Currents In Nav1 Research Articles

NaV1.8/NaV1.9 double deletion mildly affects acute pain responses in mice.

The 2 tetrodotoxin-resistant (TTXr) voltage-gated sodium channel subtypes NaV1.8 and NaV1.9 are important for peripheral pain signaling. As determinants of sensory neuron excitability, they are essential for the initial transduction of sensory stimuli, the electrogenesis of the action potential, and the release of neurotransmitters from sensory neuron terminals. NaV1.8 and NaV1.9, which are encoded by SCN10A and SCN11A, respectively, are predominantly expressed in pain-sensitive (nociceptive) neurons localized in the dorsal root ganglia (DRG) along the spinal cord and in the trigeminal ganglia. Mutations in these genes cause various pain disorders in humans. Gain-of-function missense variants in SCN10A result in small fiber neuropathy, while distinct SCN11A mutations cause, i. a., congenital insensitivity to pain, episodic pain, painful neuropathy, and cold-induced pain. To determine the impact of loss-of-function of both channels, we generated NaV1.8/NaV1.9 double knockout (DKO) mice using clustered regularly interspaced short palindromic repeats/Cas-mediated gene editing to achieve simultaneous gene disruption. Successful knockout of both channels was verified by whole-cell recordings demonstrating the absence of NaV1.8- and NaV1.9-mediated Na+ currents in NaV1.8/NaV1.9 DKO DRG neurons. Global RNA sequencing identified significant deregulation of C-LTMR marker genes as well as of pain-modulating neuropeptides in NaV1.8/NaV1.9 DKO DRG neurons, which fits to the overall only moderately impaired acute pain behavior observed in DKO mice. Besides addressing the function of both sodium channels in pain perception, we further demonstrate that the null-background is a very valuable tool for investigations on the functional properties of individual human disease-causing variants in NaV1.8 or NaV1.9 in their native physiological environment.

Increased Resurgent Sodium Currents in Nav1.8 Contribute to Nociceptive Sensory Neuron Hyperexcitability Associated with Peripheral Neuropathies.

Neuropathic pain is a significant public health challenge, yet the underlying mechanisms remain poorly understood. Painful small fiber neuropathy (SFN) may be caused by gain-of-function mutations in Nav1.8, a sodium channel subtype predominantly expressed in peripheral nociceptive neurons. However, it is not clear how Nav1.8 disease mutations induce sensory neuron hyperexcitability. Here we studied two mutations in Nav1.8 associated with hypersensitive sensory neurons: G1662S reported in painful SFN; and T790A, which underlies increased pain behaviors in the Possum transgenic mouse strain. We show that, in male DRG neurons, these mutations, which impair inactivation, significantly increase TTX-resistant resurgent sodium currents mediated by Nav1.8. The G1662S mutation doubled resurgent currents, and the T790A mutation increased them fourfold. These unusual currents are typically evoked during the repolarization phase of action potentials. We show that the T790A mutation greatly enhances DRG neuron excitability by reducing current threshold and increasing firing frequency. Interestingly, the mutation endows DRG neurons with multiple early afterdepolarizations and leads to substantial prolongation of action potential duration. In DRG neurons, siRNA knockdown of sodium channel β4 subunits fails to significantly alter T790A current density but reduces TTX-resistant resurgent currents by 56%. Furthermore, DRG neurons expressing T790A channels exhibited reduced excitability with fewer early afterdepolarizations and narrower action potentials after β4 knockdown. Together, our data demonstrate that open-channel block of TTX-resistant currents, enhanced by gain-of-function mutations in Nav1.8, can make major contributions to the hyperexcitability of nociceptive neurons, likely leading to altered sensory phenotypes including neuropathic pain in SFN.SIGNIFICANCE STATEMENT This work demonstrates that two disease mutations in the voltage-gated sodium channel Nav1.8 that induce nociceptor hyperexcitability increase resurgent currents. Nav1.8 is crucial for pain sensations. Because resurgent currents are evoked during action potential repolarization, they can be crucial regulators of action potential activity. Our data indicate that increased Nav1.8 resurgent currents in DRG neurons greatly prolong action potential duration and enhance repetitive firing. We propose that Nav1.8 open-channel block is a major factor in Nav1.8-associated pain mechanisms and that targeting the molecular mechanism underlying these unique resurgent currents represents a novel therapeutic target for the treatment of aberrant pain sensations.

