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.
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
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
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
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
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
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.
Diabetic neuropathy is a common form of peripheral neuropathy, yet the mechanisms responsible for pain in this disease are poorly understood. Alterations in the expression and function of voltage-gated tetrodotoxin-resistant (TTX-R) sodium channels have been implicated in animal models of neuropathic pain, including models of diabetic neuropathy. We investigated the expression and function of TTX-sensitive (TTX-S) and TTX-R sodium channels in dorsal root ganglion (DRG) neurons and the responses to thermal hyperalgesia and mechanical allodynia in streptozotocin-treated rats between 4–8 weeks after onset of diabetes. Diabetic rats demonstrated a significant reduction in the threshold for escape from innocuous mechanical pressure (allodynia) and a reduction in the latency to withdrawal from a noxious thermal stimulus (hyperalgesia). Both TTX-S and TTX-R sodium currents increased significantly in small DRG neurons isolated from diabetic rats. The voltage-dependent activation and steady-state inactivation curves for these currents were shifted negatively. TTX-S currents induced by fast or slow voltage ramps increased markedly in neurons from diabetic rats. Immunoblots and immunofluorescence staining demonstrated significant increases in the expression of Nav1.3 (TTX-S) and Nav1.7 (TTX-S) and decreases in the expression of Nav1.6 (TTX-S) and Nav1.8 (TTX-R) in diabetic rats. The level of serine/threonine phosphorylation of Nav1.6 and Nav1.8 increased in response to diabetes. In addition, increased tyrosine phosphorylation of Nav1.6 and Nav1.7 was observed in DRGs from diabetic rats. These results suggest that both TTX-S and TTX-R sodium channels play important roles and that differential phosphorylation of sodium channels involving both serine/threonine and tyrosine sites contributes to painful diabetic neuropathy.
Increased neuronal excitability and spontaneous firing are hallmark characteristics of injured sensory neurons. Changes in expression of various voltage-gated Na+ channels (VGSCs) have been observed under neuropathic conditions and there is evidence for the involvement of protein kinase C (PKC) in sensory hyperexcitability. Here we demonstrate the contribution of PKC to P2X-evoked VGSC activation in dorsal root ganglion (DRG) neurons in neuropathic conditions.
Using the spinal nerve ligation (SNL) model of neuropathic pain and whole-cell patch clamp recordings of dissociated DRG neurons, we examined changes in excitability of sensory neurons after nerve injury and observed that P2X3 purinoceptor-mediated currents induced by α,β-meATP triggered activation of TTX-sensitive VGSCs in neuropathic nociceptors only. Treatment of neuropathic DRGs with the PKC blocker staurosporine or calphostin C decreased the α,β-meATP-induced Na+ channels activity and reversed neuronal hypersensitivity. In current clamp mode, α,β-meATP was able to evoke action-potentials more frequently in neuropathic neurons than in controls. Pretreatment with calphostin C significantly decreased the proportion of sensitized neurons that generated action potentials in response to α,β-meATP. Recordings measuring VGSC activity in neuropathic neurons show significant change in amplitude and voltage dependence of sodium currents. In situ hybridization data indicate a dramatic increase in expression of embryonic Nav1.3 channels in neuropathic DRG neurons. In a CHO cell line stably expressing the Nav1.3 subunit, PKC inhibition caused both a significant shift in voltage-dependence of the channel in the depolarizing direction and a decrease in current amplitude.
Neuropathic injury causes primary sensory neurons to become hyperexcitable to ATP-evoked P2X receptor-mediated depolarization, a phenotypic switch sensitive to PKC modulation and mediated by increased activity of TTX-sensitive VGSCs. Upregulation in VGSC activity after injury is likely mediated by increased expression of the Nav1.3 subunit, and the function of the Nav1.3 channel is regulated by PKC.
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
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.
Despite increasing evidence for the presence of voltage-gated Na+ channels (Nav) isoforms and measurements of Nav channel currents with the patch-clamp technique in arterial myocytes, no information is available to date as to whether or not Nav channels play a functional role in arteries. The aim of the present work was to look for a physiological role of Nav channels in the control of rat aortic contraction.
