The sodium channel Nav1.6, encoded by the gene SCN8A, is one of the major voltage-gated channels in human brain. The sequences of sodium channels have been highly conserved during evolution, and minor changes in biophysical properties can have a major impact in vivo. Insight into the role of Nav1.6 has come from analysis of spontaneous and induced mutations of mouse Scn8a during the past 18 years. Only within the past year has the role of SCN8A in human disease become apparent from whole exome and genome sequences of patients with sporadic disease. Unique features of Nav1.6 include its contribution to persistent current, resurgent current, repetitive neuronal firing, and subcellular localization at the axon initial segment (AIS) and nodes of Ranvier. Loss of Nav1.6 activity results in reduced neuronal excitability, while gain-of-function mutations can increase neuronal excitability. Mouse Scn8a (med) mutants exhibit movement disorders including ataxia, tremor and dystonia. Thus far, more than ten human de novo mutations have been identified in patients with two types of disorders, epileptic encephalopathy and intellectual disability. We review these human mutations as well as the unique features of Nav1.6 that contribute to its role in determining neuronal excitability in vivo. A supplemental figure illustrating the positions of amino acid residues within the four domains and 24 transmembrane segments of Nav1.6 is provided to facilitate the location of novel mutations within the channel protein.
voltage-gated sodium channels; epilepsy; intellectual disability; SCN8A; Nav1.6; neurogenetics; genetics; exomes
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
Indoxacarb (DPX-JW062) was recently developed as a new oxadiazine insecticide with high insecticidal activity and low mammalian toxicity. Previous studies showed that indoxacarb and its bioactive metabolite, N-decarbomethoxyllated JW062 (DCJW), block insect sodium channels in nerve preparations and isolated neurons. However, the molecular mechanism of indoxacarb/DCJW action on insect sodium channels is not well understood. In this study, we identified two cockroach sodium channel variants, BgNav1-1 and BgNav1-4, which differ in voltage dependence of fast and slow inactivation, and channel sensitivity to DCJW. The voltage dependence of fast inactivation and slow inactivation of BgNav1-4 were shifted in the hyperpolarizing direction compared with those of BgNav1-1 channels. At the holding potential of −90 mV, 20 μM of DCJW reduced the peak current of BgNav1-4 by about 40%, but had no effect on BgNav1-1. However, at the holding potential of −60 mV, DCJW also reduced the peak currents of BgNav1-1 by about 50%. Furthermore, DCJW delayed the recovery from slow inactivation of both variants. Substitution of E1689 in segment 4 of domain four (IVS4) of BgNav1-4 with a K, which is present in BgNav1-1, was sufficient to shift the voltage dependence of fast and slow inactivation of BgNav1-4 channels to the more depolarizing membrane potential close to that of BgNav1-1 channels. The E1689K change also eliminated the DCJW inhibition of BgNav1-4 at the hyperpolarizing holding potentials. These results show that the E1689K change is responsible for the difference in channel gating and sensitivity to DCJW between BgNav1-4 and BgNav1-1. Our results support the notion that DCJW preferably acts on the inactivated state of the sodium channel and demonstrate that K1689E is a major molecular determinant of the voltage-dependent inactivation and state-dependent action of DCJW.