Lack of influence of sex hormones on Brugada syndrome-associated mutant Nav1.5 sodium channel

Brugada syndrome (BS) is an autosomal dominant disease. The most common causes of BS are loss-of-function mutations occur in the SCN5A gene which encodes the sodium channel protein Nav1.5. BS has a higher incidence rate in males and the underlying mechanisms of the gender inequality are not yet fully understood. Considering sex hormones are among the most important factors behind gender differences and have previously been shown to regulate the activity of multiple cardiac ion channels, we hypothesized that sex hormones also affect Nav1.5 function which lead to BS predominantly affecting males. In this study, we investigate the protein expression level and current of Nav1.5 in the HEK293 cells cotransfected with SCN5A and sex hormone receptor plasmids using both wild-type SCN5A and BS-associated SCN5A channel mutants R878C and R104W. Our findings showed that sex hormones have no effects on the protein expression level and current of the wild-type Nav1.5, neither does it affect the protein expression level and current of BS-associated Nav1.5 mutants R878C and R104W, regardless of homozygous or heterozygous state. Our results suggest that the male preponderance of BS does not arise from the effects of the sex hormones on Nav1.5. Further studies are needed to explain the male preponderance of this disease.

PRRT2 controls neuronal excitability by negatively modulating Na+ channel 1.2/1.6 activity.

See Lerche (doi:10.1093/brain/awy073) for a scientific commentary on this article.Proline-rich transmembrane protein 2 (PRRT2) is the causative gene for a heterogeneous group of familial paroxysmal neurological disorders that include seizures with onset in the first year of life (benign familial infantile seizures), paroxysmal kinesigenic dyskinesia or a combination of both. Most of the PRRT2 mutations are loss-of-function leading to haploinsufficiency and 80% of the patients carry the same frameshift mutation (c.649dupC; p.Arg217Profs*8), which leads to a premature stop codon. To model the disease and dissect the physiological role of PRRT2, we studied the phenotype of neurons differentiated from induced pluripotent stem cells from previously described heterozygous and homozygous siblings carrying the c.649dupC mutation. Single-cell patch-clamp experiments on induced pluripotent stem cell-derived neurons from homozygous patients showed increased Na+ currents that were fully rescued by expression of wild-type PRRT2. Closely similar electrophysiological features were observed in primary neurons obtained from the recently characterized PRRT2 knockout mouse. This phenotype was associated with an increased length of the axon initial segment and with markedly augmented spontaneous and evoked firing and bursting activities evaluated, at the network level, by multi-electrode array electrophysiology. Using HEK-293 cells stably expressing Nav channel subtypes, we demonstrated that the expression of PRRT2 decreases the membrane exposure and Na+ current of Nav1.2/Nav1.6, but not Nav1.1, channels. Moreover, PRRT2 directly interacted with Nav1.2/Nav1.6 channels and induced a negative shift in the voltage-dependence of inactivation and a slow-down in the recovery from inactivation. In addition, by co-immunoprecipitation assays, we showed that the PRRT2-Nav interaction also occurs in brain tissue. The study demonstrates that the lack of PRRT2 leads to a hyperactivity of voltage-dependent Na+ channels in homozygous PRRT2 knockout human and mouse neurons and that, in addition to the reported synaptic functions, PRRT2 is an important negative modulator of Nav1.2 and Nav1.6 channels. Given the predominant paroxysmal character of PRRT2-linked diseases, the disturbance in cellular excitability by lack of negative modulation of Na+ channels appears as the key pathogenetic mechanism.

'Neonatal' Nav1.2 reduces neuronal excitability and affects seizure susceptibility and behaviour.

Developmentally regulated alternative splicing produces 'neonatal' and 'adult' isoforms of four Na(+) channels in human brain, NaV1.1, NaV1.2, NaV1.3 and NaV1.6. Heterologously expressed 'neonatal' NaV1.2 channels are less excitable than 'adult' channels; however, functional importance of this difference is unknown. We hypothesized that the 'neonatal' NaV1.2 may reduce neuronal excitability and have a seizure-protective role during early brain development. To test this hypothesis, we generated NaV1.2(adult) mice expressing only the 'adult' NaV1.2, and compared the firing properties of pyramidal cortical neurons, as well as seizure susceptibility, between the NaV1.2(adult) and wild-type (WT) mice at postnatal day 3 (P3), when the 'neonatal' isoform represents 65% of the WT NaV1.2. We show significant increases in action potential firing in NaV1.2(adult) neurons and in seizure susceptibility of NaV1.2(adult) mice, supporting our hypothesis. At postnatal day 15 (P15), when 17% of the WT NaV1.2 is 'neonatal', the firing properties of NaV1.2(adult) and WT neurons converged. However, inhibitory postsynaptic currents in NaV1.2(adult) neurons were larger and the expression level of Scn2a mRNA was 24% lower compared with the WT. The enhanced seizure susceptibility of the NaV1.2(adult) mice persisted into adult age. The adult NaV1.2(adult) mice also exhibited greater risk-taking behaviour. Overall, our data reveal a significant impact of 'neonatal' NaV1.2 on neuronal excitability, seizure susceptibility and behaviour and may contribute to our understanding of NaV1.2 roles in health and diseases such as epilepsy and autism.

