Exploring the mechanism of Nav1.3 in the ION-CCI rat model based on the TLR4/TRAF6/NF-κB pathway.

BACKGROUND: Trigeminal neuralgia (TN) is a common and difficult-to-treat neuropathic pain disorder in clinical practice. Previous studies have shown that Toll-like receptor 4 (TLR4) modulates the activation of the NF-κB pathway to affect neuropathic pain in rats. Voltage-gated sodium channels (VGSCs) are known to play an important role in neuropathic pain electrical activity. OBJECTIVE: To investigate whether TLR4 can regulate Nav1.3 through the TRAF6/NF-κB p65 pathway after infraorbital nerve chronic constriction injury (ION-CCI). STUDY DESIGN: ION-CCI modeling was performed on SD (Sprague Dawley) rats. To verify the success of the modeling, we need to detect the mechanical pain threshold and ATF3. Then, detecting the expression of TLR4, TRAF6, NF-κB p65, p-p65, and Nav1.3 in rat TG. Subsequently, investigate the role of TLR4/TRAF6/NF-κB pathway in ION-CCI model by intrathecal injections of LPS-rs (TLR4 antagonist), C25-140 (TRAF6 inhibitor), and PDTC (NF-κB p65 inhibitor). RESULTS: ION-CCI surgery decreased the mechanical pain threshold of rats and increased the expression of ATF3, TLR4, TRAF6, NF-κB p-p65 and Nav1.3, but there was no difference in NF-κB p65 expression. After inject antagonist or inhibitor of the TLR4/TRAF6/NF-κB pathway, the expression of Nav1.3 was decreased and mechanical pain threshold was increased. CONCLUSION: In the rat model of ION-CCI, TLR4 in the rat trigeminal ganglion regulates Nav1.3 through the TRAF6/NF-κB p65 pathway, and TLR4 antagonist alleviates neuropathic pain in ION-CCI rats.

miR-9a-5p expression is decreased in the hippocampus of rats resistant to lamotrigine: A behavioural, molecular and bioinformatics assessment.

The major obstacle in developing new treatment strategies for refractory epilepsy is the complexity and poor understanding of its mechanisms. Utilizing the model of lamotrigine-resistant seizures, we evaluated whether changes in the expression of sodium channel subunits are responsible for the diminished responsiveness to lamotrigine (LTG) and if miRNAs, may also be associated. Male rats were administered LTG (5 mg/kg) before each stimulation during kindling acquisition. Challenge stimulation following LTG exposure (30 mg/kg) was performed to confirm resistance in fully kindled rats. RT-PCR was used to measure the mRNA levels of sodium channel subunits (SCN1A, SCN2A, and SCN3A) and miRNAs (miR-155-5p, miR-30b-5p, miR-137-3p, miR-342-5p, miR-301a-3p, miR-212-3p, miR-9a-5p, and miR-133a-3p). Western blot analysis was utilized to measure Nav1.2 protein, and bioinformatics tools were used to perform target prediction and enrichment analysis for miR-9a-5p, the only affected miRNA according to the responsiveness to LTG. Amygdala kindling seizures downregulated Nav1.2, miR-137-3p, miR-342-5p, miR-155-5p, and miR-9a-5p as well as upregulated miR-212-3p. miR-9a-5p was the only molecule decreased in rats resistant to LTG. The bioinformatic assessment and disease enrichment analysis revealed that miR-9a-5p targets expressed with high confidence in the hippocampus are the most significantly associated with epilepsy. Due to the miR-9a-5p dysregulation, major pathways affected are neurotrophic processes, neurotransmission, inflammatory response, cell proliferation and apoptosis. Interaction network analysis identified LTG target SCN2A as interacting with highest number of genes regulated by miR-9-5p. Further studies are needed to propose specific genes and miRNAs responsible for diminished responsiveness to LTG. miR-9a-5p targets, like KCNA4, KCNA2, CACNB2, SCN4B, KCNC1, should receive special attention in them.

MiR-214-3p plays a protective role in diabetic neuropathic rats by regulating Nav1.3 and TLR4.

