Sodium Channel Research Articles

In vivo imaging in mouse spinal cord reveals that microglia prevent degeneration of injured axons

Microglia, the primary immune cells in the central nervous system, play a critical role in regulating neuronal function and fate through their interaction with neurons. Despite extensive research, the specific functions and mechanisms of microglia-neuron interactions remain incompletely understood. In this study, we demonstrate that microglia establish direct contact with myelinated axons at Nodes of Ranvier in the spinal cord of mice. The contact associated with neuronal activity occurs in a random scanning pattern. In response to axonal injury, microglia rapidly transform their contact into a robust wrapping form, preventing acute axonal degeneration from extending beyond the nodes. This wrapping behavior is dependent on the function of microglial P2Y12 receptors, which may be activated by ATP released through axonal volume-activated anion channels at the nodes. Additionally, voltage-gated sodium channels (NaV) and two-pore-domain potassium (K2P) channels contribute to the interaction between nodes and glial cells following injury, and inhibition of NaV delays axonal degeneration. Through in vivo imaging, our findings reveal a neuroprotective role of microglia during the acute phase of single spinal cord axon injury, achieved through neuron-glia interaction.

A Rare Noncoding Enhancer Variant in SCN5A Contributes to the High Prevalence of Brugada Syndrome in Thailand.

Brugada syndrome (BrS) is a cardiac arrhythmia disorder that causes sudden death in young adults. Rare genetic variants in the SCN5A gene encoding the Nav1.5 sodium channel and common noncoding variants at this locus are robustly associated with the condition. BrS is particularly prevalent in Southeast Asia but the underlying ancestry-specific factors remain largely unknown. Genome sequencing of BrS probands and population-matched controls from Thailand was performed to identify rare noncoding variants at the SCN5A-SCN10A locus that were enriched in patients with BrS. A likely causal variant was prioritized by computational methods and introduced into human induced pluripotent stem cell (hiPSC) lines using CRISPR-Cas9. The effect of the variant on SCN5A expression and Nav1.5 sodium channel current was then assessed in hiPSC-derived cardiomyocytes (hiPSC-CMs). A rare noncoding variant in an SCN5A intronic enhancer region was highly enriched in patients with BrS (detected in 3.9% of cases with a case-control odds ratio of 45.2). The variant affects a nucleotide conserved across all mammalian species and predicted to disrupt a Mef2 transcription factor binding site. Heterozygous introduction of the enhancer variant in hiPSC-CMs caused significantly reduced SCN5A expression from the variant-containing allele and a 30% reduction in Nav1.5-mediated sodium current density compared with isogenic controls, confirming its pathogenicity. Patients with the variant had severe phenotypes, with 89% experiencing cardiac arrest. This is the first example of a functionally validated rare noncoding variant at the SCN5A locus and highlights how genome sequencing in understudied populations can identify novel disease mechanisms. The variant partly explains the increased prevalence of BrS in this region and enables the identification of at-risk variant carriers to reduce the burden of sudden cardiac death in Thailand.

Pharmacologic and Nonpharmacologic Management of Neuropathic Pain.

Disorders of the somatosensory nervous system that cause neuropathic pain are treated in a variety of ways. Herein, we introduce a stepwise approach to treating neuropathic pain. We then summarize the available data and guidelines for treating neuropathic pain, both with pharmacologic and nonpharmacologic methods, and provide a synthesized algorithm highlighting the similarities and differences between recent guidelines on the management of neuropathic pain. Pharmacologic treatments are primarily antiseizure medications (e.g., gabapentinoids, sodium channel blockers) and antidepressant medications (e.g., tricyclic antidepressants, serotonin-norepinephrine reuptake inhibitors), though other medications and interventional pharmacologic therapies can also be considered. There are a wide variety of nonpharmacologic treatments for neuropathic pain including neuromodulation, nerve stimulation, physiotherapy, movement therapies, lifestyle modification, nutritional supplements, acupuncture, and mind-body techniques.

