We expressed rat Nav1.6 sodium channels in combination with the rat β1 and β2 auxiliary subunits in Xenopus laevis oocytes and evaluated the effects of the pyrethroid insecticides S-bioallethrin, deltamethrin and tefluthrin on expressed sodium currents using the two-electrode voltage clamp technique. S-Bioallethrin, a Type I structure, produced transient modification evident in the induction of rapidly-decaying sodium tail currents, weak resting modification (5.7% modification at 100 μM), and no further enhancement of modification upon repetitive activation by high-frequency trains of depolarizing pulses. By contrast deltamethrin, a Type II structure, produced sodium tail currents that were ~9-fold more persistent than those caused by S-bioallethrin, barely detectable resting modification (2.5% modification at 100 μM), and 3.7-fold enhancement of modification upon repetitive activation. Tefluthrin, a Type I structure with high mammalian toxicity, exhibited properties intermediate between S-bioallethrin and deltamethrin: intermediate tail current decay kinetics, much greater resting modification (14.1% at 100 μM), and 2.8-fold enhancement of resting modification upon repetitive activation. Comparison of concentration–effect data showed that repetitive depolarization increased the potency of tefluthrin ~15-fold and that tefluthrin was ~10-fold more potent than deltamethrin as a use-dependent modifier of Nav1.6 sodium channels. Concentration–effect data from parallel experiments with the rat Nav1.2 sodium channel co-expressed with the rat β1 and β2 subunits in oocytes showed that the Nav1.6 isoform was at least 15-fold more sensitive to tefluthrin and deltamethrin than the Nav1.2 isoform. These results implicate sodium channels containing the Nav1.6 isoform as potential targets for the central neurotoxic effects of pyrethroids.
voltage-gated sodium channel; Nav1.6 isoform; pyrethroid; S-bioallethrin; deltamethrin; tefluthrin
We expressed the rat Nav1.3 and Nav1.6 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 Nav1.6 channels to tefluthrin. Modification by tefluthrin of Nav1.6 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 Nav1.6 channel with the β1 subunit enhanced the extent of channel modification twofold following repeated depolarization. Coexpression of Nav1.6 with the β2 subunit also slightly enhanced modification following repeated depolarization, but coexpression of Nav1.6 with both β subunits caused enhanced modification following repeated depolarization that was indistinguishable from that found with Nav1.6+β1 channels. In contrast to Nav1.6, the resting modification by tefluthrin of Nav1.3 channels expressed in the absence of β subunits was reduced by repeated depolarization. However, tefluthrin modification of the Nav1.3 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 Nav1.6 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.
voltage-gated sodium channels; Nav1.6 isoform; Nav1.3 isoform; β subunit; voltage clamp; tefluthrin
Voltage-gated sodium channels are important sites for the neurotoxic actions of pyrethroid insecticides in mammals. The pore-forming α subunits of mammalian sodium channels are encoded by a family of 9 genes, designated Nav1.1 - Nav1.9. Native sodium channels in the adult central nervous system (CNS) are heterotrimeric complexes of one of these 9 α subunits and two auxiliary (β) subunits. Here we compare the functional properties and pyrethroid sensitivity of the rat and human Nav1.3 isoforms, which are abundantly expressed in the developing CNS. Coexpression of the rat Nav1.3 and human Nav1.3 α subunits in combination with their conspecific β1 and β2 subunits in Xenopus laevis oocytes gave channels with markedly different inactivation properties and sensitivities to the pyrethroid insecticide tefluthrin. Rat Nav1.3 channels inactivated more slowly than human Nav1.3 channels during a depolarizing pulse. The rat and human channels also differed in their voltage dependence of steady-state inactivation. Exposure of rat and human Nav1.3 channels to 100 μM tefluthrin in the resting state produced populations of channels that activated, inactivated and deactivated more slowly than unmodified channels. For both rat and human channels, application of trains of depolarizing prepulses enhanced the extent of tefluthrin modification approximately twofold; this result implies that tefluthrin may bind to both the resting and open states of the channel. Modification of rat Nav1.3 channels by 100 μM tefluthrin was four-fold greater than that measured in parallel assays with human Nav1.3 channels. Human Nav1.3 channels were also less sensitive to tefluthrin than rat Nav1.2 channels, which are considered to be relatively insensitive to pyrethroids. These data provide the first direct comparison of the functional and pharmacological properties of orthologous rat and human sodium channels and demonstrate that orthologous channels with a high degree of amino acid sequence conservation differ in both their functional properties and their sensitivities to pyrethroid insecticides.
