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 ß4 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 ß4 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.

FHF2 isoforms differentially regulate Nav1.6-mediated resurgent sodium currents in dorsal root ganglion neurons.

Nav1.6 and Nav1.6-mediated resurgent currents have been implicated in several pain pathologies. However, our knowledge of how fast resurgent currents are modulated in neurons is limited. Our study explored the potential regulation of Nav1.6-mediated resurgent currents by isoforms of fibroblast growth factor homologous factor 2 (FHF2) in an effort to address the gap in our knowledge. FHF2 isoforms colocalize with Nav1.6 in peripheral sensory neurons. Cell line studies suggest that these proteins differentially regulate inactivation. In particular, FHF2A mediates long-term inactivation, a mechanism proposed to compete with the open-channel blocker mechanism that mediates resurgent currents. On the other hand, FHF2B lacks the ability to mediate long-term inactivation and may delay inactivation favoring open-channel block. Based on these observations, we hypothesized that FHF2A limits resurgent currents, whereas FHF2B enhances resurgent currents. Overall, our results suggest that FHF2A negatively regulates fast resurgent current by enhancing long-term inactivation and delaying recovery. In contrast, FHF2B positively regulated resurgent current and did not alter long-term inactivation. Chimeric constructs of FHF2A and Navß4 (likely the endogenous open channel blocker in sensory neurons) exhibited differential effects on resurgent currents, suggesting that specific regions within FHF2A and Navß4 have important regulatory functions. Our data also indicate that FHFAs and FHF2B isoform expression are differentially regulated in a radicular pain model and that associated neuronal hyperexcitability is substantially attenuated by a FHFA peptide. As such, these findings suggest that FHF2A and FHF2B regulate resurgent current in sensory neurons and may contribute to hyperexcitability associated with some pain pathologies.

Aberrant epilepsy-associated mutant Nav1.6 sodium channel activity can be targeted with cannabidiol.

Mutations in brain isoforms of voltage-gated sodium channels have been identified in patients with distinct epileptic phenotypes. Clinically, these patients often do not respond well to classic anti-epileptics and many remain refractory to treatment. Exogenous as well as endogenous cannabinoids have been shown to target voltage-gated sodium channels and cannabidiol has recently received attention for its potential efficacy in the treatment of childhood epilepsies. In this study, we further investigated the ability of cannabinoids to modulate sodium currents from wild-type and epilepsy-associated mutant voltage-gated sodium channels. We first determined the biophysical consequences of epilepsy-associated missense mutations in both Nav1.1 (arginine 1648 to histidine and asparagine 1788 to lysine) and Nav1.6 (asparagine 1768 to aspartic acid and leucine 1331 to valine) by obtaining whole-cell patch clamp recordings in human embryonic kidney 293T cells with 200 µM Navß4 peptide in the pipette solution to induce resurgent sodium currents. Resurgent sodium current is an atypical near threshold current predicted to increase neuronal excitability and has been implicated in multiple disorders of excitability. We found that both mutations in Nav1.6 dramatically increased resurgent currents while mutations in Nav1.1 did not. We then examined the effects of anandamide and cannabidiol on peak transient and resurgent currents from wild-type and mutant channels. Interestingly, we found that cannabidiol can preferentially target resurgent sodium currents over peak transient currents generated by wild-type Nav1.6 as well as the aberrant resurgent and persistent current generated by Nav1.6 mutant channels. To further validate our findings, we examined the effects of cannabidiol on endogenous sodium currents from striatal neurons, and similarly we found an inhibition of resurgent and persistent current by cannabidiol. Moreover, current clamp recordings show that cannabidiol reduces overall action potential firing of striatal neurons. These findings suggest that cannabidiol could be exerting its anticonvulsant effects, at least in part, through its actions on voltage-gated sodium channels, and resurgent current may be a promising therapeutic target for the treatment of epilepsy syndromes.

Navß4 regulates fast resurgent sodium currents and excitability in sensory neurons.