Scorpion Neurotoxin: An Analgesic Antitumor Peptide Down-Regulates VGSCs and Exhibits Local Anesthetic Potency during Sciatic Nerve Block

Background: Some neurotoxins inhibit nerves strongly. Saxitoxins and tetrodotoxin exhibit strong affinity for various voltage-gated sodium channels (VGSCs), including Nav 1.7, but low affinity for Nav 1.8. Emerging data suggests AGAP mediate analgesia by binding voltage-independently to sodium channel site-3. Objective: This study examined AGAP's mechanism and analgesic efficacy during sciatic nerve block. Methods: In rats, partial sciatic nerve ligation caused rapid and persistent mechanical hyperalgesia. Mechanical hyperalgesia after medication delivery was assessed with von Frey filaments. Western blot and immunohistochemical staining assessed VGSC expression following drug treatment. Results: The data showed that lidocaine and AGAP increased paw withdrawal threshold compared to control rats. Compared to the control group, lidocaine or AGAP inhibited VGSCs. Conclusion: This study suggested that AGAP inhibits VGSCs to elicit lidocaine-like analgesia.

Efficacy of add-on Cenobamate treatment in refractory epilepsy due to Rasmussen's encephalitis.

To assess antiseizure effects of cenobamate, a new antiseizure medication with at least two mechanisms of action, in the rare, highly pharmacoresistant and progressive epilepsy syndrome related to Rasmussen's encephalitis. Three patients from the epilepsy centers in Freiburg, Kork, and Valencia are reported with focal epilepsy which had been pharmacoresistant to more than 10 prior treatment regimens. Assessment included at least 1 year of follow-up after cenobamate introduction and included seizure frequency, seizure severity (in particular status epilepticus) and changes in co-medication. In the three patients, cenobamate add on treatment proved superior to all prior antiseizure and immunomodulatory treatments which had been individually applied. Not only were focal to bilateral tonic-clonic seizure completely controlled, but also focal motor status epilepticus no longer occurred. Co-medication could be reduced in all patients. This case series in a rare and highly pharmacoresistant epilepsy syndrome suggests high efficacy of cenobamate add-on treatment for seizure control. This may be a valuable information in epilepsy related to Rasmussen encephalitis and calls for further elucidation of the mechanism involved in superior seizure control also compared to prior treatments including sodium channel blockers and benzodiazepines. Rasmussen's encephalitis is a rare type of epilepsy that gets worse over time and doesn't respond well to most seizure medications. We describe three patients who tried many treatments without much success, but when they added cenobamate to their treatment, it worked better than the other medications. This also let them lower the overall amount of medication they were taking.

Alzheimer's disease induced neurons bearing PSEN1 mutations exhibit reduced excitability.

Alzheimer's disease (AD) is a devastating neurodegenerative condition that affects memory and cognition, characterized by neuronal loss and currently lacking a cure. Mutations in PSEN1 (Presenilin 1) are among the most common causes of early-onset familial AD (fAD). While changes in neuronal excitability are believed to be early indicators of AD progression, the link between PSEN1 mutations and neuronal excitability remains to be fully elucidated. This study examined iPSC-derived neurons (iNs) from fAD patients with PSEN1 mutations S290C or A246E, alongside CRISPR-corrected isogenic cell lines, to investigate early changes in excitability. Electrophysiological profiling revealed reduced excitability in both PSEN1 mutant iNs compared to their isogenic controls. Neurons bearing S290C and A246E mutations exhibited divergent passive membrane properties compared to isogenic controls, suggesting distinct effects of PSEN1 mutations on neuronal excitability. Additionally, both PSEN1 backgrounds exhibited higher current density of voltage-gated potassium (Kv) channels relative to their isogenic iNs, while displaying comparable voltage-gated sodium (Nav) channel current density. This suggests that the Nav/Kv imbalance contributes to impaired neuronal firing in fAD iNs. Deciphering these early cellular and molecular changes in AD is crucial for understanding disease pathogenesis.

Macrophages enhance sodium channel expression in cardiomyocytes.