This study aimed to investigate the functions of miR-214-3p in diabetic neuropathic rodents. The diabetic neuropathy was induced by intraperitoneal injection of streptozotocin (STZ) in rats, and miR-214-3p was delivered via tail vein injection of lentivirus. Hot or cold stimulus tests demonstrated that STZ induced thermal hyperalgesia. Neurophysiological measurements revealed that motor and sensory nerve conduction velocity and nerve blood flow were decreased in diabetic neuropathic rats. However, the STZ-induced hyperalgesia, and reduced nerve conduction velocity and nerve blood flow were all significantly reversed by miR-214-3p administration. HE staining, TUNEL, ELISA, and immunoblotting demonstrated that STZ led to obvious pathological lesion, cell apoptosis, and inflammation in dorsal root ganglion (DRG), evidenced by altered levels of apoptosis-related protein molecules and inflammatory factors, and activation of Toll-like receptor 4 (TLR4)/myeloid differentiation primary response gene 88/nuclear factor kappa B signaling. The pathological alterations in diabetic neuropathic rats in DRG were significantly ameliorated by miR-214-3p application. In addition, sodium channel protein type 3 subunit alpha isoform 1 (Nav1.3) and TLR4 were identified as targets of miR-214-3p via dual-luciferase reporter assay. MiR-214-3p may play its roles by downregulating Nav1.3 and TLR4. In summary, miR-214-3p alleviated diabetes-induced nerve injury, and pathological lesion, cell apoptosis, and inflammation in DRG by regulating Nav1.3 and TLR4 in STZ-induced rats. These findings may provide novel therapeutic targets for clinical treatment of diabetic neuropathy.

Slow recovery from the inactivation of voltage-gated sodium channel Nav1.3 in mouse taste receptor cells.

Action potentials play an important role in neurotransmitter release in response to taste. Here, I have investigated voltage-gated Na+ channels, a primary component of action potentials, in respective cell types of mouse fungiform taste bud cells (TBCs) with in situ whole-cell clamping and single-cell RT-PCR techniques. The cell types of TBCs electrophysiologically examined were determined immunohistochemically using the type III inositol 1,4,5-triphoshate receptor as a type II cell marker and synaptosomal-associated protein 25 as a type III cell marker. I show that type II cells, type III cells, and TBCs not immunoreactive to these markers (likely type I cells) generate voltage-gated Na+ currents. The recovery following inactivation of these currents was well fitted with double exponential curves. The time constants in type III cells (~20 ms and ~ 1 s) were significantly slower than respective time constants in other cell types. RT-PCR analysis indicated the expression of Nav1.3, Nav1.5, Nav1.6, and ß1 subunit mRNAs in TBCs. Pharmacological inhibition and single-cell RT-PCR studies demonstrated that type II and type III cells principally express tetrodotoxin (TTX)-sensitive Nav1.3 channels and that ~ 30% of type I cells express TTX-resistant Nav1.5 channels. The auxiliary ß1 subunit that modulates gating kinetics was rarely detected in TBCs. As the ß1 subunit co-expressed with an α subunit is known to accelerate the recovery from inactivation, it is likely that voltage-gated Na+ channels in TBCs may function without ß subunits. Slow recovery from inactivation, especially in type III cells, may limit high-frequency firing in response to taste substances.

IL-6 contributes to Nav1.3 up-regulation in trigeminal nerve following chronic constriction injury.

Background: To verify the hypothesis that the nature of trigeminal neuralgia (TN) is an ectopic impulse induced by sodium channel modulated by cytokines, we conducted an animal study using the infraorbital nerve chronic constriction injury (CCI) model in rats.Method: The expression of Nav1.3 or IL-6 in the infraorbital nerve (ION) and trigeminal ganglion (TG) were detected by western blot and immunocytochemistry after administration of antisense oligodeoxynucleotide sequence (AS), IL-6 or Anti-IL-6.Results: With intrathecal administration of AS or mismatch oligodeoxynucleotide sequence (MM) in the CCI rats, the Nav1.3-IR in ION and TG accounted for 2.2 ± 0.51% and 8.5 ± 3.1% in AS+CCI group vs. 6.9 ± 1.3% and 38.7 ± 4.8% in MM+CCI group (p < 0.05), respectively. While with local administration of IL-6 in those with sham operation, it accounted for 7.4 ± 2.1% and 45.5 ± 3.4% in IL-6+ sham group vs. 1.9 ± 0.67% and 8.1 ± 1.3% in vehicle+sham group (p < 0.05); with local administration of anti-IL-6 in CCI rats, 4.5 ± 0.78% and 32.1 ± 9.6% in Anti-IL-6+ CCI group vs 8.9 ± 2.1% and 61.4 ± 11.2% in vehicle+CCI group (p < 0.05).Discussion: We believe that the emergence of Nav1.3 from the compressed trigeminal nerve might be an important structural basis for the development of the ectopic excitability on the axon and IL-6 may play a role of necessary precondition.

