Sodium Current Amplitude Research Articles

GPD1L-A306del modifies sodium current in a family carrying the dysfunctional SCN5A-G1661R mutation associated with Brugada syndrome

Loss-of-function variants of SCN5A, encoding the sodium channel alpha subunit Nav1.5 are associated with high phenotypic variability and multiple cardiac presentations, while underlying mechanisms are incompletely understood. Here we investigated a family with individuals affected by Brugada Syndrome (BrS) of different severity and aimed to unravel the underlying genetic and electrophysiological basis.Next-generation sequencing was used to identify the genetic variants carried by family members. The index patient, who was severely affected by arrhythmogenic BrS, carried previously uncharacterized variants of Nav1.5 (SCN5A-G1661R) and glycerol-3-phosphate dehydrogenase-1-like protein (GPD1L-A306del) in a double heterozygous conformation. Family members exclusively carrying SCN5A-G1661R showed asymptomatic Brugada ECG patterns, while another patient solely carrying GPD1L-A306del lacked any clinical phenotype.To assess functional mechanisms, Nav1.5 channels were transiently expressed in HEK-293 cells in the presence and absence of GPD1L. Whole-cell patch-clamp recordings revealed loss of sodium currents after homozygous expression of SCN5A-G1661R, and reduction of current amplitude to ~ 50% in cells transfected with equal amounts of wildtype and mutant Nav1.5. Co-expression of wildtype Nav1.5 and GPD1L showed a trend towards increased sodium current amplitudes and a hyperpolarizing shift in steady-state activation and -inactivation compared to sole SCN5A expression. Application of the GPD1L-A306del variant shifted steady-state activation to more hyperpolarized and inactivation to more depolarized potentials.In conclusion, SCN5A-G1661R produces dysfunctional channels and associates with BrS. SCN5A mediated currents are modulated by co-expression of GDP1L and this interaction is altered by mutations in both proteins. Thus, additive genetic burden may aggravate disease severity, explaining higher arrhythmogenicity in double mutation carriers.

Functional diversity of sodium channel variants in common eastern bumblebee, Bombus impatiens.

For the past decade, Colony Collapse Disorder has been reported worldwide. Insecticides containing pyrethroids may be responsible for a decline in bees, which are more sensitive to pyrethroids compared with other insects. Voltage-gated sodium channels (Nav ) are the major target sites of pyrethroids, and the sodium channel diversity is generated through extensive alternative splicing and RNA editing. In this study, we cloned and analyzed the function of variants of the Nav channel, BiNav , from Bombus impatiens. BiNav covers a 46 kb genome region including 30 exons. Sequence analysis of 56 clones showed that the clones can be grouped into 22 splice types with 11 optional exons (exons j, w, p, q, r, b, e, t, l/k, and z). Here, a special alternative exon w is identified, encoding a stretch of 31 amino acid resides in domain I between S3 and S4. RNA editing generates 18 amino acid changes in different positions in individual variants. Among 56 variants examined, only six variants generated sufficient sodium currents for functional characterization in Xenopus oocytes. In the presence of B. impatiens TipE and TEH1, the sodium current amplitude of BiNav 1-1 increased by fourfold, while TipE of other insect species had no effect on the expression. Abundant alternative splicing and RNA editing of BiNav suggests the molecular and functional pharmacology diversity of the Nav channel for bumblebees. This study provides a theoretical basis for the design of insecticides that specifically target pests without affecting beneficial insects.

A de novo arrhythmogenic Nav1.5 variant, E171Q, causes multiple biophysical defects.