Nav channels were detected in the aortic media by Western blot analysis and double immunofluorescence labeling for Nav channels and smooth muscle α-actin using specific antibodies. In parallel, using real time RT-PCR, we identified three Nav transcripts: Nav1.2, Nav1.3, and Nav1.5. Only the Nav1.2 isoform was found in the intact media and in freshly isolated myocytes excluding contamination by other cell types. Using the specific Nav channel agonist veratridine and antagonist tetrodotoxin (TTX), we unmasked a contribution of these channels in the response to the depolarizing agent KCl on rat aortic isometric tension recorded from endothelium-denuded aortic rings. Experimental conditions excluded a contribution of Nav channels from the perivascular sympathetic nerve terminals. Addition of low concentrations of KCl (2–10 mM), which induced moderate membrane depolarization (e.g., from −55.9±1.4 mV to −45.9±1.2 mV at 10 mmol/L as measured with microelectrodes), triggered a contraction potentiated by veratridine (100 µM) and blocked by TTX (1 µM). KB-R7943, an inhibitor of the reverse mode of the Na+/Ca2+ exchanger, mimicked the effect of TTX and had no additive effect in presence of TTX.
These results define a new role for Nav channels in arterial physiology, and suggest that the TTX-sensitive Nav1.2 isoform, together with the Na+/Ca2+ exchanger, contributes to the contractile response of aortic myocytes at physiological range of membrane depolarization.
Substance P (SP), which mainly exists in a subtype of small-diameter dorsal root ganglion (DRG) neurons, is an important signal molecule in pain processing in the spinal cord. Our previous results have proved the expression of SP receptor neurokinin-1 (NK-1) on DRG neurons and its interaction with transient receptor potential vanilloid 1 (TRPV1) receptor.
In this study we investigated the effect of NK-1 receptor agonist on Nav1.8, a tetrodotoxin (TTX)-resistant sodium channel, in rat small-diameter DRG neurons employing whole-cell patch clamp recordings. NK-1 agonist [Sar9, Met(O2)11]-substance P (Sar-SP) significantly enhanced the Nav1.8 currents in a subgroup of small-diameter DRG neurons under both the normal and inflammatory situation, and the enhancement was blocked by NK-1 antagonist Win51708 and protein kinase C (PKC) inhibitor bisindolylmaleimide (BIM), but not the protein kinase A (PKA) inhibitor H89. In particular, the inhibitor of PKCε, a PKC isoform, completely blocked this effect. Under current clamp model, Sar-SP reduced the amount of current required to evoke action potentials and increased the firing rate in a subgroup of DRG neurons.
These data suggest that activation of NK-1 receptor potentiates Nav1.8 sodium current via PKCε-dependent signaling pathway, probably participating in the generation of inflammatory hyperalgesia.
Multiple adverse events are associated with the use of morphine for the treatment of chronic non-cancer pain, including opioid-induced hyperalgesia (OIH). Mechanisms of OIH are independent of opioid tolerance and may involve the morphine metabolite morphine-3-glucuronide (M3G). M3G exhibits limited affinity for opioid receptors and no analgesic effect. Previous reports suggest that M3G can act via the Toll-like receptor 4 (TLR4)/myeloid differentiation protein-2 (MD-2) heterodimer in the central nervous system to elicit pain.
Immunoblot and immunocytochemistry methods were used to characterize the protein expression of TLR4 present in lumbar dorsal root ganglion (DRG). Using in vitro intracellular calcium and current clamp techniques, we determined whether TLR4 activation as elicited by the prototypical agonists of TLR4, lipopolysaccharide (LPS) and M3G, contributed to changes in intracellular calcium and increased excitation. Rodents were also injected with M3G to determine the degree to which M3G-induced tactile hyperalgesia could be diminished using either a small molecule inhibitor of the MD-2/TLR4 complex in rats or TLR4 knockout mice. Whole cell voltage-clamp recordings were made from small- and medium-diameter DRG neurons (25 μm < DRG diameter <45 μm) for both control and M3G-treated neurons to determine the potential influence on voltage-gated sodium channels (NaVs).
