Action Potentials In Cells Research Articles

Dimethyloxalylglycine (DMOG), a Hypoxia Mimetic Agent, Does Not Replicate a Rat Pheochromocytoma (PC12) Cell Biological Response to Reduced Oxygen Culture.

Cells respond to reduced oxygen availability predominately by activation of the hypoxia-inducible factor (HIF) pathway. HIF activation upregulates hundreds of genes that help cells survive in the reduced oxygen environment. The aim of this study is to determine whether chemical-induced HIF accumulation mimics all aspects of the hypoxic response of cells. We compared the effects of dimethyloxalylglycine (DMOG) (a HIF stabiliser) on PC12 cells cultured in air oxygen (20.9% O2, AO) with those cultured in either intermittent 20.9% O2 to 2% O2 (IH) or constant 2% O2 (CN). Cell viability, cell cycle, HIF accumulation, reactive oxygen species (ROS) formation, mitochondrial function and differentiation were used to characterise the PC12 cells and evaluate the impact of DMOG. IH and CN culture reduced the increase in cell numbers after 72 and 96 h and MTT activity after 48 h compared to AO culture. Further, DMOG supplementation in AO induced a dose-dependent reduction in the increase in PC12 cell numbers and MTT activity. IH-cultured PC12 cells displayed increased and sustained HIF-1 expression over 96 h. This was accompanied by increased ROS and mitochondrial burden. PC12 cells in CN displayed little changes in HIF-1 expression or ROS levels. DMOG (0.1 mM) supplementation resulted in an IH-like HIF-1 profile. The mitochondrial burden and action potential of DMOG-supplemented PC12 cells did not mirror those seen in other conditions. DMOG significantly increased S phase cell populations after 72 and 96 h. No significant effect on PC12 cell differentiation was noted with IH and CN culture without induction by nerve growth factor (NGF), while DMOG significantly increased PC12 cell differentiation with and without NGF. In conclusion, DMOG and reduced oxygen levels stabilise HIF and affect mitochondrial activity and cell behaviour. However, DMOG does not provide an accurate replication of the reduced oxygen environments.

Reducing Nav1.6 expression attenuates the pathogenesis of Alzheimer's disease by suppressing BACE1 transcription.

Aberrant increases in neuronal network excitability may contribute to cognitive deficits in Alzheimer's disease (AD). However, the mechanisms underlying hyperexcitability of neurons are not fully understood. Voltage‐gated sodium channels (VGSC or Nav), which are involved in the formation of excitable cell's action potential and can directly influence the excitability of neural networks, have been implicated in AD‐related abnormal neuronal hyperactivity and higher incidence of spontaneous non‐convulsive seizures. Here, we have shown that the reduction of VGSC α‐subunit Nav1.6 (by injecting adeno‐associated virus (AAV) with short hairpin RNA (shRNA) into the hippocampus) rescues cognitive impairments and attenuates synaptic deficits in APP/PS1 transgenic mice. Concurrently, amyloid plaques in the hippocampus and levels of soluble Aβ are significantly reduced. Interfering with Nav1.6 reduces the transcription level of β‐site APP‐cleaving enzyme 1 (BACE1), which is Aβ‐dependent. In the presence of Aβ oligomers, knockdown of Nav1.6 reduces intracellular calcium overload by suppressing reverse sodium–calcium exchange channel, consequently increasing inactive NFAT1 (the nuclear factor of activated T cells) levels and thus reducing BACE1 transcription. This mechanism leads to a reduction in the levels of Aβ in APP/PS1 transgenic mice, alleviates synaptic loss, improves learning and memory disorders in APP/PS1 mice after downregulating Nav1.6 in the hippocampus. Our study offers a new potential therapeutic strategy to counteract hippocampal hyperexcitability and subsequently rescue cognitive deficits in AD by selective blockade of Nav1.6 overexpression and/or hyperactivity.

Structure and Function of Sodium Channel Nav1.3 in Neurological Disorders.