An infant presenting antenatally with cardiac conduction disease and junctional tachycardia reminiscent of the multi-focal ectopic Purkinje-related premature contractions (MEPPC) syndrome was found to harbour the de novo E171Q variant of the cardiac voltage-gated sodium channel, Nav1.5. Functional analysis was performed using whole cell patch clamp and current clamp to compare WT Nav1.5 with E171Q expressed in HEK293 cells and iPSC-CMs. We found E171Q significantly right-shifted the voltage-dependence of both activation and steady-state fast inactivation, and increased the amplitude of late sodium current. These changes were reversed upon perfusion with 100 μM ranolazine. Incorporating the shifts in voltage-dependence and late current into the Ohara-Rudy cardiac action potential model produced a delay in action potential repolarization. Further investigation revealed a significant outward gating pore (omega) current in E171Q that was mitigated upon perfusion of 3 μM flecainide or with K+-free internal solution. Current clamp experiments in iPSC-CMs revealed action potential variability with delayed afterdepolarizations in myocytes expressing E171Q. The increase in gating pore current is consistent with disrupting the interaction between E171 and a corresponding positive charge in the S4 voltage sensor. This disruption could result in the completion of an ion-permeable pore, consistent with that previously described for Nav1.5 R222Q - a variant also associated with MEPPC - and Nav1.4 R222W.

Enhanced sodium channel inactivation by temperature and FHF2 deficiency blocks heat nociception.

Transient voltage-gated sodium currents are essential for the initiation and conduction of action potentials in neurons and cardiomyocytes. The amplitude and duration of sodium currents are tuned by intracellular fibroblast growth factor homologous factors (FHFs/iFGFs) that associate with the cytoplasmic tails of voltage-gated sodium channels (Na v s), and genetic ablation of Fhf genes disturbs neurological and cardiac functions. Among reported phenotypes, Fhf2null mice undergo lethal hyperthermia-induced cardiac conduction block attributable to the combined effects of FHF2 deficiency and elevated temperature on the cardiac sodium channel (Na v 1.5) inactivation rate. Fhf2null mice also display a lack of heat nociception, while retaining other somatosensory capabilities. Here, we use electrophysiological and computational methods to show that the heat nociception deficit can be explained by the combined effects of elevated temperature and FHF2 deficiency on the fast inactivation gating of Na v 1.7 and tetrodotoxin-resistant sodium channels expressed in dorsal root ganglion C fibers. Hence, neurological and cardiac heat-associated deficits in Fhf2null mice derive from shared impacts of FHF deficiency and temperature towards Na v inactivation gating kinetics in distinct tissues.

Epilepsy-Induced High Affinity Blockade of the Cardiac Sodium Current INa by Lamotrigine; A Potential for Acquired Arrythmias.

Lamotrigine is widely prescribed to treat bipolar neurological disorder and epilepsy. It exerts its antiepileptic action by blocking voltage-gated sodium channels in neurons. Recently, the US Food and Drug Administration issued a warning on the use of Lamotrigine after observations of conduction anomalies and Brugada syndrome patterns on the electrocardiograms of epileptic patients treated with the drug. Brugada syndrome and conduction disturbance are both associated with alterations of the cardiac sodium current (INa) kinetics and amplitude. In this study, we used the patch clamp technique on cardiomyocytes from epileptic rats to test the hypothesis that Lamotrigine also blocks INa in the heart. We found that Lamotrigine inhibited 60% of INa peak amplitude and reduced cardiac excitability in epileptic rats but had little effect in sham animals. Moreover, Lamotrigine inhibited 67% of INaL and, more importantly, prolonged the action potential refractory period in epileptic animals. Our results suggest that enhanced affinity of Lamotrigine for INa may in part explain the clinical phenotypes observed in epileptic patients.

Mechanistic insights into the interaction of cardiac sodium channel Nav1.5 with MOG1 and a new molecular mechanism for Brugada syndrome.