Cardiac macrophages facilitate electrical conduction through the atrioventricular-node (AV) in mice. A possible role for cardiomyocyte-macrophage coupling on the effect of antiarrhythmic therapy has not been investigated yet. Holter monitoring was conducted in LysMCrexCsf1rLsL-DTR mice (MMDTR) under baseline conditions and after an elctrophysiological stress test by flecainide. In vivo effects were recapitulated in vitro by patch-clamp experiments. The underlying mechanism was characterized by expression and localization analysis of connexin43 (Cx43) and voltage-gated-sodium-channel-5 (Nav1.5). ECG monitoring in MMDTR mice did not show any significant conduction abnormalities but a significantly attenuated flecainide-induced extension of RR- and PP-intervals. Patch-clamp analysis revealed that the application of flecainide to neonatal rat ventricular cardiomyocytes (CMs) changed their resting-membrane-potential (RMP) to more negative potentials and decreased action-potential-duration (APD50). Coupling of macrophages to CMs significantly enhances the effects of flecainide, with a further reduction of the RMP and APD50, mediated by an upregulation of Cx43 and Nav1.5 surface expression. Macrophage depletion in mice does not correlate with cardiac electric conduction delay. Cardiac macrophages amplify the effects of flecainide on electrophysiological properties of cardiomyocytes in vivo and in vitro. Mechanistically, formation of macrophage-cardiomyocyte cell-cell-contacts via Cx43 facilitates the recruitment of Nav1.5 to the cell membrane increasing flecainide effects.

The negative chronotropic effects of (±)-propranolol and (±)-4-NO2-propranolol in the rat isolated right atrium are due to blockade of the 6-nitrodopamine receptor.

The positive chronotropic action induced by 6-nitrodopamine (6-ND) is selectively blocked by β1-adrenoceptor antagonists at concentrations that do not affect the positive chronotropic effect induced by dopamine, noradrenaline, and adrenaline. Here, the effects of ( ±)-propranolol, ( ±)-4-NO2-propranolol, and ( ±)-7-NO2-propranolol were investigated in the rat isolated right atrium. The atrium was mounted in glass chambers containing gassed (95%O2:5%CO2) and warmed (37°C) Krebs-Henseleit's solution, and the isometric tension registered (PowerLab system). ( ±)-Propranolol, ( ±)-4-NO2-propranolol, and ( ±)-7-NO2-propranolol caused concentration-dependent falls in the spontaneous atrial frequency (pIC50: 4.80 ± 0.10, 4.64 ± 0.10, and 4.95 ± 0.10, respectively). The calculated pA2 values for ( ±)-propranolol, ( ±)-4-NO2-propranolol, and ( ±)-7-NO2-propranol on noradrenaline-induced positive chronotropism were 8.44 ± 0.08, 6.41 ± 0.07, and 9.21 ± 0.29, respectively. The positive chronotropism induced by 6-ND (10pM) was blocked by ( ±)-propranolol (1µM) and ( ±)-4-NO2-propranolol (30nM), whereas ( ±)-7-NO2-propranolol (1µM) had no effect on 6-ND-induced responses. The pIC50 of ( ±)-propranolol, ( ±)-4-NO2-propranolol, and ( ±)-7-NO2-propranolol were significantly shifted to the right in L-NAME-treated atria. The discrepancy between pA2 values of ( ±)-propranolol and its respective pIC50 indicates that the falls in atrial rate induced by ( ±)-propranolol should not be attributed to b-adrenergic antagonism. The reduced chronotropism by ( ±)-propranolol was unaffected by the sodium channel inhibitors tetrodotoxin and lidocaine but that was abolished in atria pre-treated with ( ±)-4-NO2-propranolol. The finding that ( ±)-propranolol reduces spontaneous atrial rate only in concentrations that affect 6-ND-induced positive chronotropism confirms the role of this catecholamine as an endogenous modulator of heart chronotropism. ( ±)-4-NO2-Propranolol behaves as a selective antagonist of 6-ND in the rat isolated atrium.