We observed that TLR4 immunoreactivity was present in peptidergic and non-peptidergic sensory neurons in the DRG. Non-neuronal cells in the DRG lacked evidence of TLR4 expression. Approximately 15% of assayed small- and medium-diameter sensory neurons exhibited a change in intracellular calcium following LPS administration. Both nociceptive and non-nociceptive neurons were observed to respond, and approximately 40% of these cells were capsaicin-insensitive. Increased excitability observed in sensory neurons following LPS or M3G could be eliminated using Compound 15, a small molecule inhibitor of the TLR4/MD-2 complex. Likewise, systemic injection of M3G induced rapid tactile, but not thermal, nociceptive behavioral changes in the rat, which were prevented by pre-treating animals with Compound 15. Unlike TLR4 wild-type mice, TLR4 knockout mice did not exhibit M3G-induced hyperalgesia. As abnormal pain sensitivity is often associated with NaVs, we predicted that M3G acting via the MD-2/TLR4 complex may affect the density and gating of NaVs in sensory neurons. We show that M3G increases tetrodotoxin-sensitive and tetrodotoxin-resistant (NaV1.9) current densities.
These outcomes provide evidence that M3G may play a role in OIH via the TLR4/MD-2 heterodimer complex and biophysical properties of tetrodotoxin-sensitive and tetrodotoxin-resistant NaV currents.
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
Human and animal studies have shown that Nav1.7 sodium channels, which are preferentially expressed within nociceptors and sympathetic neurons, play a major role in inflammatory and neuropathic pain. Inherited erythromelalgia (IEM) has been linked to gain-of-function mutations of Nav1.7. We now report a novel mutation (V400M) in a three-generation Canadian family in which pain is relieved by carbamazepine (CBZ).
We extracted genomic DNA from blood samples of eight members of the family, and the sequence of SCN9A coding exons was compared with the reference Nav1.7 complementary DNA. Wild-type Nav1.7 and V400M cell lines were then analyzed using whole-cell patch-clamp recording for changes in activation, deactivation, steady-state inactivation, and ramp currents.
Whole-cell patch-clamp studies of V400M demonstrate changes in activation, deactivation, steady-state inactivation, and ramp currents that can produce dorsal root ganglia neuron hyperexcitability that underlies pain in these patients. We show that CBZ, at concentrations in the human therapeutic range, normalizes the voltage dependence of activation and inactivation of this inherited erythromelalgia mutation in Nav1.7 but does not affect these parameters in wild-type Nav1.7.
Our results demonstrate a normalizing effect of CBZ on mutant Nav1.7 channels in this kindred with CBZ-responsive inherited erythromelalgia. The selective effect of CBZ on the mutant Nav1.7 channel appears to explain the ameliorative response to treatment in this kindred. Our results suggest that functional expression and pharmacological studies may provide mechanistic insights into hereditary painful disorders.
Inflammation is known to be responsible for the sensitization of peripheral sensory neurons, leading to spontaneous pain and invalidating pain hypersensitivity. Given its role in regulating neuronal excitability, the voltage-gated Nav1.9 channel is a potential target for the treatment of pathological pain, but its implication in inflammatory pain is yet not fully described. In the present study, we examined the role of the Nav1.9 channel in acute, subacute and chronic inflammatory pain using Nav1.9-null mice and Nav1.9 knock-down rats. In mice we found that, although the Nav1.9 channel does not contribute to basal pain thresholds, it plays an important role in heat pain hypersensitivity induced by subacute paw inflammation (intraplantar carrageenan) and chronic ankle inflammation (complete Freund's adjuvant-induced monoarthritis). We showed for the first time that Nav1.9 also contributes to mechanical hypersensitivity in both models, as assessed using von Frey and dynamic weight bearing tests. Consistently, antisense-based Nav1.9 gene silencing in rats reduced carrageenan-induced heat and mechanical pain hypersensitivity. While no changes in Nav1.9 mRNA levels were detected in dorsal root ganglia (DRGs) during subacute and chronic inflammation, a significant increase in Nav1.9 immunoreactivity was observed in ipsilateral DRGs 24 hours following carrageenan injection. This was correlated with an increase in Nav1.9 immunolabeling in nerve fibers surrounding the inflamed area. No change in Nav1.9 current density could be detected in the soma of retrolabeled DRG neurons innervating inflamed tissues, suggesting that newly produced channels may be non-functional at this level and rather contribute to the observed increase in axonal transport. Our results provide evidence that Nav1.9 plays a crucial role in the generation of heat and mechanical pain hypersensitivity, both in subacute and chronic inflammatory pain models, and bring new elements for the understanding of its regulation in those models.