BACKGROUND: Increased electrical activity in peripheral sensory neurons including dorsal root ganglia (DRG) and trigeminal ganglia neurons is an important mechanism underlying pain. Voltage gated sodium channels (VGSC) contribute to the excitability of sensory neurons and are essential for the upstroke of action potentials. A unique type of VGSC current, resurgent current (INaR), generates an inward current at repolarizing voltages through an alternate mechanism of inactivation referred to as open-channel block. INaRs are proposed to enable high frequency firing and increased INaRs in sensory neurons are associated with pain pathologies. While Nav1.6 has been identified as the main carrier of fast INaR, our understanding of the mechanisms that contribute to INaR generation is limited. Specifically, the open-channel blocker in sensory neurons has not been identified. Previous studies suggest Navß4 subunit mediates INaR in central nervous system neurons. The goal of this study was to determine whether Navß4 regulates INaR in DRG sensory neurons. RESULTS: Our immunocytochemistry studies show that Navß4 expression is highly correlated with Nav1.6 expression predominantly in medium-large diameter rat DRG neurons. Navß4 knockdown decreased endogenous fast INaR in medium-large diameter neurons as measured with whole-cell voltage clamp. Using a reduced expression system in DRG neurons, we isolated recombinant human Nav1.6 sodium currents in rat DRG neurons and found that overexpression of Navß4 enhanced Nav1.6 INaR generation. By contrast neither overexpression of Navß2 nor overexpression of a Navß4-mutant, predicted to be an inactive form of Navß4, enhanced Nav1.6 INaR generation. DRG neurons transfected with wild-type Navß4 exhibited increased excitability with increases in both spontaneous activity and evoked activity. Thus, Navß4 overexpression enhanced INaR and excitability, whereas knockdown or expression of mutant Navß4 decreased INaR generation. CONCLUSION: INaRs are associated with inherited and acquired pain disorders. However, our ability to selectively target and study this current has been hindered due to limited understanding of how it is generated in sensory neurons. This study identified Navß4 as an important regulator of INaR and excitability in sensory neurons. As such, Navß4 is a potential target for the manipulation of pain sensations.

Mechanisms controlling neurite outgrowth in a pheochromocytoma cell line: the role of TRPC channels.

Transient Receptor Potential Canonical (TRPC) channels are implicated in modulating neurite outgrowth. The expression pattern of TRPCs changes significantly during brain development, suggesting that fine-tuning TRPC expression may be important for orchestrating neuritogenesis. To study how alterations in the TRPC expression pattern affect neurite outgrowth, we used nerve growth factor (NGF)-differentiated rat pheochromocytoma 12 (PC12) cells, a model system for neuritogenesis. In PC12 cells, NGF markedly up-regulated TRPC1 and TRPC6 expression, but down-regulated TRPC5 expression while promoting neurite outgrowth. Overexpression of TRPC1 augmented, whereas TRPC5 overexpression decelerated NGF-induced neurite outgrowth. Conversely, shRNA-mediated knockdown of TRPC1 decreased, whereas shRNA-mediated knockdown of TRPC5 increased NGF-induced neurite extension. Endogenous TRPC1 attenuated the anti-neuritogenic effect of overexpressed TRPC5 in part by forming the heteromeric TRPC1-TRPC5 channels. Previous reports suggested that TRPC6 may facilitate neurite outgrowth. However, we found that TRPC6 overexpression slowed down neuritogenesis, whereas dominant negative TRPC6 (DN-TRPC6) facilitated neurite outgrowth in NGF-differentiated PC12 cells. Consistent with these findings, hyperforin, a neurite outgrowth promoting factor, decreased TRPC6 expression in NGF-differentiated PC12 cells. Using pharmacological and molecular biological approaches, we determined that NGF up-regulated TRPC1 and TRPC6 expression via a p75(NTR)-IKK(2)-dependent pathway that did not involve TrkA receptor signaling in PC12 cells. Similarly, NGF up-regulated TRPC1 and TRPC6 via an IKK(2) dependent pathway in primary cultured hippocampal neurons. Thus, our data suggest that a balance of TRPC1, TRPC5, and TRPC6 expression determines neurite extension rate in neural cells, with TRPC6 emerging as an NGF-dependent "molecular damper" maintaining a submaximal velocity of neurite extension.