Mutations in cardiac sodium channel Nav1.5 cause Brugada syndrome (BrS). MOG1 is a chaperone that binds to Nav1.5, facilitates Nav1.5 trafficking to the cell surface, and enhances the amplitude of sodium current INa. The purpose of this study was to identify structural elements involved in MOG1-Nav1.5 interaction and their relevance to the pathogenesis of BrS. Systematic analyses of large deletions, microdeletions, and point mutations, and glutathione S-transferases pull-down, co-immunoprecipitation, cell surface protein quantification, and patch-clamping of INa were performed. Large deletion analysis defined the MOG1-Nav1.5 interaction domain to amino acids S476-H585 of Nav1.5 Loop I connecting transmembrane domains I and II. Microdeletion and point mutation analyses further defined the domain to F530T531F532R533R534R535. Mutations F530A, F532A, R533A, and R534A, but not T531A and R535A, significantly reduced MOG1-Nav1.5 interaction and eliminated MOG1-enhanced INa. Mutagenesis analysis identified D24, E36, D44, E53, and E101A of MOG1 as critical residues for interaction with Nav1.5 Loop I. We then characterized 3 mutations at the MOG1-Nav1.5 interaction domain: p.F530V, p.F532C, and p.R535Q reported from patients with long QT syndrome and BrS. We found that p.F532C reduced MOG1-Nav1.5 interaction and eliminated MOG1 function on INa; p.R535Q is also a loss-of-function mutation that reduces INa amplitude in a MOG1-independent manner, whereas p.F530V is benign as it does not have an apparent effect on MOG1 and INa. Our findings define the MOG1-Nav1.5 interaction domain to a 5-amino-acid motif of F530T531F532R533R534 in Loop I. Mutation p.F532C associated with BrS abolishes Nav1.5 interaction with MOG1 and reduces MOG1-enhanced INa density, thereby uncovering a novel molecular mechanism for the pathogenesis of BrS.

Determination of the toxicity equivalency factors for ciguatoxins using human sodium channels

Ciguatoxins (CTXs) which are produced by dinoflagellates of the genus Gambierdiscus and Fukuyoa and share a ladder-shaped polyether structure, are causative compounds of one of the most frequent foodborne illness disease known as ciguatera fish poisoning (CFP). CFP was initially found in tropical and subtropical areas but nowadays the dinoflagellates producers of ciguatoxins had spread to European coasts. Therefore, this raises the need of establishing toxicity equivalency factors for the different compounds that can contribute to ciguatera fish poisoning, since biological methods have been replaced by analytical techniques. Thus, in this work, the effects of six compounds causative of ciguatera, on their main target, the human voltage-gated sodium channels have been analyzed for the first time. The results presented here led to the conclusion that the order of potency was CTX1B, CTX3B, CTX4A, gambierol, gambierone and MTX3. Furthermore, the data indicate that the activation voltage of sodium channels is more sensitive to detect ciguatoxins than their effect on the peak sodium current amplitude.

Prefrontal cortex pyramidal neurons express functional Nav1.8 tetrodotoxin-resistant sodium currents.

It has been repeatedly proved that Nav1.8tetrodotoxin (TTX)-resistant sodium currents are expressed in peripheral sensory neurons where they play important role in nociception. There are very few publications that show the presence of TTX-resistant sodium currents in central neurons. The aim of this study was to assess if functional Nav1.8TTX-resistant sodium currents are expressed in prefrontal cortex pyramidal neurons. All recordings were performed in the presence of TTX in the extracellular solution to block TTX-sensitive sodium currents. The TTX-resistant sodium current recorded in this study was mainly carried by the Nav1.8sodium channel isoform because the Nav1.9 current was inhibited by the -65mV holding potential that we used throughout the study. Moreover, the sodium current that we recorded was inhibited by treatment with the selective Nav1.8 inhibitor A-803467. Confocal microscopy experiments confirmed the presence of the Nav1.8 α subunit in prefrontal cortex pyramidal neurons. Activation and steady state inactivation properties of TTX-resistant sodium currents were also assessed in this study and they were similar to activation and inactivation properties of TTX-resistant sodium currents expressed in dorsal root ganglia (DRG) neurons. Moreover, this study showed that carbamazepine (60µM) inhibited the maximal amplitude of the TTX-resistant sodium current. Furthermore, we found that carbamazepine shifts steady state inactivation curve of TTX-resistant sodium currents toward hyperpolarization. This study suggests that the Nav1.8TTX-resistant sodium channel is expressed not only in DRG neurons, but also in cortical neurons and may be molecular target for antiepileptic drugs such as carbamazepine.