Insect sodium channel; Insecticide; Indoxacarb; DCJW; Xenopus oocyte
Insect voltage-gated sodium (Nav) channels are formed by a well-known pore-forming α-subunit encoded by para-like gene and ancillary subunits related to TipE from the mutation “temperature-induced-paralysis locus E.” The role of these ancillary subunits in the modulation of biophysical and pharmacological properties of Na+ currents are not enough documented. The unique neuronal ancillary subunit TipE-homologous protein 1 of Drosophila melanogaster (DmTEH1) strongly enhances the expression of insect Nav channels when heterologously expressed in Xenopus oocytes. Here we report the cloning and functional expression of two neuronal DmTEH1-homologs of the cockroach, Periplaneta americana, PaTEH1A and PaTEH1B, encoded by a single bicistronic gene. In PaTEH1B, the second exon encoding the last 11-amino-acid residues of PaTEH1A is shifted to 3′UTR by the retention of a 96-bp intron-containing coding-message, thus generating a new C-terminal end. We investigated the gating and pharmacological properties of the Drosophila Nav channel variant (DmNav1-1) co-expressed with DmTEH1, PaTEH1A, PaTEH1B or a truncated mutant PaTEH1Δ(270-280) in Xenopus oocytes. PaTEH1B caused a 2.2-fold current density decrease, concomitant with an equivalent α-subunit incorporation decrease in the plasma membrane, compared to PaTEH1A and PaTEH1Δ(270-280). PaTEH1B positively shifted the voltage-dependences of activation and slow inactivation of DmNav1-1 channels to more positive potentials compared to PaTEH1A, suggesting that the C-terminal end of both proteins may influence the function of the voltage-sensor and the pore of Nav channel. Interestingly, our findings showed that the sensitivity of DmNav1-1 channels to lidocaine and to the pyrazoline-type insecticide metabolite DCJW depends on associated TEH1-like subunits. In conclusion, our work demonstrates for the first time that density, gating and pharmacological properties of Nav channels expressed in Xenopus oocytes can be modulated by an intron retention process in the transcription of the neuronal TEH1-like ancillary subunits of P. americana.
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 channels) play an important role in nociceptive transmission. They are intimately tied to the genesis and transmission of neuronal firing. Five different isoforms (Nav1.3, Nav1.6, Nav1.7, Nav1.8, and Nav1.9) have been linked to nociceptive responses. A change in the biophysical properties of these channels or in their expression levels occurs in different pathological pain states. However, the precise involvement of the isoforms in the genesis and transmission of nociceptive responses is unknown. The aim of the present study was to investigate the synergy between the different populations of Nav channels that give individual neurons a unique electrophysical profile. We used the patch-clamp technique in the whole-cell configuration to record Nav currents and action potentials from acutely dissociated small diameter DRG neurons (<30 μm) from adult rats. We also performed single cell qPCR on the same neurons. Our results revealed that there is a strong correlation between Nav currents and mRNA transcripts in individual neurons. A cluster analysis showed that subgroups formed by Nav channel transcripts by mRNA quantification have different biophysical properties. In addition, the firing frequency of the neurons was not affected by the relative populations of Nav channel. The synergy between populations of Nav channel in individual small diameter DRG neurons gives each neuron a unique electrophysiological profile. The Nav channel remodeling that occurs in different pathological pain states may be responsible for the sensitization of the neurons.
voltage-gated sodium channel; neuronal excitability; pain; biophysical properties; dorsal root ganglia neurons
β subunits of mammalian sodium channels play important roles in modulating the expression and gating of mammalian sodium channels. However, there are no orthologs of β subunits in insects. Instead, an unrelated protein, TipE in Drosophila melanogaster and its orthologs in other insects, is thought to be a sodium channel auxiliary subunit. In addition, there are four TipE-homologous genes (TEH1-4) in D. melanogaster and three to four orthologs in other insect species. TipE and TEH1-3 have been shown to enhance the peak current of various insect sodium channels expressed in Xenopus oocytes. However, limited information is available on how these proteins modulate the gating of sodium channels, particularly sodium channel variants generated by alternative splicing and RNA editing. In this study, we compared the effects of TEH1 and TipE on the function of three Drosophila sodium channel splice variants, DmNav9-1, DmNav22, and DmNav26, in Xenopus oocytes. Both TipE and TEH1 enhanced the amplitude of sodium current and accelerated current decay of all three sodium channels tested. Strikingly, TEH1 caused hyperpolarizing shifts in the voltage-dependence of activation, fast inactivation and slow inactivation of all three variants. In contrast, TipE did not alter these gating properties except for a hyperpolarizing shift in the voltage-dependence of fast inactivation of DmNav26. Further analysis of the gating kinetics of DmNav9-1 revealed that TEH1 accelerated the entry of sodium channels into the fast inactivated state and slowed the recovery from both fast- and slow-inactivated states, thereby, enhancing both fast and slow inactivation. These results highlight the differential effects of TipE and TEH1 on the gating of insect sodium channels and suggest that TEH1 may play a broader role than TipE in regulating sodium channel function and neuronal excitability in vivo.