The voltage-gated sodium channel Nav1.8 is only expressed in subsets of neurons in dorsal root ganglia (DRG) and trigeminal and nodose ganglia. We have isolated mouse partial length Nav1.8 cDNA clones spanning the exon 17 sequence, which have 17 nucleotide substitutions and 12 predicted amino acid differences from the published sequence. The absence of a mutually exclusive alternative exon 17 was confirmed by sequencing 4.1 kilobases of genomic DNA spanning exons 16–18 of Scn10a. A novel cDNA isoform was identified, designated Nav1.8c, which results from alternative 3′-splice site selection at a CAG/CAG motif to exclude the codon for glutamine 1031 within the interdomain cytoplasmic loop IDII/III. The ratio of Nav1.8c (CAG-skipped) to Nav1.8 (CAG-inclusive) mRNA in mouse is ~2:1 in adult DRG, trigeminal ganglion, and neonatal DRG. A Nav1.8c isoform also occurs in rat DRG, but is less common. Of the two other tetrodotoxin-resistant channels, no analogous alternative splicing of mouse Nav1.9 was detected, whereas rare alternative splicing of Nav1.5 at a CAG/CAG motif resulted in the introduction of a CAG trinucleotide. This isoform, designated Nav1.5c, is conserved in rat and encodes an additional glutamine residue that disrupts a putative CK2 phosphorylation site. In summary, novel isoforms of Nav1.8 and Nav1.5 are each generated by alternative splicing at CAG/CAG motifs, which result in the absence or presence of predicted glutamine residues within the interdomain cytoplasmic loop IDII/III. Mutations of sodium channels within this cytoplasmic loop have previously been demonstrated to alter electrophysiological properties and cause cardiac arrhythmias and epilepsy.
Spermidine and spermine, are endogenous polyamines (PAs) that regulate cell growth and modulate the activity of numerous ion channel proteins. In particular, intracellular PAs are potent blockers of many different cation channels and are responsible for strong suppression of outward K+ current, a phenomenon known as inward rectification characteristic of a major class of KIR K+ channels. We previously described block of heterologously expressed voltage-gated Na+ channels (NaV) of rat muscle by intracellular PAs and PAs have recently been found to modulate excitability of brain neocortical neurons by blocking neuronal NaV channels. In this study, we compared the sensitivity of four different cloned mammalian NaV isoforms to PAs to investigate whether PA block is a common feature of NaV channel pharmacology. We find that outward Na+ current of muscle (NaV1.4), heart (NaV1.5), and neuronal (NaV1.2, NaV1.7) NaV isoforms is blocked by PAs, suggesting that PA metabolism may be linked to modulation of action potential firing in numerous excitable tissues. Interestingly, the cardiac NaV1.5 channel is more sensitive to PA block than other isoforms. Our results also indicate that rapid binding of PAs to blocking sites in the NaV1.4 channel is restricted to access from the cytoplasmic side of the channel, but plasma membrane transport pathways for PA uptake may contribute to long-term NaV channel modulation. PAs may also play a role in drug interactions since spermine attenuates the use-dependent effect of the lidocaine, a typical local anesthetic and anti-arrhythmic drug.
inward rectification; lidocaine; local anesthetics; Polyamines; sodium channels; spermidine; spermine; use-dependence; voltage-gated Na+ channels
Neuropathic pain caused by peripheral nerve injury is a chronic disorder that represents a significant clinical challenge because the pathological mechanisms have not been fully elucidated. Several studies have suggested the involvement of various sodium channels, including tetrodotoxin-resistant NaV1.8, in affected dorsal root ganglion (DRG) neurons. We have hypothesized that altered local expression of NaV1.8 in the peripheral axons of DRG neurons could facilitate nociceptive signal generation and propagation after neuropathic injury.