V1848I Mutation in the Voltage-Gated Sodium Channel Confers High-Level Resistance to Indoxacarb and Metaflumizone in Spodoptera exigua.

Spodoptera exigua is one of the most serious lepidopteran pests of global importance. With the intensive use of insecticides, S. exigua has evolved resistance to many insecticides, including the sodium channel blocker insecticides (SCBIs) indoxacarb and metaflumizone. In this study, we investigated the role of the V1848I mutation in the voltage-gated sodium channel (VGSC) in SCBI resistance and its inheritance patterns in S. exigua through the development and characterization of a near-isogenic resistant strain. The AQ-23 strain of S. exigua, collected in 2023 from Anqing, Anhui province of China, shows 165-fold resistance to indoxacarb compared with the susceptible WH-S strain. A frequency of 44.6% for the V1848I mutation was detected in the SeVGSC of the AQ-23 strain, while no F1845Y mutation was found. Through repeated backcrossing and marker-assisted selection, the V1848I mutation in the AQ-23 strain was introgressed into the susceptible WH-S strain, creating a near-isogenic strain named WH-1848I. This WH-1848I strain exhibits high levels of resistance to indoxacarb (146-fold) and metaflumizone (431-fold) but remains susceptible to broflanilide and spinosad compared with the WH-S strain. Inheritance analysis revealed that SCBI resistance in the WH-1848I strain is autosomal, nonrecessive, and genetically linked to the V1848I mutation. These findings establish a clear link between the V1848I mutation and SCBI resistance in S. exigua, offering valuable insights for developing molecular detection tools and resistance management strategies.

Analgesia and peripheral c-fiber modulation by selective Nav1.8 inhibition in rhesus.

Voltage-gated sodium (Nav) channels present untapped therapeutic value for better and safer pain medications. The Nav1.8 channel isoform is of particular interest because of its location on peripheral pain fibers and demonstrated role in rodent preclinical pain and neurophysiological assays. To-date, no inhibitors of this channel have been approved as drugs for treating painful conditions in human, possibly because of challenges in developing a sufficiently selective drug-like molecule with necessary potency not only in human but also across preclinical species critical to the preclinical development path of drug discovery. In addition, the relevance of rodent pain assays to the human condition is under increasing scrutiny as a number of mechanisms (or at the very least molecules) that are active in rodents have not translated to humans, and direct impact on pain fibers has not been confirmed in vivo. In this report, we have leveraged numerous physiological end points in nonhuman primates to evaluate the analgesic and pharmacodynamic activity of a novel, potent, and selective Nav1.8 inhibitor compound, MSD199. These pharmacodynamic biomarkers provide important confirmation of the in vivo impact of Nav1.8 inhibition on peripheral pain fibers in primates and have high translational potential to the clinical setting. These findings may thus greatly improve success of translational drug discovery efforts toward better and safer pain medications, as well as the understanding of primate biology of Nav1.8 inhibition broadly.

Interplay of Nav1.8 and Nav1.7 channels drives neuronal hyperexcitability in neuropathic pain.

While voltage-gated sodium channels Nav1.7 and Nav1.8 both contribute to electrogenesis in dorsal root ganglion (DRG) neurons, details of their interactions have remained unexplored. Here, we studied the functional contribution of Nav1.8 in DRG neurons using a dynamic clamp to express Nav1.7L848H, a gain-of-function Nav1.7 mutation that causes inherited erythromelalgia (IEM), a human genetic model of neuropathic pain, and demonstrate a profound functional interaction of Nav1.8 with Nav1.7 close to the threshold for AP generation. At the voltage threshold of -21.9 mV, we observed that Nav1.8 channel open-probability exceeded Nav1.7WT channel open-probability ninefold. Using a kinetic model of Nav1.8, we showed that a reduction of Nav1.8 current by even 25-50% increases rheobase and reduces firing probability in small DRG neurons expressing Nav1.7L848H. Nav1.8 subtraction also reduces the amplitudes of subthreshold membrane potential oscillations in these cells. Our results show that within DRG neurons that express peripheral sodium channel Nav1.7, the Nav1.8 channel amplifies excitability at a broad range of membrane voltages with a predominant effect close to the AP voltage threshold, while Nav1.7 plays a major role at voltages closer to resting membrane potential. Our data show that dynamic-clamp reduction of Nav1.8 conductance by 25-50% can reverse hyperexcitability of DRG neurons expressing a gain-of-function Nav1.7 mutation that causes pain in humans and suggests, more generally, that full inhibition of Nav1.8 may not be required for relief of pain due to DRG neuron hyperexcitability.