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
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.
Voltage-gated sodium (Nav) channels are required for impulse conductance in excitable tissues. Navs have been linked to human cancers, including prostate. The expression and distribution of Nav isoforms (Nav1.1-Nav1.9) in human prostate cancer are not well established. Here, we evaluated the expression of these isoforms and investigated the expression of Nav1.8 in human prostate cancer tissues. Nav1.8 was highly expressed in all examined cells. Expression of Nav1.1, Nav1.2, and Nav1.9 were high in DU-145, PC-3 and PC-3M cells compared to LNCaP (hormone-dependent), C4-2, C4-2B, and CWR22Rv-1 cells. Nav1.5 and Nav1.6 were expressed in all cells examined. Nav1.7 expression was absent in PC-3M and CWR22Rv-1, but expressed in the other cells examined. Immunohistochemistry revealed intensive Nav1.8 staining correlated with more advanced pathologic stage of disease. Increased intensity of nuclear Nav1.8 correlated with increased Gleason grade. Our results revealed that Nav1.8 is universally expressed in human prostate cancer cells. Nav1.8 expression statistically correlated with pathologic stage (P=0.04) and Gleason score (P=0.01) of human prostate tissue specimens. The aberrant nuclear localization of Nav1.8 with advanced prostate cancer tissues warrant further investigation into use of Nav1.8 as a potential biomarker to differentiate between early and advanced disease.
Voltage-gated sodium channel; Prostate cancer; Prostate biomarker; Gleason score
Voltage-gated sodium channels are responsible for the rising phase of the action potential in cardiac muscle. Previously, both TTX-sensitive neuronal sodium channels (NaV1.1, NaV1.2, NaV1.3, NaV1.4 and NaV1.6) and the TTX-resistant cardiac sodium channel (NaV1.5) have been detected in cardiac myocytes, but relative levels of protein expression of the isoforms were not determined. Using a quantitative approach, we analyzed z-series of confocal microscopy images from individual mouse myocytes stained with either anti-NaV1.1, anti-NaV1.2, anti-NaV1.3, anti-NaV1.4, anti-NaV1.5, or anti-NaV1.6 antibodies and calculated the relative intensity of staining for these sodium channel isoforms. Our results indicate that the TTX-sensitive channels represented approximately 23% of the total channels, whereas the TTX-resistant NaV1.5 channel represented 77% of the total channel staining in mouse ventricular myocytes. These ratios are consistent with previous electrophysiological studies in mouse ventricular myocytes. NaV1.5 was located at the cell surface, with high density at the intercalated disc, but was absent from the transverse (t)-tubular system, suggesting that these channels support surface conduction and inter-myocyte transmission. Low-level cell surface staining of NaV1.4 and NaV1.6 channels suggest a minor role in surface excitation and conduction. Conversely, NaV1.1 and NaV1.3 channels are localized to the t-tubules and are likely to support t-tubular transmission of the action potential to the myocyte interior. This quantitative immunocytochemical approach for assessing sodium channel density and localization provides a more precise view of the relative importance and possible roles of these individual sodium channel protein isoforms in mouse ventricular myocytes and may be applicable to other species and cardiac tissue types.
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
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
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 voltage-gated sodium channel (Nav) plays a key role in regulation of neuronal excitability. Aberrant regulation of Nav expression and/or function can result in an imbalance in neuronal activity which can progress to epilepsy. Regulation of Nav activity is achieved by coordination of a multitude of mechanisms including RNA alternative splicing and translational repression. Understanding of these regulatory mechanisms is complicated by extensive genetic redundancy: the mammalian genome encodes ten Navs. By contrast, the genome of the fruitfly, Drosophila melanogaster, contains just one Nav homologue, encoded by paralytic (DmNav). Analysis of splicing in DmNav shows variants exhibit distinct gating properties including varying magnitudes of persistent sodium current (INaP). Splicing by Pasilla, an identified RNA splicing factor, alters INaP magnitude as part of an activity-dependent mechanism. Enhanced INaP promotes membrane hyperexcitability that is associated with seizure-like behaviour in Drosophila. Nova-2, a mammalian Pasilla homologue, has also been linked to splicing of Navs and, moreover, mouse gene knockouts display seizure-like behaviour.