Abstract 14308: Lamotrigine Inhibits the Inward Fast Sodium Current (I Na ) in Epileptic Rat Cardiomyocytes

Introduction: Lamotrigine (LTG) is widely prescribed to treat neurological disorders such as migraine, neurophatic pain, and epilepsy. Recently, LTG has been linked to Brugada síndrome (BrS) ECG associated with a reduction in sodium current (I Na ) amplitude. Experimental studies have revealed that LTG blocks voltage gated sodium channels (VGSC). Hypothesis: Therefore, disrupting the depolarizing fast sodium inward current is a potential cause for arrhythmias. Recently our laboratory has shown that epilepsy induces molecular remodeling of the cardiac ventricle characterized by neuronal sodium channels overexpression, so it would be very plausible that LTG can cause important alterations on I Na in epileptic individuals. Methods: We used Patch-Clamp technique to test the effect of LTG on I Na in sham and epileptic rat ventricular cardiomyocytes. Results: We found that LTG inhibited I Na in a single dose-response manner with an IC 50 =326.35μM in sham rats. In epileptic rat cardiomyocytes the inhibitory effect was biphasic way with an IC 50 (1)=8.09 μM, which is close to the therapeutic range (between 12 and 50 μM), and a IC 50 (2)=333.66 μM. Moreover, LTG also altered sodium channels availability in biphasic manner with Vh(1)=-76.36 ± 0.31 mV and Vh(2)=-121.42 ± 1.38 mV, suggesting that LTG reduced the number of ion channels contributing to I Na . Conclusions: These findings may explain in part how LTG induce a Brugada pattern on the ECG of epileptic patients.

Modulation of Neuronal Excitability by Low- Intensity Ultrasound in Two Principal Neurons of Rat Anteroventral Cochlear Nucleus.

Ultrasonic neuromodulation has proved to be a promising new approach for direct neuromodulation or potential noninvasive deep brain stimulation technology for treating various neurological disorders. Previous studies have demonstrated that ultrasonic waves can noninvasively diffuse through the intact skull and thus precisely target specific brain regions with high spatial resolution. However, its neuromodulatory effects over different cell types of target nuclei have not been fully elucidated. In the present study, we investigated the neuronal excitability resulted from ultrasound stimulation on the two major neurons of anteroventral cochlear nucleus (AVCN) in vitro. Our results demonstrated that bushy cells (BCs) were well maintaining one action potential (AP) in response to the pairing of a sequence of depolarizing current pulses and 60-s continuous low-intensity ultrasound (LIUS), and meanwhile, stellate cells (SCs) significantly increased the firing rate. The ultrasonic waves with an acoustic pressure of 0.13 MPa were elicited by an on-chip ultrasonic stimulation system compatible with patch-clamp recording. Furthermore, LIUS significantly improved the neuronal excitability in both BCs and SCs based on their intrinsic excitability. Modulation of membrane properties among cell types was due to the LIUS-induced increase in the total inward sodium currents ( INa ) and outward potassium currents ( IKv ). LIUS significantly, at a similar rate, increased the amplitude of total inward sodium currents in both cell types. Meanwhile, LIUS induces a higher rate of the outward potassium currents in the BCs compared with SCs. Therefore, this study could provide new evidence for safe use of ultrasonic neuromodulation and its potential therapy for many auditory diseases, such as the central auditory processing disorder.