Nav1.3, encoded by the SCN3A gene, is a voltage-gated sodium channel on the cell membrane. It is expressed abundantly in the fetal brain but little in the normal adult brain. It is involved in the generation and conduction of action potentials in excitable cells. Nav1.3 plays an important role in many neurological diseases. The aim of this review is to summarize new findings about Nav1.3 in the field of neurology. Many mutations of SCN3A can lead to neuronal hyperexcitability and then cause epilepsy. The rapid recovery from inactivation and slow closed-state inactivation kinetics of Nav1.3 leads to a reduced activation threshold of the channel and a high frequency of firing of neurons. Hyperactivity of Nav1.3 also induces increased excitability of sensory neurons, a lower nociceptive threshold, and neuropathic pain. This review summarizes the structure and the function of Nav1.3 and focuses on its relationship with epilepsy and neuropathic pain.

Chemical and Biological Tools for the Study of Voltage-Gated Sodium Channels in Electrogenesis and Nociception.

The malfunction and misregulation of voltage-gated sodium channels (NaV s) underlie in large part the electrical hyperexcitability characteristic of chronic inflammatory and neuropathic pain. NaV s are responsible for the initiation and propagation of electrical impulses (action potentials) in cells. Tissue and nerve injury alter the expression and localization of multiple NaV isoforms, including NaV 1.1, 1.3, and 1.6-1.9, resulting in aberrant action potential firing patterns. To better understand the role of NaV regulation, localization, and trafficking in electrogenesis and pain pathogenesis, a number of chemical and biological reagents for interrogating NaV function have been advanced. The development and application of such tools for understanding NaV physiology are the focus of this review.

Activation of Voltage-Gated Na+ Current by GV-58, a Known Activator of CaV Channels.

GV-58 ((2R)-2-[(6-{[(5-methylthiophen-2-yl)methyl]amino}-9-propyl-9H-purin-2-yl)amino]butan-1-ol) is recognized to be an activator of N- and P/Q-type Ca2+ currents. However, its modulatory actions on other types of ionic currents in electrically excitable cells remain largely unanswered. This study was undertaken to explore the possible modifications caused by GV-58 in ionic currents (e.g., voltage-gated Na+ current [INa], A-type K+ current [IK(A)], and erg-mediated K+ current [IK(erg)]) identified from pituitary GH3 lactotrophs. GH3 cell exposure to GV-58 enhanced the transient and late components of INa with varying potencies; consequently, the EC50 values of GV-58 required for its differential increase in peak and late INa in GH3 cells were estimated to be 8.9 and 2.6 μM, respectively. The INa in response to brief depolarizing pulse was respectively stimulated or suppressed by GV-58 or tetrodotoxin, but it failed to be altered by ω-conotoxin MVIID. Cell exposure to this compound increased the recovery of INa inactivation evoked by two-pulse protocol based on a geometrics progression; however, in its presence, there was a slowing in the inactivation rate of current decay evoked by a train of depolarizing pulses. The existence of GV-58 also resulted in an increase in the amplitude of ramp-induced resurgent and window INa. The presence of this compound inhibited IK(A) magnitude, accompanied by a shortening in inactivation time course of the current; however, it mildly decreased IK(erg). Under current-clamp conditions, GV-58 increased the frequency of spontaneous action potentials in GH3 cells. Moreover, in NSC-34 motor neuron-like cells, the presence of GV-58 not only raised INa amplitude but also reduced current inactivation. Taken together, the overall work provides a noticeable yet unidentified finding which implies that, in addition to its agonistic effect on Ca2+ currents, GV-58 may concertedly modify the amplitude and gating kinetics of INa in electrically excitable cells, hence modifiying functional activities in these cells.