Expression level of Navs is also regulated through a mechanism of translational repression in both flies and mammals. The translational repressor Pumilio (Pum) can bind to Nav transcripts and repress the normal process of translation, thus regulating sodium current (INa) density in neurons. Pum2-deficient mice exhibit spontaneous EEG abnormalities. Taken together, aberrant regulation of Nav function and/or expression is often epileptogenic. As such, a better understanding of regulation of membrane excitability through RNA alternative splicing and translational repression of Navs should provide new leads to treat epilepsy.
Excitability; Drosophila; Epilepsy; Paralytic; Splicing; Translational repression
A direct role of sodium channels in pain has recently been confirmed by establishing a monogenic link between SCN9A, the gene which encodes sodium channel Nav1.7, and pain disorders in humans, with gain-of-function mutations causing severe pain syndromes, and loss-of-function mutations causing congenital indifference to pain. Expression of sodium channel Nav1.8 in DRG neurons has also been shown to be essential for the manifestation of mutant Nav1.7-induced neuronal hyperexcitability. These findings have confirmed key roles of Nav1.7 and Nav1.8 in pain and identify these channels as novel targets for pain therapeutic development. Ranolazine preferentially blocks cardiac late sodium currents at concentrations that do not significantly reduce peak sodium current. Ranolazine also blocks wild-type Nav1.7 and Nav1.8 channels in a use-dependent manner. However, ranolazine's effects on gain-of-function mutations of Nav1.7 and on DRG neuron excitability have not been investigated. We used voltage- and current-clamp recordings to evaluate the hypothesis that ranolazine may be effective in regulating Nav1.7-induced DRG neuron hyperexcitability.
We show that ranolazine produces comparable block of peak and ramp currents of wild-type Nav1.7 and mutant Nav1.7 channels linked to Inherited Erythromelalgia and Paroxysmal Extreme Pain Disorder. We also show that ranolazine, at a clinically-relevant concentration, blocks high-frequency firing of DRG neurons expressing wild-type but not mutant channels.
Our data suggest that ranalozine can attenuate hyperexcitability of DRG neurons over-expressing wild-type Nav1.7 channels, as occurs in acquired neuropathic and inflammatory pain, and thus merits further study as an alternative to existing non-selective sodium channel blockers.
Ranolazine is an antianginal agent that targets a number of ion channels in the heart, including cardiac voltage-gated Na+ channels. However, ranolazine block of muscle and neuronal Na+ channel isoforms has not been examined. We compared the state- and use-dependent ranolazine block of Na+ currents carried by muscle Nav1.4, cardiac Nav1.5, and neuronal Nav1.7 isoforms expressed in human embryonic kidney 293T cells. Resting and inactivated block of Na+ channels by ranolazine were generally weak, with a 50% inhibitory concentration (IC50) ≥ 60 μM. Use-dependent block of Na+ channel isoforms by ranolazine during repetitive pulses (+50 mV/10 ms at 5 Hz) was strong at 100 μM, up to 77% peak current reduction for Nav1.4, 67% for Nav1.5, and 83% for Nav1.7. In addition, we found conspicuous time-dependent block of inactivation-deficient Nav1.4, Nav1.5, and Nav1.7 Na+ currents by ranolazine with estimated IC50 values of 2.4, 6.2, and 1.7 μM, respectively. On- and off-rates of ranolazine were 8.2 μM−1 s−1 and 22 s−1, respectively, for Nav1.4 open channels and 7.1 μM−1 s−1 and 14 s−1, respectively, for Nav1.7 counterparts. A F1579K mutation at the local anesthetic receptor of inactivation-deficient Nav1.4 Na+ channels reduced the potency of ranolazine ~17-fold. We conclude that: 1) both muscle and neuronal Na+ channels are as sensitive to ranolazine block as their cardiac counterparts; 2) at its therapeutic plasma concentrations, ranolazine interacts predominantly with the open but not resting or inactivated Na+ channels; and 3) ranolazine block of open Na+ channels is via the conserved local anesthetic receptor albeit with a relatively slow on-rate.