After unilateral sciatic nerve entrapment injury in rats, compound action potential amplitudes were increased in both myelinated and unmyelinated fibers of the ipsilateral sciatic nerve. Tetrodotoxin resistance of both fiber populations and sciatic nerve NaV1.8 immunoreactivity were also increased. Further analysis of NaV1.8 distribution revealed that immunoreactivity and mRNA levels were decreased and unaffected, respectively, in the ipsilateral L4 and L5 DRG; however sciatic nerve NaV1.8 mRNA showed nearly an 11-fold ipsilateral increase. Nav1.8 mRNA observed in the sciatic nerve was likely of axonal origin since it was not detected in non-neuronal cells cultured from nerve tissue. Absence of changes in NaV1.8 mRNA polyadenylation suggests that increased mRNA stability was not responsible for the selective peripheral mRNA increase. Furthermore, mRNA levels of NaV1.3, NaV1.5, NaV1.6, NaV1.7, and NaV1.9 were not significantly different between ipsilateral and contralateral nerves. We therefore propose that selective NaV1.8 mRNA axonal transport and local up-regulation could contribute to the hyperexcitability of peripheral nerves in some neuropathic pain states.
Cuff entrapment injury resulted in significantly elevated axonal excitability and increased NaV1.8 immunoreactivity in rat sciatic nerves. The concomitant axonal accumulation of NaV1.8 mRNA may play a role in the pathogenesis of this model of neuropathic pain.
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.
NaV channels play a crucial role in neuronal and muscle excitability. Using whole-cell recordings we studied effects of low extracellular pH on the biophysical properties of NaV1.2, NaV1.4, and NaV1.5, expressed in cultured mammalian cells. Low pH produced different effects on different channel subtypes. Whereas NaV1.4 exhibited very low sensitivity to acidosis, primarily limited to partial block of macroscopic currents, the effects of low pH on gating in NaV1.2 and NaV1.5 were profound. In NaV1.2 low pH reduced apparent valence of steady-state fast inactivation, shifted the τ(V) to depolarizing potentials and decreased channels availability during onset to slow and use-dependent inactivation (UDI). In contrast, low pH delayed open-state inactivation in NaV1.5, right-shifted the voltage-dependence of window current, and increased channel availability during onset to slow and UDI. These results suggest that protons affect channel availability in an isoform-specific manner. A computer model incorporating these results demonstrates their effects on membrane excitability.
gating; activation; fast inactivation; slow inactivation; patch-clamp; sodium channels
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
Intracellular Ca2+ ([Ca2+]i) can trigger dual-mode regulation of the voltage gated cardiac sodium channel (NaV1.5). The channel components of the Ca2+ regulatory system are the calmodulin (CaM)-binding IQ motif and the Ca2+ sensing EF hand–like (EFL) motif in the carboxyl terminus of the channel. Mutations in either motif have been associated with arrhythmogenic changes in expressed NaV1.5 currents. Increases in [Ca2+]i shift the steady-state inactivation of NaV1.5 in the depolarizing direction and slow entry into inactivated states. Mutation of the EFL (NaV1.54X) shifts inactivation in the hyperpolarizing direction compared with the wild-type channel and eliminates the Ca2+ sensitivity of inactivation gating. Modulation of the steady-state availability of NaV1.5 by [Ca2+]i is more pronounced after the truncation of the carboxyl terminus proximal to the IQ motif (NaV1.5Δ1885), which retains the EFL. Mutating the EFL (NaV1.54X) unmasks CaM-mediated regulation of the kinetics and voltage dependence of inactivation. This latent CaM modulation of inactivation is eliminated by mutation of the IQ motif (NaV1.54X-IQ/AA). The LQT3 EFL mutant channel NaV1.5D1790G exhibits Ca2+ insensitivity and unmasking of CaM regulation of inactivation gating. The enhanced effect of CaM on NaV1.54X gating is associated with significantly greater fluorescence resonance energy transfer between enhanced cyan fluorescent protein–CaM and NaV1.54X channels than is observed with wild-type NaV1.5. Unlike other isoforms of the Na channel, the IQ-CaM interaction in the carboxyl terminus of NaV1.5 is latent under physiological conditions but may become manifest in the presence of disease causing mutations in the CT of NaV1.5 (particularly in the EFL), contributing to the production of potentially lethal ventricular arrhythmias.
voltage-gated sodium channel; EF hand motif; IQ motif; calmodulin; FRET