Inhibition of voltage-gated Na⁺ channels by the synthetic cannabinoid ajulemic acid.

The synthetic cannabinoid ajulemic acid has been demonstrated to alleviate pain in patients suffering from chronic neuropathic pain. Cannabinoids interact with several molecules within the pain circuit, including a potent inhibition of voltage-gated sodium channels. In this study, we closely characterized this property on neuronal and nonneuronal sodium channels. The inhibition of sodium inward currents by ajulemic acid was studied in vitro. Human embryonic kidney 293t cells were used as the expression system for Nav1.2, 1.3, 1.4, 1.5, 1.5N406K, 1.5F1760A, and 1.7; Nav1.8 was transiently expressed in ND7/23 cells. Nav1.2, Nav1.3, and Nav 1.8 were from rats, and Nav1.4, Nav1.5, and Nav1.7 were of human origin. Sodium currents were analyzed by means of the whole cell patch-clamp technique. The investigated concentrations of ajulemic acid were 0.1, 0.3, 1, 3, 10, and 30 μmol/L. Ajulemic acid reversibly and concentration-dependently inhibited all voltage-gated sodium channel (Nav) isoforms investigated in this study, including Nav1.2, 1.3, 1.4, 1.5, 1.7, and 1.8. Tonic block of resting channels yielded half-maximal inhibitory concentration values between 2 and 9 μmol/L and was strongly enhanced on inactivated channels, suggesting state-dependent inhibition by ajulemic acid. Tonic block did not differ significantly when comparing Nav1.2 and Nav1.3, Nav1.4 and Nav1.5, and Nav1.7 and Nav1.8. Statistical analysis of other combinations of subunits (e.g., Nav1.2 and Nav1.4) by analysis of variance yielded a significant difference in block. Although we did not observe any relevant use-dependent block, ajulemic acid induced a strong hyperpolarizing shift of the voltage dependency of fast inactivation and modest shift of slow inactivation. The local anesthetic-insensitive Nav1.5 constructs N406K and F1760A displayed a preserved sensitivity to block by ajulemic acid. Finally, we found that low concentrations of ajulemic acid efficiently inhibited Navβ4 peptide-mediated resurgent currents in Nav1.5. Our data suggest that block of sodium channels can be a relevant mechanism by which ajulemic acid alleviates neuropathic pain. The potent inhibition of resurgent currents and the preserved block on local anesthetic-insensitive channels indicates that ajulemic acid interacts with a conserved but yet unknown site of sodium channels.

Functional Analysis of Stably Expressed Human Nav1.9

The tetrodotoxin (TTX)-resistant voltage gated sodium channels, NaV1.8 and NaV1.9, are important for neuronal pain pathways. NaV1.9 (a.k.a. NaN) is preferentially expressed in nociceptive neurons of dorsal root ganglia. The role of NaV1.9 in inflammatory and neuropathic pain along with a restricted cellular localization makes it an attractive target for novel analgesics. Most of our knowledge about NaV1.9 function comes from studies of rodent sensory neurons using internal solutions and recording protocols to differentiate NaV1.8 and NaV1.9 currents, as well as recent studies using neurons isolated from NaV1.8-null mice. To further elucidate the functional properties of NaV1.9, we developed a stable cell line expressing full length human NaV1.9. ND7/23 cells were stably transfected using a novel transposon system in combination with human β1 and β2 sodium channel accessory subunits. Whole-cell currents were recorded from a holding potential of −120 mV and elicited with 20 ms pulses from −80 to +50 mV in the continuous presence of 200 nM TTX to block an endogenous TTX-sensitive sodium current. We recorded slow-activating, persistent sodium currents in NaV1.9 expressing cells following 24 h incubation at 28°C (to boost cell surface expression). The whole-cell current peaked at −30 mV (15.2 ± 1.8 pA/pF, n=26) and exhibited a voltage-dependence of activation with V½ of −50.5 ± 1 mV and slope factor (k) of 7.2 ± 0.3 (n=26). Activation kinetics was much slower than that observed for neuronal, TTX-sensitive NaV channels; time to peak current (at −30 mV) was 12.6 ± 0.7 ms, n=26. Preliminary single channel recording demonstrated frequent late re-openings and a single channel conductance of ∼16 pS. These results demonstrate the biophysical properties of stably expressed human NaV1.9 channel and provide a cell based platform for the analysis of potential NaV1.9 therapeutic agents.