Nav1.3; oocyte; sodium channel; pyrethroid; tefluthrin; rat; human
We expressed rat Nav1.6 sodium channels in combination with the rat β1 and β2 auxiliary subunits in human embryonic kidney (HEK293) cells and evaluated the effects of the pyrethroid insecticides tefluthrin and deltamethrin on expressed sodium currents using the whole-cell patch clamp technique. Both pyrethroids produced concentration-dependent, resting modification of Nav1.6 channels, prolonging the kinetics of channel inactivation and deactivation to produce persistent “late” currents during depolarization and tail currents following repolarization. Both pyrethroids also produced concentration dependent hyperpolarizing shifts in the voltage dependence of channel activation and steady-state inactivation. Maximal shifts in activation, determined from the voltage dependence of the pyrethroid-induced late and tail currents, were ~25 mV for tefluthrin and ~20 mV for deltamethrin. The highest attainable concentrations of these compounds also caused shifts of ~5–10 mV in the voltage dependence of steady-state inactivation. In addition to their effects on the voltage dependence of inactivation, both compounds caused concentration-dependent increases in the fraction of sodium current that was resistant to inactivation following strong depolarizing prepulses. We assessed the use-dependent effects of tefluthrin and deltamethrin on Nav1.6 channels by determining the effect of trains of 1 to 100 5-ms depolarizing prepulses at frequencies of 20 or 66.7 Hz on the extent of channel modification. Repetitive depolarization at either frequency increased modification by deltamethrin by ~2.3-fold but had no effect on modification by tefluthrin. Tefluthrin and deltamethrin were equally potent as modifiers of Nav1.6 channels in HEK293 cells using the conditions producing maximal modification as the basis for comparison. These findings show that the actions of tefluthrin and deltamethrin of Nav1.6 channels in HEK293 cells differ from the effects of these compounds on Nav1.6 channels in Xenopus oocytes and more closely reflect the actions of pyrethroids on channels in their native neuronal environment.
voltage-gated sodium channel; Nav1.6 isoform; pyrethroid; deltamethrin; tefluthrin; HEK293 cells
Pyrethroid insecticides disrupt nerve function by modifying the gating kinetics of transitions between the conducting and nonconducting states of voltage-gated sodium channels. Pyrethroids modify rat Nav1.6 + β1 + β2 channels expressed in Xenopus oocytes in both the resting state and in one or more states that require channel activation by repeated depolarization. The state dependence of modification depends on the pyrethroid examined: deltamethrin modification requires repeated channel activation, tefluthrin modification is significantly enhanced by repeated channel activation, and S-bioallethrin modification is unaffected by repeated activation. Use-dependent modification by deltamethrin and tefluthrin implies that these compounds bind preferentially to open channels. We constructed the rat Nav1.6Q3 cDNA, which contained the IFM/QQQ mutation in the inactivation gate domain that prevents fast inactivation and results in a persistently open channel. We expressed Nav1.6Q3 + β1 + β2 sodium channels in Xenopus oocytes and assessed the modification of open channels by pyrethroids by determining the effect of depolarizing pulse length on the normalized conductance of the pyrethroid-induced sodium tail current. Deltamethrin caused little modification of Nav1.6Q3 following short (10 ms) depolarizations, but prolonged depolarizations (up to 150 ms) caused a progressive increase in channel modification measured as an increase in the conductance of the pyrethroid-induced sodium tail current. Modification by tefluthrin was clearly detectable following short depolarizations and was increased by long depolarizations. By contrast modification by S-bioallethrin following short depolarizations was not altered by prolonged depolarization. These studies provide direct evidence for the preferential binding of deltamethrin and tefluthrin (but not S-bioallethrin) to Nav1.6Q3 channels in the open state and imply that the pyrethroid receptor of resting and open channels occupies different conformations that exhibit distinct structure–activity relationships.