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
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
In injured neurons, “leaky” voltage-gated sodium channels (Nav) underlie dysfunctional excitability that ranges from spontaneous subthreshold oscillations (STO), to ectopic (sometimes paroxysmal) excitation, to depolarizing block. In recombinant systems, mechanical injury to Nav1.6-rich membranes causes cytoplasmic Na+-loading and “Nav-CLS”, i.e., coupled left-(hyperpolarizing)-shift of Nav activation and availability. Metabolic injury of hippocampal neurons (epileptic discharge) results in comparable impairment: left-shifted activation and availability and hence left-shifted INa-window. A recent computation study revealed that CLS-based INa-window left-shift dissipates ion gradients and impairs excitability. Here, via dynamical analyses, we focus on sustained excitability patterns in mildly damaged nodes, in particular with more realistic Gaussian-distributed Nav-CLS to mimic “smeared” injury intensity. Since our interest is axons that might survive injury, pumps (sine qua non for live axons) are included. In some simulations, pump efficacy and system volumes are varied. Impacts of current noise inputs are also characterized. The diverse modes of spontaneous rhythmic activity evident in these scenarios are studied using bifurcation analysis. For “mild CLS injury”, a prominent feature is slow pump/leak-mediated EIon oscillations. These slow oscillations yield dynamic firing thresholds that underlie complex voltage STO and bursting behaviors. Thus, Nav-CLS, a biophysically justified mode of injury, in parallel with functioning pumps, robustly engenders an emergent slow process that triggers a plethora of pathological excitability patterns. This minimalist “device” could have physiological analogs. At first nodes of Ranvier and at nociceptors, e.g., localized lipid-tuning that modulated Nav midpoints could produce Nav-CLS, as could co-expression of appropriately differing Nav isoforms.
Nerve cells damaged by trauma, stroke, epilepsy, inflammatory conditions etc, have chronically leaky sodium channels that eventually kill. The usual job of sodium channels is to make brief voltage signals –action potentials– for long distance propagation. After sodium channels open to generate action potentials, sodium pumps work harder to re-establish the intracellular/extracellular sodium imbalance that is, literally, the neuron's battery for firing action potentials. Wherever tissue damage renders membranes overly fluid, we hypothesize, sodium channels become chronically leaky. Our experimental findings justify this. In fluidized membranes, sodium channel voltage sensors respond too easily, letting channels spend too much time open. Channels leak, pumps respond. By mathematical modeling, we show that in damaged channel-rich membranes the continual pump/leak counterplay would trigger the kinds of bizarre intermittent action potential bursts typical of injured neurons. Arising ectopically from injury regions, such neuropathic firing is unrelated to events in the external world. Drugs that can silence these deleterious electrical barrages without blocking healthy action potentials are needed. If fluidized membranes house the problematic leaky sodium channels, then drug side effects could be diminished by using drugs that accumulate most avidly into fluidized membranes, and that bind their targets with highest affinity there.