Biological concepts in human sodium channel epilepsies and their relevance in clinical practice.

OBJECTIVE: Voltage-gated sodium channels (SCNs) share similar amino acid sequence, structure, and function. Genetic variants in the four human brain-expressed SCN genes SCN1A/2A/3A/8A have been associated with heterogeneous epilepsy phenotypes and neurodevelopmental disorders. To better understand the biology of seizure susceptibility in SCN-related epilepsies, our aim was to determine similarities and differences between sodium channel disorders, allowing us to develop a broader perspective on precision treatment than on an individual gene level alone. METHODS: We analyzed genotype-phenotype correlations in large SCN-patient cohorts and applied variant constraint analysis to identify severe sodium channel disease. We examined temporal patterns of human SCN expression and correlated functional data from in vitro studies with clinical phenotypes across different sodium channel disorders. RESULTS: Comparing 865 epilepsy patients (504 SCN1A, 140 SCN2A, 171 SCN8A, four SCN3A, 46 copy number variation [CNV] cases) and analysis of 114 functional studies allowed us to identify common patterns of presentation. All four epilepsy-associated SCN genes demonstrated significant constraint in both protein truncating and missense variation when compared to other SCN genes. We observed that age at seizure onset is related to SCN gene expression over time. Individuals with gain-of-function SCN2A/3A/8A missense variants or CNV duplications share similar characteristics, most frequently present with early onset epilepsy (<3 months), and demonstrate good response to sodium channel blockers (SCBs). Direct comparison of corresponding SCN variants across different SCN subtypes illustrates that the functional effects of variants in corresponding channel locations are similar; however, their clinical manifestation differs, depending on their role in different types of neurons in which they are expressed. SIGNIFICANCE: Variant function and location within one channel can serve as a surrogate for variant effects across related sodium channels. Taking a broader view on precision treatment suggests that in those patients with a suspected underlying genetic epilepsy presenting with neonatal or early onset seizures (<3 months), SCBs should be considered.

Neurodevelopmental disorder associated with de novo SCN3A pathogenic variants: two new cases and review of the literature.

SCN3A was recently recognized as a gene associated with neurodevelopmental disorder and epilepsy. We present two additional patients with a novel de novo SCN3A pathogenic variant, and a review of all published cases of de novo variants. In one of our patients brain magnetic resonance imaging (MRI) disclosed a severe polymicrogyria and in the other it was normal. The clinical phenotype was characterized by a severe developmental delay and refractory epilepsy in the patient with polymicrogyria and intellectual disability with autistic features and pharmacoresponsive epilepsy in the subject with normal MRI. Polymicrogyria, a disorder of progenitor cells proliferation and migration, is an unanticipated finding for an ion channel dysfunction.

MicroRNA-212-3p Attenuates Neuropathic Pain via Targeting Sodium Voltage-gated Channel Alpha Subunit 3 (NaV 1.3).

PURPOSE: To explore the role and potential mechanism of miR-212-3p in neuropathic pain regulation. METHODS: Adult male rats were used to establish chronic constriction injury (CCI) model to mimic the neuropathic pain. Then, paw withdrawal threshold (PWT) and paw withdrawal thermal latency (PWL) were determined. The concentrations of interleukin 1 beta (IL-1ß), interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-α) were measured with enzyme-linked immune sorbent assay (ELISA) kit and the expression of miR-212-3p was measured by real time quantitative PCR (RTqPCR). Besides, miR-212-3p agomir was intrathecally injected into CCI rats and the expression of key apoptotic proteins was determined by western blot. Furthermore, dual-luciferase reporter assay was used to determine the binding of miR-212-3p and 3' untranslated regions (3'UTR) of NaV1.3 and the expression levels of NaV1.3 were measured by western blot and RT-qPCR. RESULTS: In the CCI group, the PWT and PWL were significantly decreased and IL-1ß, IL-6 and TNF-α were increased. miR-212-3p was decreased in response to CCI. The intrathecal injection of miR-212-3p agomir into CCI rats improved the PWT and PWL, decreased the IL-1ß, IL-6 and TNF-α, decreased the expression levels of BCL2 associated X, apoptosis regulator (Bax), cleaved caspase-3 and increased the expression levels of BCL2 apoptosis regulator (Bcl-2). The results of dual--luciferase reporter assay showed that miR-212-3p could directly bind with 3'UTR of NaV1.3. The expression of NaV1.3 was up-regulated in CCI rats who were intrathecally injected with miRctrl, whereas it decreased in CCI rats intrathecally injected with miR-212-3p agomir. CONCLUSION: The expression of miR-212a-3p attenuates neuropathic pain by targeting NaV1.3.