Luminescence rigidochromism and redox chemistry of pyrazolate-bridged binuclear platinum(II) diimine complex intercalated into zirconium phosphate layers.

The direct intercalation of a pyrazolate-bridged platinum(II) bipyridyl dimer ([{Pt(dmbpy)(µ-pz)}(2)](2+); dmbpy = 4,4'-dimethyl-2,2'-bipyridine, pz(-) = pyrazolate) within a zirconium phosphate (ZrP) framework has been accomplished. The physical and spectroscopic properties of [{Pt(dmbpy)(µ-pz)}(2)](2+) intercalated in ZrP were investigated by X-ray powder diffraction and X-ray photoelectron, infrared, absorption, and luminescence spectroscopies. Zirconium phosphate layers have a special microenvironment that is capable of supporting a variety of platinum oxidation states. Diffuse reflectance spectra from powders of the blue-gray intercalated materials show the formation of a low-energy band at 600 nm that is not present in the platinum dimer salt. The nonintercalated complex is nonemissive in room-temperature fluid solution, but gives rise to intense blue-green emission in a 4:1 ethanol/methanol 77 K frozen glassy solution. Powders and colloidal suspensions of [{Pt(dmbpy)(µ-pz)}(2)](2+)-exchanged ZrP materials exhibit intense emissions at room-temperature.

Evaluation of actions to address acquired syphilis between 2016 and 2019, in a southeast brazilian state capital city / Avaliação das ações de enfrentamento da sífilis adquirida no período de 2016 a 2019, numa capital do sudeste brasileiro

Introdução: as Infecções Sexualmente Transmissíveis (IST) representam um problema de saúde pública global, sendo que são responsáveis por efeitos diretos sobre a saúde reprodutiva e infantil, ocasionando consequências como infertilidade e complicações na gestação e no parto, morte fetal e diversos agravos à saúde da criança.Objetivo: avaliar os indicadores de resultado relacionados à sífilis adquirida no quadriênio 2016-2019, em uma capital do sudeste brasileiro. Método: estudo de avaliação para a gestão, descritivo, de abordagem quantitativa, que avaliou os indicadores de resultado "realização de tratamento adequado para a sífilis na população geral" e "monitoramento da sífilis adquirida". Os dados foram coletados no Sistema de Informação de Agravos de Notificação (SINAN), referentes ao período de 1.º de janeiro de 2016 a 31 de dezembro de 2019. As informações referentes ao tratamento e monitoramento dos casos de sífilis congênita ocorreram mediante busca em prontuário eletrônico, no período de 1.º de agosto de 2020 a 31 de março de 2021. Resultados: o município de Vitória teve 2.647 casos de sífilis adquirida, pelo critério ano de diagnóstico. Quanto à variável sexo, notificaram-se 1.641 homens (61,99%) e 1.006 mulheres (38,01%). A faixa etária predominante foi de 20 a 29 anos, e a raça/cor de maior frequência, f a parda. O nível de escolaridade mais frequente foi médio completo. O teste não treponêmico foi realizado em 84,93% dos casos e o treponêmico 47,22%. Com relação ao indicador "oferta de tratamento adequado para a sífilis", constatou-se que o percentual de tratamento adequado, dentre os casos da rede SEMUS, foi de 90,08%, no total do quadriênio: 87,25% em 2016; 85,27% em 2017; 91,79 em 2018; e 94,23% em 2019. Referente ao indicador "monitoramento dos casos de sífilis na população geral", verificou-se que o percentual de monitoramento adequado, dentre os casos da rede SEMUS, foi de 35,72% no total do quadriênio: 33,33% em 2016; 36,83% em 2017; 34,53% em 2018; e 38,20% em 2019. Conclusão: Os indicadores de resultado avaliados em Vitória, no quadriênio 2016-2019, foram: percentual de tratamento adequado (90,08%) e percentual de monitoramento adequado (35,72%).