Defining Cardiomyocyte Repolarization Response to Pharmacotherapy in Long-QT Syndrome Type 3.

Long-QT syndrome is a primary cardiac ion channelopathy predisposing a patient to ventricular arrhythmia through delayed repolarization on the resting ECG. We aimed to establish a patient-specific, human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes model of long-QT syndrome type 3 (LQT3) using clustered regularly interspaced palindromic repeats (CRISPR/Cas9), for disease modeling and drug challenge. HiPSCswere generated from a patient with LQT3 harboring an SCN5A pathogenic variant (c.1231G>A; p.Val411Met), and an unrelated healthy control. The same SCN5A pathogenic variant was engineered into the background healthy control hiPSCs via CRISPR/Cas9gene editing to generate a second disease model of LQT3 for comparison with an isogenic control. All 3 hiPSC lines were differentiated into cardiomyocytes. Both the patient-derived LQT3 (SCN5A+/-) and genetically engineered LQT3 (SCN5A+/-) hiPSC-derived cardiomyocytesshowed significantly prolonged cardiomyocyte repolarization compared with the healthy control. Mexiletine, a cardiac voltage-gated sodium channel (NaV1.5) blocker, shortened repolarization in both patient-derived LQT3 and geneticallyengineered LQT3 hiPSC-derived cardiomyocytes, but had no effect in the control. Notably, calcium channel blockers nifedipine and verapamil showed a dose-dependent shortening of repolarization, rescuing the phenotype. Additionally, therapeutic drugs known to prolong the corrected QT in humans (ondansetron, clarithromycin, and sotalol) demonstrated this effect invitro, but the LQT3 clones were not more disproportionately affected compared with the control. We demonstrated that patient-derived and genetically engineered LQT3 hiPSC-derived cardiomyocytesfaithfully recapitulate pathologic characteristics of LQT3. The clinical significance of such an invitro model is in the exploration of novel therapeutic strategies, stratifying drug adverse reaction risk and potentially facilitating a more targeted, patient-specific approach in high-risk patients with LQT3.

The Initial Experience of Eslicarbazepine in Children at Three Canadian Tertiary Pediatric Care Centers.

Eslicarbazepine (ESL) is a once-daily, third-generation antiseizure medication for focal-onset seizures. The primary mechanism of action is enhancing the slow inactivation of voltage-gated sodium channels. The study objective was to review real-world experience regarding retention rate, efficacy, and tolerability of eslicarbazepine, soon after it became available for children in Canada. A retrospective review was performed on all patients prescribed eslicarbazepine from September 2017 to June 2020, with at least 3 years of follow-up data, at 3 Canadian tertiary care pediatric centers. Fifty patients were identified, and the mean age of eslicarbazepine initiation was 12.4 years (range 3-19 years). Most patients had drug-resistant epilepsy, trying a mean of 5.04 (range 0-14) antiseizure medications before the initiation of eslicarbazepine. Twenty-four patients (48.0%) experienced adverse effects, including dizziness (n = 10), drowsiness (n = 6), dizziness and drowsiness (n = 1), nausea and abdominal pain (n = 4), transient unsteadiness and diplopia (n = 1), and negative mood changes (n = 2). None had serious adverse effects, including rash. The retention rate of eslicarbazepine at last follow-up was 70%. Fifteen (30%) had ≥50% seizure reduction, with 2 of these patients becoming seizure free. Ten (20%) had 25% to 50% reduction, 2 (4%) had worsening of seizures, and 17 (34%) had no change in seizure frequency. The study results support the long-term effectiveness and tolerability of eslicarbazepine in a cohort of children with predominantly drug-resistant epilepsy in a real-life setting from 3 Canadian centers with initial use after approval. Adverse effects were nonserious, infrequently leading to eslicarbazepine discontinuation.