Genome-Wide Transcriptome Landscape of Embryonic Brain-Derived Neural Stem Cells Exposed to Alcohol with Strain-Specific Cross-Examination in BL6 and CD1 Mice.

We have previously reported the deregulatory impact of ethanol on global DNA methylation of brain-derived neural stem cells (NSC). Here, we conducted a genome-wide RNA-seq analysis in differentiating NSC exposed to different modes of ethanol exposure. RNA-seq results showed distinct gene expression patterns and canonical pathways induced by ethanol exposure and withdrawal. Short-term ethanol exposure caused abnormal up-regulation of synaptic pathways, while continuous ethanol treatment profoundly affected brain cells' morphology. Ethanol withdrawal restored the gene expression profile of differentiating NSC without rescuing impaired expression of epigenetics factors. Ingenuity Pathway Analysis (IPA) analysis predicated that ethanol may impact synaptic functions via GABA receptor signalling pathway and affects neural system and brain morphology. We identified Sptbn2, Dcc, and Scn3a as candidate genes which may link alcohol-induced neuronal morphology to brain structural abnormalities, predicted by IPA analysis. Cross-examination of Scn3a and As3mt in differentiated NSC from two different mouse strains (BL6 and CD1) showed a consistent pattern of induction and reduction, respectively. Collectively, our study identifies genetic networks, which may contribute to alcohol-mediated cellular and brain structural dysmorphology, contributing to our knowledge of alcohol-mediated damage to central nervous system, paving the path for better understanding of FASD pathobiology.

Deep sequencing and miRNA profiles in alcohol-induced neuroinflammation and the TLR4 response in mice cerebral cortex.

Alcohol abuse can induce brain injury and neurodegeneration, and recent evidence shows the participation of immune receptors toll-like in the neuroinflammation and brain damage. We evaluated the role of miRNAs as potential modulators of the neuroinflammation associated with alcohol abuse and the influence of the TLR4 response. Using mice cerebral cortex and next-generation sequencing (NGS), we identified miRNAs that were differentially expressed in the chronic alcohol-treated versus untreated WT or TLR4-KO mice. We observed a differentially expression of miR-183 Cluster (C) (miR-96/-182/-183), miR-200a and miR-200b, which were down-regulated, while mirR-125b was up-regulated in alcohol-treated WT versus (vs.) untreated mice. These miRNAs modulate targets genes related to the voltage-gated sodium channel, neuron hyperexcitability (Nav1.3, Trpv1, Smad3 and PP1-γ), as well as genes associated with innate immune TLR4 signaling response (Il1r1, Mapk14, Sirt1, Lrp6 and Bdnf). Functional enrichment of the miR-183C and miR-200a/b family target genes, revealed neuroinflammatory pathways networks involved in TLR4 signaling and alcohol abuse. The changes in the neuroinflammatory targets genes associated with alcohol abuse were mostly abolished in the TLR4-KO mice. Our results show the relationship between alcohol intake and miRNAs expression and open up new therapeutically targets to prevent deleterious effects of alcohol on the brain.

Re-expression of voltage-gated sodium channel subtype Nav1.3 in the substantia nigra after dopamine depletion.