Mechanical, ischemic, and inflammatory injuries to voltage-gated sodium channel (Nav)-rich membranes of axon initial segments and nodes of Ranvier render Nav channels dangerously leaky. By what means? The behavior of recombinant Nav1.6 (Wang et al., 2009) leads us to postulate that, in neuropathologic conditions, structural degradation of axolemmal bilayer fosters chronically left-shifted Nav channel operation, resulting in ENa rundown. This “sick excitable cell Nav-leak” would encompass left-shifted fast- and slow-mode based persistent INa (i.e., Iwindow and slow-inactivating INa). Bilayer-damage-induced electrophysiological dysfunctions of native-Nav channels, and effects on inhibitors on those channels, should, we suggest, be studied in myelinated axons, exploiting INa(V,t) hysteresis data from sawtooth ramp clamp. We hypothesize that (like dihydropyridines for Ca channels), protective lipophilic Nav antagonists would partition more avidly into disorderly bilayers than into the well-packed bilayers characteristic of undamaged, healthy plasma membrane. Whereas inhibitors using aqueous routes would access all Navs equally, differential partitioning into “sick bilayer” would co-localize lipophilic antagonists with “sick-Nav channels,” allowing for more specific targeting of impaired cells. Molecular fine-tuning of Nav antagonists to favor more avid partitioning into damaged than into intact bilayers could reduce side effects. In potentially salvageable neurons of traumatic and/or ischemic penumbras, in inflammatory neuropathies, in muscular dystrophy, in myocytes of cardiac infarct borders, Nav-leak driven excitotoxicity overwhelms cellular repair mechanisms. Precision-tuning of a lipophilic Nav antagonist for greatest efficacy in mildly damaged membranes could render it suitable for the prolonged continuous administration needed to allow for the remodeling of the excitable membranes, and thus functional recovery.
traumatic brain injury; spinal; riluzole; ranolazine; simulation; modeling
Inherited mutations in voltage-gated sodium channels (VGSCs; or Nav) cause many
disorders of excitability, including epilepsy, chronic pain, myotonia, and cardiac
arrhythmias. Understanding the functional consequences of the disease-causing
mutations is likely to provide invaluable insight into the roles that VGSCs play in
normal and abnormal excitability. Here, we sought to test the hypothesis that
disease-causing mutations lead to increased resurgent currents, unusual sodium
currents that have not previously been implicated in disorders of excitability. We
demonstrated that a paroxysmal extreme pain disorder (PEPD) mutation in the human
peripheral neuronal sodium channel Nav1.7, a paramyotonia congenita (PMC) mutation in
the human skeletal muscle sodium channel Nav1.4, and a long-QT3/SIDS mutation in the
human cardiac sodium channel Nav1.5 all substantially increased the amplitude of
resurgent sodium currents in an optimized adult rat–derived dorsal root
ganglion neuronal expression system. Computer simulations indicated that resurgent
currents associated with the Nav1.7 mutation could induce high-frequency action
potential firing in nociceptive neurons and that resurgent currents associated with
the Nav1.5 mutation could broaden the action potential in cardiac myocytes. These
effects are consistent with the pathophysiology associated with the respective
channelopathies. Our results indicate that resurgent currents are associated with
multiple channelopathies and are likely to be important contributors to neuronal and
muscle disorders of excitability.
Rapid energy-efficient signaling along vertebrate axons is achieved through intricate subcellular arrangements of voltage-gated ion channels and myelination. One recently appreciated example is the tight colocalization of Kv7 potassium channels and voltage-gated sodium (Nav) channels in the axonal initial segment and nodes of Ranvier. The local biophysical properties of these Kv7 channels and the functional impact of colocalization with Nav channels remain poorly understood. Here, we quantitatively examined Kv7 channels in myelinated axons of rat neocortical pyramidal neurons using high-resolution confocal imaging and patch-clamp recording. Kv7.2 and 7.3 immunoreactivity steeply increased within the distal two-thirds of the axon initial segment and was mirrored by the conductance density estimates, which increased from ∼12 (proximal) to 150 pS μm−2 (distal). The axonal initial segment and nodal M-currents were similar in voltage dependence and kinetics, carried by Kv7.2/7.3 heterotetramers, 4% activated at the resting membrane potential and rapidly activated with single-exponential time constants (∼15 ms at 28 mV). Experiments and computational modeling showed that while somatodendritic Kv7 channels are strongly activated by the backpropagating action potential to attenuate the afterdepolarization and repetitive firing, axonal Kv7 channels are minimally recruited by the forward-propagating action potential. Instead, in nodal domains Kv7.2/7.3 channels were found to increase Nav channel availability and action potential amplitude by stabilizing the resting membrane potential. Thus, Kv7 clustering near axonal Nav channels serves specific and context-dependent roles, both restraining initiation and enhancing conduction of the action potential.
axon; excitability; Kv7
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
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)