Introduction: sexually Transmitted Infections (STIs) represent a global public health problem, and are responsible for direct effects on reproductive and child health, causing consequences such as infertility and complications during pregnancy and delivery, fetal death, as well as a variety of health problems in affected children.Objective: to evaluate outcome indicators related to acquired syphilis in the period 2016-2019 in a southeast Brazilian state capital.Methods: this is a descriptive management evaluation study with a quantitative approach, which evaluated the outcome indicators "provision of adequate syphilis treatment in the general population" and "monitoring of acquired syphilis". Data were collected on the Notifiable Health Conditions Information System (SINAN), for the period from January 1st 2016 to December 31st 2019. Information regarding treatment and monitoring of cases of acquired syphilis was obtained by searching electronic medical records between August 1st 2020 and March 31st 2021.Results: the municipality of Vitória had 2,647 cases of acquired syphilis, using the criterion of year of diagnosis. The notified cases related to 1,641 men (61.99%) and 1,006 women (38.01%). The predominant age group was 20 to 29 years, while race / skin color was predominantly brown. The most frequent level of education was complete high school. The non-treponemal test was performed in 84.93% of the cases and the treponemal test in 47.22%. Regarding the "provision of adequate syphilis treatment" indicator, we found a total of 90.08% for adequate treatment of cases in Vitória's public health service network for the four-year period: 87.25% in 2016; 85.27% in 2017; 91.79% in 2018; and 94.23% in 2019. Regarding the "monitoring of syphilis cases in the general population" indicator, we found a total of 35.72% for adequate monitoring of cases in Vitória's public health service network for the four-year period: 33.33% in 2016; 36.83% in 2017; 34.53% in 2018; and 38.20% in 2019.Conclusion: the outcome indicators evaluated in Vitória, in the 2016-2019 four-year period, were: adequate treatment 90.08% and adequate monitoring 35.72.

Upregulation of the sodium channel NaVß4 subunit and its contributions to mechanical hypersensitivity and neuronal hyperexcitability in a rat model of radicular pain induced by local dorsal root ganglion inflammation.

High-frequency spontaneous firing in myelinated sensory neurons plays a key role in initiating pain behaviors in several different models, including the radicular pain model in which the rat lumbar dorsal root ganglia (DRG) are locally inflamed. The sodium channel isoform NaV1.6 contributes to pain behaviors and spontaneous activity in this model. Among all isoforms in adult DRG, NaV1.6 is the main carrier of tetrodotoxin-sensitive resurgent Na currents that allow high-frequency firing. Resurgent currents flow after a depolarization or action potential, as a blocking particle exits the pore. In most neurons, the regulatory ß4 subunit is potentially the endogenous blocker. We used in vivo siRNA-mediated knockdown of NaVß4 to examine its role in the DRG inflammation model. NaVß4 but not control siRNA almost completely blocked mechanical hypersensitivity induced by DRG inflammation. Microelectrode recordings in isolated whole DRG showed that NaVß4 siRNA blocked the inflammation-induced increase in spontaneous activity of Aß neurons and reduced repetitive firing and other measures of excitability. NaVß4 was preferentially expressed in larger diameter cells; DRG inflammation increased its expression, and this was reversed by NaVß4 siRNA, based on immunohistochemistry and Western blotting. NaVß4 siRNA also reduced immunohistochemical NaV1.6 expression. Patch-clamp recordings of tetrodotoxin-sensitive Na currents in acutely cultured medium diameter DRG neurons showed that DRG inflammation increased transient and especially resurgent current, effects blocked by NaVß4 siRNA. NaVß4 may represent a more specific target for pain conditions that depend on myelinated neurons expressing NaV1.6.

Human Nav1.6 Channels Generate Larger Resurgent Currents than Human Nav1.1 Channels, but the Navß4 Peptide Does Not Protect Either Isoform from Use-Dependent Reduction.