146 Human Interictal Epileptiform Discharges are Traveling Waves Reflecting Ictal Self-organization

INTRODUCTION: Interictal epileptiform discharges (IEDs), are large intermittent electrophysiological events observed between seizures in epileptic patients. While seizures are infrequent and unpredictable, IEDs are far more common, occurring several times per minute (0.67 IEDs per minute in this dataset). The relative frequency of IEDs makes them an alluring indicator of epileptogenic brain, especially when compared to more scarce and unpredictable seizures. Yet despite the relative abundance of IEDs, it remains unknown how they relate to seizures. METHODS: To better understand this relationship, we examined semi-chronic, multi-day recordings of 96-channel microelectrode arrays implanted in human epilepsy patients. These recordings—spanning single cell action potentials to local field potentials—allowed us to study the microscale spatiotemporal organization of over 45,000 IEDs across 10 patients. We used ordinary multivariate linear regression to estimiate IED speed and direction, and vonMises Mixture models to cluster bimodal IED directions in to sub-distributions. RESULTS: These recordings showed that the majority (66.4%) of IEDs propagate across neocortex as traveling waves with a dominant direction (Hermans-Rasson Tests, all p < 10-3). IED traveling wave direction distributions were bimodal in 8 of the 10 subjects, with a median difference between sub-distributions of 178 degrees. Firing rates and voltages of IEDs in each sub distribution were not significantly different, however, the proportion of IEDs in each sub-distrbution and their speeds and directions were different among sub-distributions, and mimicked sub-distributions of ictal discharges. CONCLUSION: These results show that IEDs propagate across the surface of the brain as traveling waves, and the that their directions mimic those of discharges occuring during seizures. Furthermore, these results provide a new understanding of epileptic brian tissue, and suggest that spatial features of IEDs may be used to help diagnose and treat medically refractory epilepsy.

Exploring Venom Toxins as Molecular Models for Chronic Pain Treatment


 
 
 The purpose of this project is to propose venomous animal toxins as molecular models for pain medication. Chronic pain is a prevalent health problem among the general population, and current pharmacological treatments are oftentimes ineffective or limited due to undesirable side-effects. This project explores the role of specific Voltage- Gated Sodium Channels, such as Sodium Channel 1.7 (NaV 1.7), in setting the stage for proposing analgesics with binding properties in peripheral pain-sensing neurons. Sodium Channels, notably NaV 1.7, play a major role in human pain signaling pathways that propagate action potentials in excitable cells. By inhibiting and blocking them, analgesic effects are known to be achievable. Through means of bioinformatic tools, we explore amino acid sequence alignments, Motif scans, tertiary structure modeling, and molecular docking of venomous animal toxins for in-silico new drug discovery research. Cysteine residues in toxins were reviewed as a possible link between acting upon the receptor and their analgesic effects. This led to the questioning of cysteine's role in the search for potential antagonists of NaV 1.7. Eventually, the attempt for a further investigation prompted the consideration for molecular docking between selected toxins and the receptor, aiming to seize chronic pain.
 
 

Spike-timing-dependent plasticity rewards synchrony rather than causality.

Spike-timing-dependent plasticity (STDP) is a candidate mechanism for information storage in the brain, but the whole-cell recordings required for the experimental induction of STDP are typically limited to 1 h. This mismatch of time scales is a long-standing weakness in synaptic theories of memory. Here we use spectrally separated optogenetic stimulation to fire precisely timed action potentials (spikes) in CA3 and CA1 pyramidal cells. Twenty minutes after optogenetic induction of STDP (oSTDP), we observed timing-dependent depression (tLTD) and timing-dependent potentiation (tLTP), depending on the sequence of spiking. As oSTDP does not require electrodes, we could also assess the strength of these paired connections three days later. At this late time point, late tLTP was observed for both causal (CA3 before CA1) and anticausal (CA1 before CA3) timing, but not for asynchronous activity patterns (Δt = 50 ms). Blocking activity after induction of oSTDP prevented stable potentiation. Our results confirm that neurons wire together if they fire together, but suggest that synaptic depression after anticausal activation (tLTD) is a transient phenomenon.

Fenestropathy of Voltage-Gated Sodium Channels.