Abnormal synchronized oscillatory bursts occurring in the basal ganglia (BG) are suggested to be correlated with motor symptoms in Parkinson's disease (PD) patients and animal models of PD. Voltage-gated sodium channels (VGSCs) have been demonstrated to play an important role in the abnormal electrical activity of neurons in the BG. Nav1.3, a VGSCs subtype, is predominantly expressed in embryonic and neonatal nervous system but barely detected in the normal adult nervous system in rodents. Here we investigated the expression patterns of Nav1.3 in the BG of 6-OHDA lesioned Sprague Dawley rats. The results showed that Nav1.3 at mRNA and protein levels was abundantly re-expressed in the ipsilateral and contralateral SN at 49 days postlesion, but was rarely detected in the other nuclei of the BG in the 6-OHDA lesioned rats. Furthermore, Nav1.3 was not only expressed in TH-positive dopaminergic neurons of the ipsilateral and contralateral SN, but also in nestin-positive neural progenitor cells surrounding the ipsilateral SN and the midline region adjacent to the ipsilateral SN in the midbrain at 49 days postlesion. These results suggested that the re-expression of Nav1.3 may influence the electrical activity of dopaminergic neurons in the SN in 6-OHDA lesioned rats.

Bulleyaconitine A attenuates hyperexcitability of dorsal root ganglion neurons induced by spared nerve injury: The role of preferably blocking Nav1.7 and Nav1.3 channels.

Background Oral administration of Bulleyaconitine A, an extracted diterpenoid alkaloid from Aconitum bulleyanum plants, is effective for treating chronic pain in rats and in human patients, but the underlying mechanisms are poorly understood. Results As the hyperexcitability of dorsal root ganglion neurons resulting from the upregulation of voltage-gated sodium (Nav) channels has been proved critical for development of chronic pain, we tested the effects of Bulleyaconitine A on Nav channels in rat spared nerve injury model of neuropathic pain. We found that Bulleyaconitine A at 5 nM increased the threshold of action potentials and reduced the firing rate of dorsal root ganglion neurons in spared nerve injury rats but not in sham rats. Bulleyaconitine A preferably blocked tetrodotoxin-sensitive Nav channels over tetrodotoxin-resistant ones in dorsal root ganglion neurons of spared nerve injury rats. Bulleyaconitine A was more potent for blocking Nav1.3 and Nav1.7 than Nav1.8 in cell lines. The half maximal inhibitory concentration (IC50) values for resting Nav1.3, Nav1.7, and Nav1.8 were 995.6 ± 139.1 nM, 125.7 ± 18.6 nM, and 151.2 ± 15.4 µM, respectively, which were much higher than those for inactivated Nav1.3 (20.3 ± 3.4 pM), Nav1.7 (132.9 ± 25.5 pM), and Nav1.8 (18.0 ± 2.5 µM). The most profound use-dependent blocking effect of Bulleyaconitine A was observed on Nav1.7, less on Nav1.3, and least on Nav1.8 at IC50 concentrations. Bulleyaconitine A facilitated the inactivation of Nav channels in each subtype. Conclusions Preferably blocking tetrodotoxin-sensitive Nav1.7 and Nav1.3 in dorsal root ganglion neurons may contribute to Bulleyaconitine A's antineuropathic pain effect.

The neurosteroid allopregnanolone sulfate inhibits Nav1.3 α subunit-containing voltage-gated sodium channels, expressed in Xenopus oocytes.

The neurosteroid allopregnanolone has potent analgesic effects, and its potential use for neuropathic pain is supported by recent reports. However, the analgesic mechanisms are obscure. The voltage-gated sodium channels (Nav) α subunit Nav1.3 is thought to play an essential role in neuropathic pain. Here, we report the effects of allopregnanolone sulfate (APAS) on sodium currents (INa) in Xenopus oocytes expressing Nav1.3 with ß1 or ß3 subunits. APAS suppressed INa of Nav1.3 with ß1 and ß3 in a concentration-dependent manner (IC50 values; 75 and 26 µmol/L). These results suggest the possible importance of Nav1.3 inhibition for the analgesic mechanisms of allopregnanolone.

Swimming Exercise Induced Reversed Expression of miR-96 and Its Target Gene NaV1.3 in Diabetic Peripheral Neuropathy in Rats.