Expression defect of the rare variant/Brugada mutation R1512W depends upon the SCN5A splice variant background and can be rescued by mexiletine and the common polymorphism H558R

ABSTRACT Background : Mutations in SCN5A that decrease Na current underlie arrhythmia syndromes such as the Brugada syndrome (BrS). SCN5A in humans has two splice variants, one lacking a glutamine at position 1077 (Q1077del) and one containing Q1077. We investigated the effect of splice variant background on loss-of-function and rescue for R1512W, a mutation reported to cause BrS. Methods and results : We made the mutation in both variants and expressed them in HEK-293 cells for voltage-clamp study. After 24 hours of transfection, the current expression level of R1512W was reduced by ~50% in both Q1077del and Q1077 compared to the wild-type (WT) channel, respectively. The activation and inactivation midpoint were not different between WT and mutant channels in both splice variant backgrounds. However, slower time constants of recovery and enhanced intermediate inactivation were observed for R1512W/Q1077 compared with WT-Q1077, while the recovery and intermediate inactivation parameters of R1512W/Q1077del were similar to WT-Q1077del. Furthermore, both mexiletine and the common polymorphism H558R restored peak sodium current (I Na) amplitude of the mutant channel by increasing the cell surface expression of SCN5A. Conclusion : These findings provide further evidence that the splice variant affects the molecular phenotype with implications for the clinical phenotype, and they provide insight into the expression defect mechanisms and potential treatment in BrS.

Partial Blockade of Human Voltage-Dependent Sodium Channels by the Marine Toxins Azaspiracids.

Azaspiracid toxins were first identified at the end of the last century in Irish mussels, and during the last two decades considerable cytotoxic and neurotoxic effects caused by these toxins have been described. Azaspiracids are synthesized by dinoflagellates and accumulate in several species of filter-feeding bivalve mollusks, thereby incorporating into the food chain and causing human intoxications. Among the cellular effects of azaspiracids, inhibition of spikes in neurons and hyperpolarization of the neuronal membrane potential have been reported; however, the underlying processes leading to these effects were never elucidated. In this regard, initial studies reported no activity of the toxin in neuronal voltage-gated sodium channels, and a recent work described no effect of azaspiracid-1 on the inactivation kinetics of voltage-gated sodium channels; however, the relationship between the known alterations of the cytoskeleton caused by these toxins and their effects on ion channels has never been evaluated. In this work, the cytotoxic effect of azaspiracids was evaluated in human cells as well as their activity on voltage-gated sodium channels and in cell morphology in order to unravel the cellular targets involved in the mechanism of action of this group of marine toxins. The data reported here demonstrate, for the first time, that both azaspiracid-1 and azaspiracid-2 caused a rapid concentration-dependent inhibition of the amplitude of voltage-gated sodium currents without affecting their inactivation kinetics, an effect that was increased after long-term treatment of the cells with the toxin. Simultaneously, long-term exposure of the cells to azaspiracids caused a profound alteration of the cell cytoskeleton and decreased the metabolic activity of human cells. Altogether, the data presented here indicate that the partial blockade of voltage-gated sodium channels by these toxins is not related with their effect on the actin cytoskeleton. However, since azaspiracids are common toxins in European waters, their effect on voltage-gated sodium channels, first reported here, should be considered to avoid synergistic toxicity with other marine toxins that are known potent blockers of sodium channels such as the saxitoxins and tetrodotoxins, but further studies are needed in order to elucidate how these compounds alter ion homeostasis.

Lemnalol Modulates the Electrophysiological Characteristics and Calcium Homeostasis of Atrial Myocytes.