Sodium channel; Nav1.6; Use-dependent modification; Deltamethrin; S-bioallethrin; Tefluthrin
The Nav1.6 voltage-gated sodium channel α subunit isoform is the most abundant isoform in the brain and is implicated in the transmission of high frequency action potentials. Purification and immunocytochemical studies imply that Nav1.6 exist predominantly as Nav1.6+β1+β2 heterotrimeric complexes. We assessed the independent and joint effects of the rat β1 and β2 subunits on the gating and kinetic properties of rat Nav1.6 channels by recording whole-cell currents in the two-electrode voltage clamp configuration following transient expression in Xenopus oocytes. The β1 subunit accelerated fast inactivation of sodium currents but had no effect on the voltage dependence of their activation and steady-state inactivation and also prevented the decline of currents following trains of high-frequency depolarizing prepulses. The β2 subunit selectively retarded the fast phase of fast inactivation and shifted the voltage dependence of activation towards depolarization without affecting other gating properties and had no effect on the decline of currents following repeated depolarization. The β1 and β2 subunits expressed together accelerated both kinetic phases of fast inactivation, shifted the voltage dependence of activation towards hyperpolarization, and gave currents with a persistent component typical of those recorded from neurons expressing Nav1.6 sodium channels. These results identify unique effects of the β1 and β2 subunits and demonstrate that joint modulation by both auxiliary subunits gives channel properties that are not predicted by the effects of individual subunits.
voltage-gated sodium channels; Nav1.6; β subunits; voltage clamp; kinetics; steady-state properties
The Nav1.6 voltage-gated sodium channel α subunit isoform is abundantly expressed in the adult rat brain. To assess the functional modulation of Nav1.6 channels by the auxiliary β1 subunit we expressed the rat Nav1.6 sodium channel α subunit by stable transformation in HEK293 cells either alone or in combination with the rat β1 subunit and assessed the properties of the reconstituted channels by recording sodium currents using the whole-cell patch clamp technique. Coexpression with the β1 subunit accelerated the inactivation of sodium currents and shifted the voltage dependence of channel activation and steady-state fast inactivation by approximately 5–7 mV in the direction of depolarization. By contrast the β1 subunit had no effect on the stability of sodium currents following repeated depolarizations at high frequencies. Our results define modulatory effects of the β1 subunit on the properties of rat Nav1.6-mediated sodium currents reconstituted in HEK293 cells that differ from effects measured previously in the Xenopus oocyte expression system. We also identify differences in the kinetic and gating properties of the rat Nav1.6 channel expressed in the absence of the β1 subunit compared to the properties of the orthologous mouse and human channels expressed in this system.
Sodium channel inhibitor (SCI) insecticides are hypothesized to inhibit voltage-gated sodium channels by binding selectively to the slow-inactivated state. Replacement of valine at position 787 in the S6 segment of homology domain II of the rat Nav1.4 sodium channel by lysine (V787K) enchances slow inactivation of this channel whereas replacement by alanine or cysteine (V787A, V787C) inhibits slow inactivation. To test the hypothesis that SCI insecticides bind selectively to the slow-inactivated state, we constructed mutated Nav1.4/V787A, Nav1.4/V787C, and Nav1.4/V787K cDNAs, expressed wildtype and mutated channels with the auxiliary β1 subunit in Xenopus oocytes, and used the two-electrode voltage clamp technique to examine the effects of these mutations on channel inhibition by four SCI insecticides (indoxacarb, its bioactivated metabolite DCJW, metaflumizone, and RH3421). Mutations at Val787 affected SCI insecticide sensitivity in a manner that was independent of mutation-induced changes in slow inactivation gating. Sensitivity to inhibition by 10 μM indoxacarb was significantly increased in all three mutated channels, whereas sensitivity to inhibition by 10 μM metaflumizone was significantly reduced in Nav1.4/V787A channels and completely abolished in Nav1.4/V787K channels. The effects of Val787 mutations on metaflumizone were correlated with the hydrophobicity of the substituted amino acid rather than the extent of slow inactivation. None of the mutations at Val787 significantly affected the sensitivity to inhibition by DCJW or RH3421. These results demonstrate that the impact of mutations at Val787 on sodium channel inhibition by SCI insecticides depends on the specific insecticide examined and is independent of mutation-induced changes in slow inactivation gating. We propose that Val787 may be a unique determinant of metaflumizone binding.