Inhibition of Navβ4 Peptide-Mediated Resurgent Sodium Currents in Nav1.7 Channels by Carbamazepine, Riluzole, and Anandamide

Paroxysmal extreme pain disorder (PEPD) and inherited erythromelalgia (IEM) are inherited pain syndromes arising from different sets of gain-of-function mutations in the sensory neuronal sodium channel isoform Nav1.7. Mutations associated with PEPD, but not IEM, result in destabilized inactivation of Nav1.7 and enhanced resurgent sodium currents. Resurgent currents arise after relief of ultra-fast open-channel block mediated by an endogenous blocking particle and are thought to influence neuronal excitability. As such, enhancement of resurgent currents may constitute a pathological mechanism contributing to sensory neuron hyperexcitability and pain hypersensitivity associated with PEPD. Furthermore, pain associated with PEPD, but not IEM, is alleviated by the sodium channel inhibitor carbamazepine. We speculated that selective attenuation of PEPD-enhanced resurgent currents might contribute to this therapeutic effect. Here we examined whether carbamazepine and two other sodium channel inhibitors, riluzole and anandamide, exhibit differential inhibition of resurgent currents. To gain further insight into the potential mechanism(s) of resurgent currents, we examined whether these inhibitors produced correlative changes in other properties of sodium channel inactivation. Using stably transfected human embryonic kidney 293 cells expressing wild-type Nav1.7 and the PEPD mutants T1464I and M1627K, we examined the effects of the three drugs on Navβ4 peptide-mediated resurgent currents. We observed a correlation between resurgent current inhibition and a drug-mediated increase in the rate of inactivation and inhibition of persistent sodium currents. Furthermore, although carbamazepine did not selectively target resurgent currents, anandamide strongly inhibited resurgent currents with minimal effects on the peak transient current amplitude, demonstrating that resurgent currents can be selectively targeted.

TNF-α enhances the currents of voltage gated sodium channels in uninjured dorsal root ganglion neurons following motor nerve injury

The ectopic discharges observed in uninjured dorsal root ganglion (DRG) neurons following various lesions of spinal nerves have been attributed to functional alterations of voltage-gated sodium channels (VGSCs). Such mechanisms may be important for the development of neuropathic pain. However, the pathophysiology underlying the functional modulation of VGSCs following nerve injury is largely unknown. Here, we studied this issue with use of a selective lumbar 5 ventral root transection (L5-VRT) model, in which dorsal root ganglion (DRG) neurons remain intact. We found that the L5-VRT increased the current densities of TTX-sensitive Na channels as well as currents in Nav1.8, but not Nav1.9 channels in uninjured DRG neurons. The thresholds of action potentials decreased and firing rates increased in DRG neurons following L5-VRT. As we found that levels of tumor necrosis factor-alpha (TNF-α) increased in cerebrospinal fluid (CSF) and in DRG tissue after L5-VRT, we tested whether the increased TNF-α might result in the changes in sodium channels. Indeed, recombinant rat TNF (rrTNF) enhanced the current densities of TTX-S and Nav1.8 in cultured DRG neurons dose-dependently. Furthermore, genetic deletion of TNF receptor 1 (TNFR-1) in mice attenuated the mechanical allodynia and prevented the increase in sodium currents in DRG neurons induced by L5-VRT. These data suggest that the increase in sodium currents in uninjured DRG neurons following nerve injury might be mediated by over-production of TNF-α.

Synthetic Ciguatoxins Selectively Activate Nav1.8-derived Chimeric Sodium Channels Expressed in HEK293 Cells

The synthetic ciguatoxin CTX3C has been shown to activate tetrodotoxin (TTX)-sensitive sodium channels (Na(v)1.2, Na(v)1.4, and Na(v)1.5) by accelerating activation kinetics and shifting the activation curve toward hyperpolarization (Yamaoka, K., Inoue, M., Miyahara, H., Miyazaki, K., and Hirama, M. (2004) Br. J. Pharmacol. 142, 879-889). In this study, we further explored the effects of CTX3C on the TTX-resistant sodium channel Na(v)1.8. TTX-resistant channels have been shown to be involved in transducing pain and related sensations (Akopian, A. N., Sivilotti, L., and Wood, J. N. (1996) Nature 379, 257-262). Thus, we hypothesized that ciguatoxin-induced activation of the Na(v)1.8 current would account for the neurological symptoms of ciguatera poisoning. We found that 0.1 mum CTX3C preferentially affected the activation process of the Na(v)1.8 channel compared with those of the Na(v)1.2 and Na(v)1.4 channels. Importantly, without stimulation, 0.1 mum CTX3C induced a large leakage current (I (L)). The conductance of the I (L) calculated relative to the maximum conductance (G (max)) was 10 times larger than that of Na(v)1.2 or Na(v)1.4. To determine the molecular domain of Na(v)1.8 responsible for conferring higher sensitivity to CTX3C, we made two chimeric constructs from Na(v)1.4 and Na(v)1.8. Chimeras containing the N-terminal half of Na(v)1.8 exhibited a large response similar to wild-type Na(v)1.8, indicating that the region conferring high sensitivity to ciguatoxin action is located in the D1 or D2 domains.