Discovery of Knock-Down Resistance in the Major African Malaria Vector Anopheles funestus.

A major insecticide resistance mechanism in insect pests is knock-down resistance (kdr) caused by mutations in the voltage-gated sodium channel (Vgsc) gene. Despite being common in most malaria Anopheles vector species, kdr mutations have never been observed in Anopheles funestus, the principal malaria vector in Eastern and Southern Africa, with resistance mainly being conferred by detoxification enzymes. In a parallel study, we monitored 10 populations of An. funestus in Tanzania for insecticide resistance unexpectedly identified resistance to a banned insecticide, DDT, in the Morogoro region. Through whole-genome sequencing of 333 An. funestus samples from these populations, we found eight novel amino acid substitutions in the Vgsc gene, including the kdr variant, L976F (equivalent to L995F in An. gambiae), in tight linkage disequilibrium with another (P1842S). The mutants were found only at high frequency in one region and were accompanied by weak signatures of a selective sweep, with a significant decline between 2017 and 2023. Notably, kdr L976F was strongly associated with survivorship to exposure to DDT insecticide, while no clear association was noted with a pyrethroid insecticide (deltamethrin). The WHO prequalifies no DDT products for vector control, and the chemical is banned in Tanzania. Widespread DDT contamination and a legacy of extensive countrywide stockpiles may have selected for this mutation. Continued monitoring is necessary to understand the origin of kdr in An. funestus, and the threat posed to insecticide-based vector control in Africa.

Differences in seasonal dynamics and pyrethroid resistance development among Anopheles Hyrcanus group species

BackgroundThe Anopheles Hyrcanus group, which transmits Plasmodium vivax, consists of six confirmed species in South Korea. An epidemiological study revealed differences in the seasonal occurrence patterns of each species. Pyrethroid resistance in An. sinensis dates back to the early 2000s, whereas information on pyrethroid resistance in other species is lacking despite their greater significance in malaria epidemiology.MethodsAnopheles mosquitoes were collected from two malaria-endemic regions in South Korea for 2 years and their knockdown resistance (kdr) mutations were genotyped. The larval susceptibility to λ-cyhalothrin was compared in six Anopheles species and its seasonal changes in three species were investigated. The full-length sequences of the voltage-sensitive sodium channel (VSSC) were compared across six species to evaluate potential target-site insensitivity. The contribution of the kdr mutation to phenotypic resistance was confirmed by comparing median lethal time (LT50) to λ-cyhalothrin between populations of Anopheles belenrae with distinct genotypes.ResultsThe composition and seasonal occurrence of rare species (Anopheles kleini, Anopheles lestri, and Anopheles sineroides) varied considerably, whereas An. sinensis occurs continuously throughout the season. A kdr mutation in the form of heterozygous allele was newly identified in An. belenrae, An. lesteri, An. pullus, and An. sineroides. The baseline susceptibility to λ-cyhalothrin was the highest in An. belenrae, followed by An. lesteri, An. sineroides, An. kleini, An. pullus, and An. sinensis, with median lethal concentration (LC50) values ranging from 6.0- to 73.5-fold higher than that of An. belenrae. The susceptibility of An. sinensis and An. pullus varied by season, whereas that of An. belenrae remained stable. The kdr-heterozygous An. belenare population exhibited 5.1 times higher LT50 than that of the susceptible population. Species-specific VSSC sequence differences were observed among the six species.ConclusionsOur findings suggest that the status and extent of pyrethroid resistance vary among Anopheles Hyrcanus group species. While An. sinensis, the predominant species, developed a considerable level of pyrethroid resistance through kdr mutation, the resistance levels of other species appeared to be less pronounced. Large-scale monitoring is crucial to fully understand species-specific seasonal occurrence and resistance status for effective management strategies, considering the ongoing impact of climate change on their vectorial capacity.Graphical