Sepsis, an inflammatory response to infection provoked by lipopolysaccharide (LPS), is associated with high mortality, as well as ischemic stroke and new-onset atrial arrhythmia. Severe bacterial infections causing sepsis always result in profound physiological changes, including fever, hypotension, arrhythmia, necrosis of tissue, systemic multi-organ dysfunction and finally death. LPS challenge-induced inflammatory responses during sepsis may increase the likelihood of the arrhythmogenesis. Lemnalol is known to possess potent anti-inflammatory effects. This study examined whether Lemnalol (0.1 μM) could modulate the electrophysiological characteristics and calcium homeostasis of atrial myocytes under the influence of LPS (1μg/mL). Under challenge with LPS, Lemnalol-treated LA myocytes, had a longer AP duration at 20%, 50% and 90% repolarization of the amplitude, compared to the LPS-treated cells. LPS-challenged LA myocytes showed increased late sodium current, Na+-Ca2+ exchanger current, transient outward current, rapid component of delayed rectifier potassium current, tumor necrosis factor-α, NF-κB and increased phosphorylation of ryanodine receptor (RyR), but a lower L-type Ca2+ current than the control LA myocytes. Exposure to Lemnalol reversed the LPS-induced effects. The LPS-treated and control groups of LA myocytes, with or without the existence of Lemnalol. showed no apparent alterations in the sodium current amplitude or Cav1.2 expression. The expression of sarcoendoplasmic reticulum calcium transport ATPase (SERCA2) was reduced by LPS treatment, while Lemnalol ameliorated the LPS-induced alterations. The phosphorylation of RyR was enhanced by LPS treatment, while Lemnalol attenuated the LPS-induced alterations. In conclusion, Lemnalol modulates LPS-induced alterations of LA calcium homeostasis and blocks the NF-κB pathways, which may contribute to the attenuation of LPS-induced arrhythmogenesis.

Electrophysiological characterization of acutely isolated spiral ganglion neurons in neonatal and mature sonic hedgehog knock-in mice

Spiral ganglion neurons (SGNs) are primary afferent auditory neurons activated by inner hair cells in mammalian cochlea. Here, for the convenience of SGN studies such as patch-clamp or single cell RNA-sequence studies, a knock-in mouse (ShhCreEGFP/+; Rosa26-Tdtomatoloxp/+) was generated for the purpose of obtaining fluorescence SGNs. Auditory brainstem response (ABR) and Tuj1 immunohistochemistry staining were performed to verify the hearing function and the morphological characteristics. The results showed that there was no significant difference between shh and wild type mice. In electrophysiological studies, we verified a series of electrophysiological characteristics including the amplitude of sodium and potassium currents and action potential characteristics of shh and wild type mice and no significant differences were found either. From the above, shh mice have the same cell function and morphology as their littermate control wild type mice and could be used as an ideal tool to study the function and characteristics of spiral ganglion neurons.Potassium channels of SGNs play an important role in resolving time accuracy. We obtained similar amplitude of IK+ in neonatal and mature mice in the aging competition experiment, however, the density of IK+ from mature mice were significantly different from those of neonatal mice, a phenomenon that may play a key role in the nervous system. Potassium channels have been shown to contribute to apoptosis induced by cisplatin administration in various cell lines. Here we used cisplatin administration to study the ototoxicity and found that the effects of a low dose of cisplatin (0.5 mM correspond to therapeutic doses) causes a decrease in currents and is reversible after a short administration time. Moreover, we propose the activated state of potassium channels has changed but the characteristic and number remain still after cisplatin administration. The excess potassium ions may accumulate in the cell body, which had affected the firing properties and induce cytotoxicity and apoptosis. We suggest that the electrophysiological properties of acutely isolated SGNs may support further research on the mechanics of auditory propagation and ion channel pharmacology.

Modified excitability and persistent sodium current amplitude in cortical pyramidal neurons from a mouse model of Amyotrophic Lateral Sclerosis

Modified excitability and persistent sodium current amplitude in cortical pyramidal neurons from a mouse model of Amyotrophic Lateral Sclerosis

Abstract 808: Functional Characterization of a Novel Scn5a Mutation Associated With the Brugada Syndrome

