Nav1.8-preferring blocker on the electrophysiological properties of Nav1.8 currents and on the mechanical sensitivity and inflammation of the hind paw in CFA-treated rats.
Our findings revealed that Nav1.8 is up-regulated in NF200-positive large sensory neurons and is subsequently anterogradely transported from the DRG cell bodies along the axons toward the periphery after CFA-induced inflammation. We also demonstrated that both total INa and Nav1.8 peak current densities are enhanced in inflamed large myelinated Aβ-fiber neurons. Persistent inflammation leading to nociception also induced time-dependent changes in Aβ-fiber neuron excitability by shifting the voltage-dependent activation of Nav1.8 in the hyperpolarizing direction, thus decreasing the current threshold for triggering action potentials. Finally, we found that ambroxol significantly reduces the potentiation of Nav1.8 currents in Aβ-fiber neurons observed following intraplantar CFA injection and concomitantly blocks CFA-induced mechanical allodynia, suggesting that Nav1.8 regulation in Aβ-fibers contributes to inflammatory pain.
Collectively, these findings support a key role for Nav1.8 in controlling the excitability of Aβ-fibers and its potential contribution to the development of mechanical allodynia under persistent inflammation.
Aβ-fibers; Allodynia; Complete Freund’s adjuvant; Electrophysiology; Sodium channel blocker
Voltage-gated sodium channels (VGSC) are critical membrane components that participate in the electrical activity of excitable cells. The type one VGSC family includes the tetrodotoxin insensitive sodium channel, Nav1.8, encoded by the Scn10a gene. Nav1.8 expression is restricted to small and medium diameter nociceptive sensory neurons of the dorsal root (DRG) and cranial sensory ganglia. In order to understand the stringent transcriptional regulation of the Scn10a gene, the sensory neuron specific promoter was functionally identified. While identifying the mRNA 5’ end, alternative splicing within the 5’ UTR was observed to create heterogeneity in the RNA transcript. Four kilobases of upstream genomic DNA was cloned and the presence of tissue specific promoter activity was tested by microinjection and adenoviral infection of fluorescent protein reporter constructs into primary mouse and rat neurons, and cell lines. The region contained many putative transcription factor binding sites and strong homology with the predicted rat ortholog. Homology to the predicted human ortholog was limited to the proximal end and several conserved cis elements were noted. Two regulatory modules were identified by microinjection of reporter constructs into DRG and superior cervical ganglia neurons: a neuron specific proximal promoter region between −1.6 and −0.2kb of the transcription start site cluster, and a distal sensory neuron switch region beyond −1.6kb that restricted fluorescent protein expression to a subset of primary sensory neurons.
tetrodotoxin-resistant sodium channel; pain; promoter; sensory neuron
Members of the degenerin/epithelial (DEG/ENaC) sodium channel family are mechanosensors in C elegans, and Nav1.7 and Nav1.8 voltage-gated sodium channel knockout mice have major deficits in mechanosensation. β and γENaC sodium channel subunits are present with acid sensing ion channels (ASICs) in mammalian sensory neurons of the dorsal root ganglia (DRG). The extent to which epithelial or voltage-gated sodium channels are involved in transduction of mechanical stimuli is unclear.
Here we show that deleting β and γENaC sodium channels in sensory neurons does not result in mechanosensory behavioural deficits. We had shown previously that Nav1.7/Nav1.8 double knockout mice have major deficits in behavioural responses to noxious mechanical pressure. However, all classes of mechanically activated currents in DRG neurons are unaffected by deletion of the two sodium channels. In contrast, the ability of Nav1.7/Nav1.8 knockout DRG neurons to generate action potentials is compromised with 50% of the small diameter sensory neurons unable to respond to electrical stimulation in vitro.
Behavioural deficits in Nav1.7/Nav1.8 knockout mice reflects a failure of action potential propagation in a mechanosensitive set of sensory neurons rather than a loss of primary transduction currents. DEG/ENaC sodium channels are not mechanosensors in mouse sensory neurons.
Mechanotransduction; Sodium channels; Pain; Nav1.7; Nav1.8; ENaCs
Sensory neurons in the dorsal root ganglia (DRG) express a subset of voltage dependent sodium channels (NaV) including NaV1.1, 1.6, 1.7, 1.8 and 1.9. Previous work supported preferential localization of NaV1.8 channels to small-medium diameter, nociceptive afferent neurons. However, we recently published evidence that NaV1.8 was the dominant NaV channel expressed in the somas of small, medium and large diameter muscle afferent neurons, which is consistent with other reports. Here, we extend those results to show that NaV1.8 expression is not correlated with afferent neuron diameter. Using immunocytochemistry, we found NaV1.8 expression in ~50% of sensory afferent neurons with diameters ranging from 20 to 70 µm. In addition, electrophysiological analysis shows that the kinetic and inactivation properties of NaV1.8 current are invariant with neuron size. These data add further support to the idea that NaV1.8 contributes to the electrical excitability of both nociceptive and non-nociceptive sensory neurons.
cutaneous afferents; muscle afferents; dorsal root ganglia neurons; Tetrodotoxin-resistant (TTX-R)