7065 Eradication Of Metastatic ER-Positive Breast, Ovarian, Endometrial And Lung Cancer In Mouse Xenografts And Patient Derived Organoids

Abstract Disclosure: D.J. Shapiro: None. D. Duraki: None. S. Ghosh: None. J. Zhu: None. C. Mao: None. J. Kim: None. M. Jabeen: None. M.W. Boudreau: None. M.P. Mulligan: None. R. Romero: None. Y. Han: None. J. Zhang: None. E.R. Nelson: None. O. Olopade: None. G. Chang: None. P.J. Hergenrother: Advisory Board Member; Self; Systems Oncology. We described the tumor protective estrogen-ER activated anticipatory Unfolded Protein Response (a-UPR) pathway. Here we report a previously undescribed multistep pathway by which very rapid estrogen-ER activation of the a-UPR authorizes and regulates subsequent transcription of the estrogen-ER transcriptome. We repurposed the tumor protective a-UPR through identification and optimization of the small molecule ErSO and its family of agents. ErSO acts non-competitively with estrogen to induce lethal hyperactivation of the a-UPR. In orthotopic mouse xenografts containing wild type or mutant ER, ErSO often induced complete regression without recurrence of large primary breast tumors and of most lung, bone and liver metastases, near complete regression of normally lethal brain metastases and complete destruction of patient derived organoids (PDOs) in Matrigel. Six research teams in four locations have confirmed the remarkable effectiveness of ErSO in ER-positive breast cancer. We next evaluated ErSO in diverse ER containing cancers in which estrogen is not the primary driver of proliferation. ErSO often induced complete regression in multiple orthotopic xenograft models of primary and metastatic ovarian cancer. Using fresh ovarian cancer organoids immediately after removal from patients by paracentesis, ErSO destroyed the organoids from some patients with advanced stage III and stage IV disease. ErSO was effective in a mouse xenograft model of endometrial cancer and, surprisingly, even in ERalpha positive lung cancer. Our ongoing studies demonstrate ErSO induces complete, or near complete regression, of primary and metastatic lung cancer in mice. Studies of ErSO’s mechanism of action using genome-wide CRISPR screens with positive selection, and other techniques, unveil a novel death pathway in which ErSO activation of the a-UPR triggers rapid calcium release into the cell body. The calcium opens the plasma membrane TRPM4 sodium channel, leading to an influx of extracellular sodium. This swells the cells, resulting in osmotic stress that sustains lethal a-UPR activation. A little-studied cytoskeleton regulator we identified, FGD3, plays a pivotal role in whether the cells rupture their membranes, inducing necrotic cell death. Notably, medium from diverse cancer cells killed by ErSO-induced necrosis robustly activates human macrophage and induces their migration - potentially facilitating immunotherapy. These studies describe a pathway from a multiprotein ER-SRC-PLC complex, through the a-UPR, to a novel cell swelling and necrosis pathway, that we are exploiting to target diverse, therapeutically challenging, ER-positive human cancers. Presentation: 6/2/2024

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.

Regulation of voltage-gated sodium channels by TNF-α during herpes simplex virus latency establishment.