Background: Brugada syndrome (BrS) is a cardiac disorder characterized by conduction abnormalities that can lead to sudden death; syncope and cardiac arrest are clinical manifestations which are often associated with an enhancement of the vagal activity. Mutations in the SCN5A gene (Na v 1.5 channel) are the most common cause of the inherited forms of BrS. Objective: To characterize the functional behavior of mutant Na v 1.5 channels expressing a novel heterozygous mutation (S805L) recently identified in an Italian family affected by the BrS. Methods: HEK cells were used as experimental model to express both the wild-type (WT) and the mutated S805L channels (alone, Homo or in combination, Hetero) and the accessory β-subunit (SCN1B). Patch-clamp and western blot experiments were carried out to assess the dysfunctional role of the mutation. Results: When compared to the WT current, the S508L mutation significantly (P&It0.05) decreases the peak current density by about 65% for the Homo condition (WT: -120.2±10.2, n=28); Homo: -40.3±4.2, n=16) and by 35% for the Hetero condition (Hetero: -78.2±8.3, n=27). Densitometric analysis carried out on western blot data further support the conclusion that S805L channels are less abundant in the plasma membrane. We also observed that the S805L mutation positively shifts the V½ values of the voltage dependence of the inactivation of both Homo and Hetero currents (V½: WT -85.5±0.2 mV, n=55; Homo -80.9±0.3 mV, n=22; Hetero -81.9±0.2 mV, n=25; P&It0.05); a positive shift of the V½ of the activation was also observed but only in the Homo condition (V½: WT -33.0±0.4 mV, n=28; Homo -30.0±0.5, n=16, P&It0.05). The kinetics of recovery from inactivation and the amplitude of the late sodium current were also evaluated but they were unaffected by the mutation. Conclusion: When expressed in the Hetero condition, the S805L mutation causes a reduction in the channel expression, however, the positive shift of the inactivation curve suggests an increase in Na channel availability. We thus believe that the precise quantitative balance between these two phenomena and their relation with vagal activity may underlie the clinical manifestation of the disease.

Modified age-dependent expression of NaV1.6 in an ALS model correlates with motor cortex excitability alterations

Cortical hyperexcitability is an early and intrinsic feature of Amyotrophic Lateral Sclerosis (ALS), but the mechanisms underlying this critical neuronal dysfunction are poorly understood. Recently, we have demonstrated that layer V pyramidal neurons (PNs) in the primary motor cortex (M1) of one-month old (P30) G93A ALS mice display an early hyperexcitability status compared to Control mice. In order to investigate the time-dependent evolution of the cortical excitability in the G93A ALS model, here we have performed an electrophysiological and immunohistochemical study at three different mouse ages. M1 PNs from 14-days old (P14) G93A mice have shown no excitability alterations, while M1 PNs from 3-months old (P90) G93A mice have shown a hypoexcitability status, compared to Control mice. These age-dependent cortical excitability dysfunctions correlate with a similar time-dependent trend of the persistent sodium current (INaP) amplitude alterations, suggesting that INaP may play a crucial role in the G93A cortical excitability aberrations. Specifically, immunohistochemistry experiments have indicated that the expression level of the NaV1.6 channel, one of the voltage-gated Na+ channels mainly distributed within the central nervous system, varies in G93A primary motor cortex during disease progression, according to the excitability and INaP alterations, but not in other cortical areas. Microfluorometry experiments, combined with electrophysiological recordings, have verified that P30 G93A PNs hyperexcitability is associated to a greater accumulation of intracellular calcium ([Ca2+]i) compared to Control PNs, and that this difference is still present when G93A and Control PNs fire action potentials at the same frequency.These results suggest that [Ca2+]i de-regulation in G93A PNs may contribute to neuronal demise and that the NaV1.6 channels could be a potential therapeutic target to ameliorate ALS disease progression.

Treating TB human neuroectodermal cell line with retinoic acid induces the appearance of neuron-like voltage-gated ionic currents