Sodium channel; inhibition; insecticide; slow inactivation; indoxacarb; metaflumizone
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
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.
Sodium channel-blocker insecticides (SCBIs), such as indoxacarb and metaflumizone, are a new class of insecticides with a mechanism of action different from those of other insecticides that target sodium channels. SCBIs block sodium channels in a manner similar to local anesthetics (LA) such as lidocaine. Several residues, particularly F1579 and Y1586, in the sixth transmembrane segment (S6) of domain IV (IV) of rat Nav1.4 sodium channels are required for the action of LAs and SCBIs and may form part of overlapping receptor sites. However, the binding site for SCBIs in insect sodium channels remains undefined. We used site-directed mutagenesis, the Xenopus laevis oocyte expression system, and the two-electrode voltage clamp technique to study the effects on SCBI activity of mutating F1817 and Y1824 (analogous to those residues identified in mammalian sodium channels) to alanine in the voltage-sensitive sodium channel of the German cockroach, Blattella germanica. The mutant channels showed no effect or a marked increase in channel sensitivity to both DCJW (the active metabolite of indoxacarb) and metaflumizone. Thus, it appeared that although the F1817 residue plays a role in the action of SCBIs and both residues are involved in LA activity in mammalian sodium channels, neither F1817 nor Y1824 are integral determinants of SCBI binding on insect sodium channels. Our results suggest that the receptor site of SCBIs on insect sodium channels may be significantly different from that on mammalian sodium channels.
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
The excitotoxic conopeptide ι-RXIA induces repetitive action potentials in frog motor axons and seizures upon intracranial injection into mice. We recently discovered that ι-RXIA shifts the voltage-dependence of activation of voltage-gated sodium channel NaV1.6 to a more hyperpolarized level. Here, we performed voltage-clamp experiments to examine its activity against rodent NaV1.1 through NaV1.7 co-expressed with the β1 subunit in Xenopus oocytes and NaV1.8 in dissociated mouse DRG neurons. The order of sensitivity to ι-RXIA was NaV1.6 > 1.2 > 1.7, and the remaining subtypes were insensitive. The time course of ι-RXIA-activity on NaV1.6 during exposure to different peptide concentrations were well fit by single-exponential curves that provided kobs. The plot of kobs versus [ι-RXIA] was linear, consistent with a bimolecular reaction with a Kd of ~3 μM, close to the steady-state EC50 of ~2 μM. ι-RXIA has an unusual residue, D-Phe, and the analog with an L-Phe instead, ι-RXIA[L-Phe44], had a two-fold lower affinity and two-fold faster off-rate than ι-RXIA on NaV1.6 and furthermore was inactive on NaV1.2. ι-RXIA induced repetitive action potentials in mouse sciatic nerve with conduction velocities of both A- and C-fibers, consistent with the presence of NaV1.6 at nodes of Ranvier as well as in unmyelinated axons. Sixteen peptides homologous to ι-RXIA have been identified from a single species of Conus, so these peptides represent a rich family of novel sodium channel-targeting ligands.