Diabetes is a common metabolic disease which leads to diabetic peripheral neuropathy. Recently, the role of microRNA-96 (miR-96) in alleviating neuropathic pain by inhibiting the expression of NaV1.3, an isoform of voltage-gated sodium channels, has been shown. Peripheral nerve injuries result in NaV1.3 elevation. Exercise has beneficial role in diabetes management and peripheral neuropathy. However, the effects of exercise on miR-96 and its target gene NaV1.3 in diabetic rats are unknown. Therefore, the present study investigated the effects of exercise training on the expression of miR-96 and NaV1.3 in diabetic rats. For this purpose, rats were randomly divided into four groups: control, exercise, diabetic and diabetic-exercise groups. Type 2 diabetes was induced by a high-fat diet and the administration of streptozotocin (STZ) (35 mg/kg, i.p.). The exercise groups were subjected to swimming exercise 5 days/week for 10 weeks. At the end of the treatment period, thermal pain threshold, determined through the tail-flick test, and the expression levels of miR-96 and its target gene NaV1.3 were determined by reverse transcription (RT)-PCR in the sciatic nerve tissues of the rats. Data of the present study indicated that diabetes diminished miR-96 expression levels, but significantly upregulated NaV1.3 expression in the sciatic nerve. On exercise training, miR-96 expression was reversed with concurrent down-regulation of the NaV1.3 expression. This study introduced a new and potential miRNA-dependent mechanism for exerciseinduced protective effects against diabetic thermal hyperalgesia.

Conditional knockout of NaV1.6 in adult mice ameliorates neuropathic pain.

Voltage-gated sodium channels NaV1.7, NaV1.8 and NaV1.9 have been the focus for pain studies because their mutations are associated with human pain disorders, but the role of NaV1.6 in pain is less understood. In this study, we selectively knocked out NaV1.6 in dorsal root ganglion (DRG) neurons, using NaV1.8-Cre directed or adeno-associated virus (AAV)-Cre mediated approaches, and examined the specific contribution of NaV1.6 to the tetrodotoxin-sensitive (TTX-S) current in these neurons and its role in neuropathic pain. We report here that NaV1.6 contributes up to 60% of the TTX-S current in large, and 34% in small DRG neurons. We also show NaV1.6 accumulates at nodes of Ranvier within the neuroma following spared nerve injury (SNI). Although NaV1.8-Cre driven NaV1.6 knockout does not alter acute, inflammatory or neuropathic pain behaviors, AAV-Cre mediated NaV1.6 knockout in adult mice partially attenuates SNI-induced mechanical allodynia. Additionally, AAV-Cre mediated NaV1.6 knockout, mostly in large DRG neurons, significantly attenuates excitability of these neurons after SNI and reduces NaV1.6 accumulation at nodes of Ranvier at the neuroma. Together, NaV1.6 in NaV1.8-positive neurons does not influence pain thresholds under normal or pathological conditions, but NaV1.6 in large NaV1.8-negative DRG neurons plays an important role in neuropathic pain.

Abnormal changes in voltage-gated sodium channels subtypes NaV1.1, NaV1.2, NaV1.3, NaV1.6 and CaM/CaMKII pathway in low-grade astrocytoma.

Epileptic seizures are the main clinical manifestation of low-grade astrocytoma. Voltage-gated sodium channels (VGSCs) play a crucial role in epilepsy. Until now, the role of VGSCs and the relationships between calmodulin (CaM)/CaM-dependent protein kinase II (CaMKII) and VGSCs in low-grade astrocytoma have not been demonstrated. In our study, the protein expression of NaV1.3, NaV1.6 and CaM was significantly increased in the tumor compared to control tissue, while the level of p-CaMKII/CaMKII was significantly decreased in the tumor group as determined by Western Blotting and immunohistochemistry. Furthermore, double-labeling immunofluorescence results showed that NaV1.3/NaV1.6 and CaM co-localization was significantly increased in the tumor group compared to control tissue. This study represents the first evidence of the abnormal changes in VGSCs subtypes and CaM/CaMKII pathway in human brain low-grade astrocytoma, providing new potential targets for molecular therapies of this disease.

Biphasic voltage-dependent inactivation of human NaV 1.3, 1.6 and 1.7 Na+ channels expressed in rodent insulin-secreting cells.