TB is a cell line derived from the cerebrospinal fluid sample of a patient with primary leptomeningeal melanomatosis. Our previous immunological and ultrastructural analysis revealed that TB cells differentiate towards a neuronal phenotype when grown in vitro up to 7 days in presence of 10 µM all-trans retinoic acid (RA). Recently, we reported that TB cells are sensitive to the cytotoxic effects of β-amyloid peptides, activating the cytosolic phospholipase A2. To date, it is not known if RA, in addition to inducing morphological changes, also causes functional modification in TB cells, by regulating voltage-gated ionic currents. To this purpose, we performed electrophysiological characterization of undifferentiated (TB) and differentiated (RA-TB) cells by means of whole-cell patch clamp recordings. Upon depolarizing stimuli, both groups displayed voltage-gated K+ outward currents of similar amplitude. By contrast, the low amplitude voltage-gated Na+ currents recorded in undifferentiated TB cells were largely up-regulated by RA exposure. This current was strongly reduced by TTX and lidocaine and completely abolished by removal of extracellular sodium. Furthermore, treatment with RA caused the appearance of a late-onset inward current carried by Ca2+ ions in a subpopulation of TB cells. This current was not affected by removal of extracellular Na+ and was completely blocked by Cd2+, a broad-spectrum blocker of Ca2+ currents. Altogether, our results indicate that RA-differentiation of TB cells induces functional changes by augmenting the amplitude of voltage-gated sodium current and by inducing, in a subpopulation of treated cells, the appearance of a voltage-gated calcium current.

6 Hz Active Anticonvulsant Fluorinated N-Benzamide Enaminones and Their Inhibitory Neuronal Activity.

A small library of novel fluorinated N-benzamide enaminones were synthesized and evaluated in a battery of acute preclinical seizure models. Three compounds (GSA 62, TTA 35, and WWB 67) were found to have good anticonvulsant activity in the 6-Hz ‘psychomotor’ 44-mA rodent model. The focus of this study was to elucidate the active analogs’ mode of action on seizure-related molecular targets. Electrophysiology studies were employed to evaluate the compounds’ ability to inhibit neuronal activity in central olfactory neurons, mitral cells, and sensory-like ND7/23 cells, which express an assortment of voltage and ligand-gated ion channels. We did not find any significant effects of the three compounds on action potential generation in mitral cells. The treatment of ND7/23 cells with 50 µM of GSA 62, TTA 35, and WWB 67 generated a significant reduction in the amplitude of whole-cell sodium currents. Similar treatment of ND7/23 cells with these compounds had no effect on T-type calcium currents, indicating that fluorinated N-benzamide enaminone analogs may have a selective effect on voltage-gated sodium channels, but not calcium channels.

Eliminating spiral wave and spatiotemporal chaos in cardiac tissues by suppressing the rotation of spiral wave tip

The variation of the function of sodium channel in cardiomyocyte is associated with multiple cardiac diseases. Increasing sodium channel availability can effectively increase sodium influx, leading to enhanced cardiomyocyte excitability, prolonged action potential duration and late sodium current activity, which may cause ventricular arrhythmia. On the other hand, enhancing cardiomyocyte excitability can effectively increase the conduction velocity of the medium in the rotation center of spiral wave, which can restrain the rotation of spiral wave, leading to the disappearance of spiral wave. However, how to increase the excitability of cardiomyocytes while avoiding arrhythmias has not yet been explored so far. In this paper, we study how to regulate the changes of sodium current in cardiac myocytes to control spiral wave and spatiotemporal chaos in a two-dimensional cardiac tissues by using the Luo-Rudy phase I model. We propose such a sodium current control scheme:when the cell is excited, the regulation of sodium current begins. If the absolute value of sodium current obtained from the model equation is less than the absolute value of sodium current control threshold, the sodium current is simply equal to the control threshold of sodium current. In other cases, the absolute value of sodium current cannot exceed the maximum value without control. When the membrane potential rises over-5 mV, the sodium current evolves naturally. This method of regulating sodium current ensures that all cells have almost the same amplitude of sodium current, while without obviously changing the excitation-time. All cells thus have the same excitability under the control of sodium current, so that the excitation of cell is less affected by spiral wave tip. The numerical simulation results show that as long as the control threshold of sodium current reaches a critical value, the rotation of spiral wave tip is effectively suppressed, causing spiral wave to move out of the system boundary and spatiotemporal chaos to disappear after it has evolved into a spiral wave. If the absolute value of sodium current control threshold is large enough, the spiral wave and spatiotemporal chaos would also disappear through conductive block. These results can provide a new idea for antiarrhythmic therapy.