channel-activation; conopeptide; excitotoxin; iota-conotoxin RXIA; neurotoxin; voltage-gated sodium channel
Sensory neurons in the dorsal root ganglion express two kinds of tetrodotoxin resistant (TTX-R) isoforms of voltage-gated sodium channels, NaV1.8 and NaV1.9. These isoforms play key roles in the pathophysiology of chronic pain. Of special interest is NaV1.9: our previous studies revealed a unique property of the NaV1.9 current, i.e., the NaV1.9 current shows a gradual and notable up-regulation of the peak amplitude during recording (“spontaneous augmentation of NaV1.9”). However, the mechanism underlying the spontaneous augmentation of NaV1.9 is still unclear. In this study, we examined the effects of protein kinases A and C (PKA and PKC), on the spontaneous augmentation of NaV1.9. The spontaneous augmentation of the NaV1.9 current was significantly suppressed by activation of PKA, whereas activation of PKA did not affect the voltage dependence of inactivation for the NaV1.9 current. On the contrary, the finding that activation of PKC can affect the voltage dependence of inactivation for NaV1.9 in the perforated patch recordings, where the augmentation does not occur, suggests that the effects of PMA are independent of the augmentation process. These results indicate that the spontaneous augmentation of NaV1.9 was regulated directly by PKA, and indirectly by PKC.
Na+ channel; tetrodotoxin; dorsal root ganglion; patch clamp; PKA; PKC
Voltage-gated sodium (Nav) channels are responsible for initiation and propagation of action potential in the neurons. To explore the mechanisms for chronic heart failure (CHF)-induced baroreflex dysfunction, we measured the expression and current density of Nav channel subunits (Nav1.7, Nav1.8, and Nav1.9) in the aortic baroreceptor neurons and investigated the role of Nav channels on aortic baroreceptor neuron excitability and baroreflex sensitivity in sham and CHF rats. CHF was induced by left coronary artery ligation. The development of CHF (6–8 weeks after the coronary ligation) was confirmed by hemodynamic and morphological characteristics. Immunofluorescent data indicated that Nav1.7 was expressed in A-type (myelinated) and C-type (unmyelinated) nodose neurons but Nav1.8 and Nav1.9 were expressed only in C-type nodose neurons. Real-time RT-PCR and western blot data showed that CHF reduced mRNA and protein expression levels of Nav channels in nodose neurons. In addition, using the whole cell patch-clamp technique, we found that Nav current density and cell excitability of the aortic baroreceptor neurons were lower in CHF rats than that in sham rats. Aortic baroreflex sensitivity was blunted in anesthetized CHF rats, compared with that in sham rats. Furthermore, Nav channel activator (rATX II, 100 nM) significantly enhanced Nav current density and cell excitability of aortic baroreceptor neurons and improved aortic baroreflex sensitivity in CHF rats. These results suggest that reduced expression and activation of the Nav channels is involved in the attenuation of baroreceptor neuron excitability, which subsequently contributes to the impairment of baroreflex in CHF state.
Aortic baroreceptor neuron; Baroreflex; Heart failure; 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.
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.
Many inhaled anesthetics inhibit voltage-gated sodium channels at clinically relevant concentrations, and suppression of neurotransmitter release by these agents results, at least partly, from decreased presynaptic sodium channel activity. Volatile aromatic anesthetics can inhibit N-methyl-D-aspartate (NMDA) receptor function and enhance γ-amino butyric acid A (GABAA) receptor function, but these effects depend strongly on the chemical properties of the aromatic ompounds. The present study tested whether diverse aromatic anesthetics consistently inhibit sodium channel function.
We studied the effect of eight aromatic anesthetics on Nav1.2 sodium channels with β1 subunits, using whole-cell, two-electrode voltage-clamp techniques in Xenopus oocytes.
All aromatic anesthetics inhibited INa (sodium currents) at a holding potential which produce half-maximal current (V1/2) (partial depolarization); inhibition was modest with 1,3,5-trifluorobenzene (8 ± 2%), pentafluorobenzene (13 ± 2%), and hexafluorobenzene (13 ± 2%), but greater with benzene (37 ± 2%), fluorobenzene (39 ± 2%), 1,2-difluorobenzene (48 ± 2%), 1,4-difluorobenzene (31 ± 3%), and 1,2,4-trifluorobenzene (33 ± 1%). Such dichotomous effects were noted by others for NMDA and GABAA receptors. Parallel, but much smaller inhibition, was found for INa at a holding potential which produced near maximal current (−90 mV) (VH-90), and hexafluorobenzene caused small (6 ± 1%) potentiation of this current. These changes in sodium channel function were correlated with effectiveness for inhibiting NMDA receptors, with lipid solubility of the compounds, with molecular volume, and with cation-π interactions.