During lytic or latent infection of sensory neurons with herpes simplex virus type 1 (HSV-1) there are significant changes in the expression of voltage-gated Na+ channels, which may disrupt the transmission of pain information. HSV-1 infection can also evoke the secretion of various pro-inflammatory cytokines, including TNF-α and IL-6. In this work, we hypothesized that TNF-α regulates the expression of Na+ channels during HSV-1 latency establishment in ND7/23 sensory-like neurons. Latency establishment was mimicked by culturing HSV-1 infected ND7/23 cells in the presence of acyclovir (ACV) for 3 days. Changes in the functional expression of voltage-gated Na+ channels were assessed by whole-cell recordings. Our results demonstrate that infection of ND7/23 cells with the HSV-1 strain McKrae with GFP expression (M-GFP) causes a significant decrease in sodium currents during latency establishment. Exposure of ND7/23 cells to TNF-α during latency establishment reverses the effect of HSV-1, resulting in a significant increase in sodium current density. However, Na+ currents were not restored by 3day-treatment with IL-6. There were no changes in the pharmacological and biophysical properties of sodium currents promoted by TNF-α, including sensitivity to tetrodotoxin and the current-voltage relationship. TNF-α stimulation of ND7/23 cells increases p38 signaling. Inhibition of p38 signaling with SB203580 or SB202190 eliminates the stimulatory effect of TNF-α on sodium currents. These results indicate that TNF-α signaling in sensory neurons during latency establishment upregulates the expression of voltage-gated Na+ channels in order to maintain the transmission of pain information.

Molecular determinants of resurgent sodium currents mediated by Navβ4 peptide and A-type FHFs.

Resurgent current (INaR ) generated by voltage-gated sodium channels (VGSCs) plays an essential role in maintaining high-frequency firing of many neurons and contributes to disease pathophysiology such as epilepsy and painful disorders. Targeting INaR may present a highly promising strategy in the treatment of these diseases. Navβ4 and A-type fibroblast growth factor homologous factors (FHFs) have been identified as two classes of important INaR mediators; however, their receptor sites in VGSCs remain unknown, which hinders the development of novel agents to effectively target INaR . Navβ4 and FHF4A can mediate INaR generation through the amino acid segment located in their C-terminus and N-terminus, respectively. We mainly employed site-directed mutagenesis, chimera construction and whole-cell patch-clamp recording to explore the receptor sites of Navβ4 peptide and FHF4A in Nav1.7 and Nav1.8. We show that the receptor of Navβ4-peptide involves four residues, N395, N945, F1737 and Y1744, in Nav1.7 DI-S6, DII-S6, and DIV-S6. We show that A-type FHFs generating INaR depends on the segment located at the very beginning, not at the distal end, of the FHF4 N-terminus domain. We show that the receptor site of A-type FHFs also resides in VGSC inner pore region. We further show that an asparagine at DIIS6, N891 in Nav1.8, is a major determinant of INaR generated by A-type FHFs in VGSCs. Cryo-EM structures reveal that the side chains of the critical residues project into the VGSC channel pore. Our findings provide additional evidence that Navβ4 peptide and A-type FHFs function as open-channel pore blockers and highlight channel inner pore region as a hotspot for development of novel agents targeting INaR .

Unraveling the dynamics of firing patterns for neurons with impairment of sodium channels.

Various factors such as mechanical trauma, chemical trauma, local ischemia, and inflammation can impair voltage-gated sodium channels (Nav) in neurons. These impairments lead to a distinctive leftward shift in the activation and inactivation curves of voltage-gated sodium channels. The resulting sodium channel impairments in neurons are known to affect firing patterns, which play a significant role in neuronal activities within the nervous system. However, the underlying dynamic mechanism for the emergence of these firing patterns remains unclear. In this study, we systematically investigated the effects of sodium channel dysfunction on individual neuronal dynamics and firing patterns. By employing codimension-1 bifurcation analysis, we revealed the underlying dynamical mechanism responsible for the generation of different firing patterns. Additionally, through codimension-2 bifurcation analysis, we theoretically determined the distribution of firing patterns on different parameter planes. Our results indicate that the firing patterns of impaired neurons are regulated by multiple parameters, with firing pattern transitions caused by the degree of sodium channel impairment being more diverse than those caused by the ratio of impaired sodium channel and current. Furthermore, we observed that the firing pattern of tonic firing is more likely to be the norm in impaired sodium channel neurons, providing valuable insights into the signaling of impaired neurons. Overall, our findings highlight the intricate relationships among sodium channel impairments, neuronal dynamics, and firing patterns, shedding light on the impact of disruptions in ion concentration gradients on neuronal function.