Voltage-gated sodium channels are responsible for the initiation and propagation of action potentials (APs). Two brain isoforms, Nav1.1 and Nav1.6, have very distinct cellular and subcellular expression. Specifically, Nav1.1 is predominantly expressed in the soma and proximal axon initial segment of fast-spiking GABAergic neurons, while Nav1.6 is found at the distal axon initial segment and nodes of Ranvier of both fast-spiking GABAergic and excitatory neurons. Interestingly, an auxiliary voltage-gated sodium channel subunit, Navß4, is also enriched in the axon initial segment of fast-spiking GABAergic neurons. The C-terminal tail of Navß4 is thought to mediate resurgent sodium current, an atypical current that occurs immediately following the action potential and is predicted to enhance excitability. To better understand the contribution of Nav1.1, Nav1.6 and Navß4 to high frequency firing, we compared the properties of these two channel isoforms in the presence and absence of a peptide corresponding to part of the C-terminal tail of Navß4. We used whole-cell patch clamp recordings to examine the biophysical properties of these two channel isoforms in HEK293T cells and found several differences between human Nav1.1 and Nav1.6 currents. Nav1.1 channels exhibited slower closed-state inactivation but faster open-state inactivation than Nav1.6 channels. We also observed a greater propensity of Nav1.6 to generate resurgent currents, most likely due to its slower kinetics of open-state inactivation, compared to Nav1.1. These two isoforms also showed differential responses to slow and fast AP waveforms, which were altered by the Navß4 peptide. Although the Navß4 peptide substantially increased the rate of recovery from apparent inactivation, Navß4 peptide did not protect either channel isoform from undergoing use-dependent reduction with 10 Hz step-pulse stimulation or trains of slow or fast AP waveforms. Overall, these two channels have distinct biophysical properties that may differentially contribute to regulating neuronal excitability.

Substituted N-(biphenyl-4'-yl)methyl (R)-2-acetamido-3-methoxypropionamides: potent anticonvulsants that affect frequency (use) dependence and slow inactivation of sodium channels.

We prepared 13 derivatives of N-(biphenyl-4'-yl)methyl (R)-2-acetamido-3-methoxypropionamide that differed in type and placement of a R-substituent in the terminal aryl unit. We demonstrated that the R-substituent impacted the compound's whole animal and cellular pharmacological activities. In rodents, select compounds exhibited excellent anticonvulsant activities and protective indices (PI=TD50/ED50) that compared favorably with clinical antiseizure drugs. Compounds with a polar, aprotic R-substituent potently promoted Na+ channel slow inactivation and displayed frequency (use) inhibition of Na+ currents at low micromolar concentrations. The possible advantage of affecting these two pathways to decrease neurological hyperexcitability is discussed.

Identification of the benzyloxyphenyl pharmacophore: a structural unit that promotes sodium channel slow inactivation.

Four compounds that contained the N-benzyl 2-amino-3-methoxypropionamide unit were evaluated for their ability to modulate Na(+) currents in catecholamine A differentiated CAD neuronal cells. The compounds differed by the absence or presence of either a terminal N-acetyl group or a (3-fluoro)benzyloxy moiety positioned at the 4'-benzylamide site. Analysis of whole-cell patch-clamp electrophysiology data showed that the incorporation of the (3-fluoro)benzyloxy unit, to give the (3-fluoro)benzyloxyphenyl pharmacophore, dramatically enhanced the magnitude of Na(+) channel slow inactivation. In addition, N-acetylation markedly increased the stereoselectivity for Na(+) channel slow inactivation. Furthermore, we observed that Na(+) channel frequency (use)-dependent block was maintained upon inclusion of this pharmacophore. Confirmation of the importance of the (3-fluoro)benzyloxyphenyl pharmacophore was shown by examining compounds where the N-benzyl 2-amino-3-methoxypropionamide unit was replaced by a N-benzyl 2-amino-3-methylpropionamide moiety, as well as examining a series of compounds that did not contain an amino acid group but retained the pharmacophore unit. Collectively, the data indicated that the (3-fluoro)benzyloxyphenyl unit is a novel pharmacophore for the modulation of Na(+) currents.