Aromatic compounds vary in their actions on the kinetics of sodium channel gating and this may underlie their variable inhibition. The range of inhibition produced by MAC concentrations of inhaled anesthetics indicates that sodium channel inhibition may underlie the action of some of these anesthetics but not others.
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
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.
Tetrodotoxin (TTX) is the quintessential ligand of voltage-gated sodium channels (NaVs). Like TTX, μ-conotoxin peptides are pore blockers, and both toxins have helped to define the properties of neurotoxin receptor Site 1 of NaVs. Here, we report unexpected results showing that the recently discovered μ-conotoxin KIIIA and TTX can simultaneously bind to Site 1 and act in concert. Results with saturating concentrations of peptide applied to voltage-clamped Xenopus oocytes expressing brain NaV1.2, and single-channel recordings from brain channels in lipid bilayers, show that KIIIA or its analog, KIIIA[K7A], block partially, with a residual current that can be completely blocked by TTX. In addition, the kinetics of block by TTX and peptide are each affected by the prior presence of the other toxin. For example, bound peptide slows subsequent binding of TTX (an antagonistic interaction) and slows TTX dissociation when both toxins are bound (a synergistic effect on block). The overall functional consequence resulting from the combined action of the toxins depends on the quantitative balance between these opposing actions. The results lead us to postulate that in the bi-liganded NaV complex, TTX is bound between the peptide and the selectivity filter. These observations refine our view of Site 1 and open new possibilities in NaV pharmacology.
conotoxin; contratoxin; NaV1.2; oocyte; sodium channel; site 1; syntoxin; tetrodotoxin; voltage clamp
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
Midbrain dopamine (DA) neurons are slow intrinsic pacemakers that undergo depolarization (DP) block upon moderate stimulation. Understanding DP block is important because it has been correlated with the clinical efficacy of chronic antipsychotic drug treatment. Here we describe how voltage-gated sodium (NaV) channels regulate DP block and pacemaker activity in DA neurons of the substantia nigra using rat brain slices. The distribution, density and gating of NaV currents were manipulated by blocking native channels with tetrodotoxin and by creating virtual channels and anti-channels with dynamic clamp. Although action potentials initiate in the axon initial segment (AIS) and NaV channels are distributed in multiple dendrites, selective reduction of NaV channel activity in the soma was sufficient to decrease pacemaker frequency and increase susceptibility to DP block. Conversely, increasing somatic NaV current density raised pacemaker frequency and lowered susceptibility to DP block. Finally, when NaV currents were restricted to the soma, pacemaker activity occurred at abnormally high rates due to excessive local subthreshold NaV current. Together with computational simulations, these data show that both the slow pacemaker rate and the sensitivity to DP block that characterizes DA neurons result from the low density of somatic NaV channels. More generally, we conclude that the somatodendritic distribution of NaV channels is a major determinant of repetitive spiking frequency.
Hypokalemic periodic paralysis (HypoPP) is associated with mutations in either the CaV1.1 calcium channel or the NaV1.4 sodium channel. Some NaV1.4 HypoPP mutations have been shown to cause an anomalous inward current that may contribute to the attacks of paralysis. Herein, we test whether disease-associated NaV1.4 mutations in previously untested homologous regions of the channel also give rise to the anomalous current.
The functional properties of mutant NaV1.4 channels were studied with voltage-clamp techniques in an oocyte expression system.
The HypoPP mutation NaV1.4-R1132Q conducts an anomalous gating pore current, but the homologous R1448C mutation in paramyotonia congenita does not.
Gating pore currents arising from missense mutations at arginine residues in the voltage sensor domains of NaV1.4 are a common feature of HypoPP mutant channels and contribute to the attacks of paralysis.
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