Ralfinamide administered orally before hindpaw neurectomy or postoperatively provided long-lasting suppression of spontaneous neuropathic pain-related behavior in the rat

Ralfinamide is analgesic when applied as a single dose in rodent models of stimulus-evoked chronic pain. However, it is unknown whether its chronic application after nerve injury can suppress spontaneous chronic pain, the main symptom driving patients to seek treatment. In this study ralfinamide was administered to rats at doses producing plasma levels similar to those causing analgesia in pain patients. The analgesic effect was tested on autotomy, a behavior of self-mutilation of a denervated paw that models spontaneous neuropathic pain. Sprague–Dawley male rats ( N = 10–20/group) underwent transection of the sciatic and saphenous nerves unilaterally. Ralfinamide or its vehicle were administered per os for 7 days preoperatively (80 mg/kg; bid), followed by the vehicle or Ralfinamide, until postoperative d42. Autotomy was scored daily until d63. Lasting ‘preemptive analgesia’ was found in rats treated with ralfinamide preoperatively, expressed by delayed autotomy onset ( P = 0.009) and reduced scores on d63 ( P = 0.01). Rats treated with ralfinamide (30 or 60 mg/kg; bid) from the operation till d42, but not preoperatively, also showed delayed autotomy ( P = 0.05, P = 0.006), and reduced autotomy scores lasting till d63 ( P = 0.02, P = 0.01), for the two doses, respectively. Combining ralfinamide treatments for 7 days preoperatively and 42 days postoperatively also resulted in significantly suppressed scores on d42 and d63 ( P = 0.005, P = 0.001, respectively). Suppression of neuropathic pain-related behavior was likely caused by a combination of mechanisms reported for ralfinamide, including inhibition of Na + and Ca ++ currents in Nav1.3, Nav1.7, Nav1.8, and Cav2.2 channels in rat DRG neurons, inhibition of substance P release from spinal cord synaptosomes, NMDA receptor antagonism and neuroprotection.

Painful Channels

Paroxysmal extreme pain disorder (PEPD), previously known as familial rectal pain (FRP, OMIM 167400), is an inherited disease causing intense burning rectal, ocular, and submandibular pain and flushing. Fertleman et al. (this issue of Neuron) show that mutations in SCN9A, the gene encoding the sodium channel Na(V)1.7 channels, are responsible for this syndrome. Together with earlier work implicating a distinct class of functional mutations in SCN9A in a distinct inherited pain syndrome, these results point to Na(V)1.7 channels as key players in signaling nociceptive information and as a potential target for drug therapy of chronic pain.

A mutation in the local anaesthetic binding site abolishes toluene effects in sodium channels

Toluene is a solvent of abuse that inhibits cardiac sodium channels in a manner that resembles the action of local anaesthetics. The purpose of this work was to analyze toluene effects on skeletal muscle sodium channels with and without β 1 subunit (Na v1.4 + β 1 and Na v1.4 − β 1, respectively) expressed in Xenopus laevis oocytes and to compare them with those produced in the F1579A mutant channel lacking a local anaesthetic binding site. Toluene inhibited Na v1.4 sodium currents (IC 50 = 2.7 mM in Na v1.4 + β 1 and 2.2 mM in Na v1.4 − β 1) in a concentration dependent way. Toluene (3 mM) blocked sodium currents in Na v1.4 channels proportionally throughout the entire current–voltage relationship producing inactivation at more negative potentials. Minimal inhibition was produced by 3 mM toluene in F1579A mutant channels. Recovery from inactivation was slower both in Na v1.4 and F1579A channels in the presence of 3 mM toluene. The solvent blocked sodium currents in a use-dependent and frequency-dependent manner in Na v1.4 channels. A single mutation in the local anaesthetic binding site of Na v1.4 channels almost abolished toluene effects. These results suggest that this site is important for toluene action.