KEY POINTS: Na+ current inactivation is biphasic in insulin-secreting cells, proceeding with two voltage dependences that are half-maximal at ∼-100 mV and -60 mV. Inactivation of voltage-gated Na+ (NaV ) channels occurs at ∼30 mV more negative voltages in insulin-secreting Ins1 and primary ß-cells than in HEK, CHO or glucagon-secreting αTC1-6 cells. The difference in inactivation between Ins1 and non-ß-cells persists in the inside-out patch configuration, discounting an involvement of a diffusible factor. In Ins1 cells and primary ß-cells, but not in HEK cells, inactivation of a single NaV subtype is biphasic and follows two voltage dependences separated by 30-40 mV. We propose that NaV channels adopt different inactivation behaviours depending on the local membrane environment. ABSTRACT: Pancreatic ß-cells are equipped with voltage-gated Na+ channels that undergo biphasic voltage-dependent steady-state inactivation. A small Na+ current component (10-15%) inactivates over physiological membrane potentials and contributes to action potential firing. However, the major Na+ channel component is completely inactivated at -90 to -80 mV and is therefore inactive in the ß-cell. It has been proposed that the biphasic inactivation reflects the contribution of different NaV α-subunits. We tested this possibility by expression of TTX-resistant variants of the NaV subunits found in ß-cells (NaV 1.3, NaV 1.6 and NaV 1.7) in insulin-secreting Ins1 cells and in non-ß-cells (including HEK and CHO cells). We found that all NaV subunits inactivated at 20-30 mV more negative membrane potentials in Ins1 cells than in HEK or CHO cells. The more negative inactivation in Ins1 cells does not involve a diffusible intracellular factor because the difference between Ins1 and CHO persisted after excision of the membrane. NaV 1.7 inactivated at 15--20 mV more negative membrane potentials than NaV 1.3 and NaV 1.6 in Ins1 cells but this small difference is insufficient to solely explain the biphasic inactivation in Ins1 cells. In Ins1 cells, but never in the other cell types, widely different components of NaV inactivation (separated by 30 mV) were also observed following expression of a single type of NaV α-subunit. The more positive component exhibited a voltage dependence of inactivation similar to that found in HEK and CHO cells. We propose that biphasic NaV inactivation in insulin-secreting cells reflects insertion of channels in membrane domains that differ with regard to lipid and/or membrane protein composition.

Sodium channel NaV1.3 is important for enterochromaffin cell excitability and serotonin release.

In the gastrointestinal (GI) epithelium, enterochromaffin (EC) cells are enteroendocrine cells responsible for producing >90% of the body's serotonin (5-hydroxytryptamine, 5-HT). However, the molecular mechanisms of EC cell function are poorly understood. Here, we found that EC cells in mouse primary cultures fired spontaneous bursts of action potentials. We examined the repertoire of voltage-gated sodium channels (NaV) in fluorescence-sorted mouse EC cells and found that Scn3a was highly expressed. Scn3a-encoded NaV1.3 was specifically and densely expressed at the basal side of both human and mouse EC cells. Using electrophysiology, we found that EC cells expressed robust NaV1.3 currents, as determined by their biophysical and pharmacologic properties. NaV1.3 was not only critical for generating action potentials in EC cells, but it was also important for regulating 5-HT release by these cells. Therefore, EC cells use Scn3a-encoded voltage-gated sodium channel NaV1.3 for electrical excitability and 5-HT release. NaV1.3-dependent electrical excitability and its contribution to 5-HT release is a novel mechanism of EC cell function.

High-throughput electrophysiological assays for voltage gated ion channels using SyncroPatch 768PE.

Ion channels regulate a variety of physiological processes and represent an important class of drug target. Among the many methods of studying ion channel function, patch clamp electrophysiology is considered the gold standard by providing the ultimate precision and flexibility. However, its utility in ion channel drug discovery is impeded by low throughput. Additionally, characterization of endogenous ion channels in primary cells remains technical challenging. In recent years, many automated patch clamp (APC) platforms have been developed to overcome these challenges, albeit with varying throughput, data quality and success rate. In this study, we utilized SyncroPatch 768PE, one of the latest generation APC platforms which conducts parallel recording from two-384 modules with giga-seal data quality, to push these 2 boundaries. By optimizing various cell patching parameters and a two-step voltage protocol, we developed a high throughput APC assay for the voltage-gated sodium channel Nav1.7. By testing a group of Nav1.7 reference compounds' IC50, this assay was proved to be highly consistent with manual patch clamp (R > 0.9). In a pilot screening of 10,000 compounds, the success rate, defined by > 500 MΩ seal resistance and >500 pA peak current, was 79%. The assay was robust with daily throughput ~ 6,000 data points and Z' factor 0.72. Using the same platform, we also successfully recorded endogenous voltage-gated potassium channel Kv1.3 in primary T cells. Together, our data suggest that SyncroPatch 768PE provides a powerful platform for ion channel research and drug discovery.