Voltage-gated sodium channels (Nav) are responsible for the initiation and propagation of action potentials in excitable cells. From pain to heartbeat, these integral membrane proteins are the ignition stations for every sensation and action in human bodies. They are large (>200 kDa, 24 transmembrane helices) multi-domain proteins that couple changes in membrane voltage to the gating cycle of the sodium-selective pore. Nav mutations lead to a multitude of diseases - including chronic pain, cardiac arrhythmia, muscle illnesses, and seizure disorders - and a wide variety of currently used therapeutics block Nav. Despite this, the mechanisms of action of Nav blocking drugs are only modestly understood at this time and many questions remain to be answered regarding their state- and voltage-dependence, as well as the role of the hydrophobic membrane access pathways, or fenestrations, in drug ingress or egress. Nav fenestrations, which are pathways that connect the plasma membrane to the central cavity in the pore domain, were discovered through functional studies more than 40 years ago and once thought to be simple pathways. A variety of recent genetic, structural, and pharmacological data, however, shows that these fenestrations are actually key functional regions of Nav that modulate drug binding, lipid binding, and influence gating behaviors. We discovered that some of the disease mutations that cause arrhythmias alter amino acid residues that line the fenestrations of Nav1.5. This indicates that fenestrations may play a critical role in channel’s gating, and that individual genetic variation may also influence drug access through the fenestrations for resting/inactivated state block. In this review, we will discuss the channelopathies associated with these fenestrations, which we collectively name “Fenestropathy,” and how changes in the fenestrations associated with the opening of the intracellular gate could modulate the state-dependent ingress and egress of drugs binding in the central cavity of voltage gated sodium channels.

Naview: A d3.js Based JavaScript Library for Drawing and Annotating Voltage-Gated Sodium Channels Membrane Diagrams.

Voltage-gated sodium channels (Nav) are membrane proteins essential to initiating and propagating action potential in neurons and other excitable cells. For a given organism there are often multiple, specialized sodium channels found in different tissues, whose mutations can cause deleterious effects observed in numerous diseases. Consequently, there is high medical and pharmacological interest in these proteins. Scientific literature often uses membrane diagrams to depict important patterns in these channels including the six transmembrane segments (S1–S6) present in four different homologous domains (D1–D4), the S4 voltage sensors, the pore-lining residue segments and the ion selectivity filter residues, glycosylation and phosphorylation residues, toxin binding sites and the inactivation loop, among others. Most of these diagrams are illustrated either digitally or by hand and programs specifically dedicated to the interactive and data-friendly generation of such visualizations are scarce or non-existing. This paper describes Naview, an open-source javascript visualization compatible with modern web browsers for the dynamic drawing and annotation of voltage-gated sodium channels membrane diagrams based on the D3.js library. By using a graphical user interface and combining user-defined annotations with optional UniProt code as inputs, Naview allows the creation and customization of membrane diagrams. In this interface, a user can also map and display important sodium channel properties, residues, regions and their relationships through symbols, colors, and edge connections. Such features can facilitate data exploration and provide fast, high-quality publication-ready graphics for this highly active area of research.

The Purkinje-myocardial junction is the anatomic origin of ventricular arrhythmia in CPVT.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmia syndrome caused by gene mutations that render RYR2 Ca release channels hyperactive, provoking spontaneous Ca release and delayed afterdepolarizations (DADs). What remains unknown is the cellular source of ventricular arrhythmia triggered by DADs: Purkinje cells in the conduction system or ventricular cardiomyocytes in the working myocardium. To answer this question, we used a genetic approach in mice to knock out cardiac calsequestrin either in Purkinje cells or in ventricular cardiomyocytes. Total loss of calsequestrin in the heart causes a severe CPVT phenotype in mice and humans. We found that loss of calsequestrin only in ventricular myocytes produced a full-blown CPVT phenotype, whereas mice with loss of calsequestrin only in Purkinje cells were comparable to WT mice. Subendocardial chemical ablation or restoration of calsequestrin expression in subendocardial cardiomyocytes neighboring Purkinje cells was sufficient to protect against catecholamine-induced arrhythmias. In silico modeling demonstrated that DADs in ventricular myocardium can trigger full action potentials in the Purkinje fiber, but not vice versa. Hence, ectopic beats in CPVT are likely generated at the Purkinje–myocardial junction via a heretofore unrecognized tissue mechanism, whereby DADs in the ventricular myocardium trigger full action potentials in adjacent Purkinje cells.

Binding mechanisms of the tarantula venom ProTx-II to the human NaV1.7 channel

Voltage-gated sodium channels are responsible for initiating and propagating action potentials in excitable cells and have been associated with numerous cardiac and neurological disorders. However, there are many channel subtypes with different roles making it a challenging task to develop therapeutics specific to certain subtypes. NaV1.7 channels, present at the endings of pain-sensing nerves, have garnered a lot of scientific interest as potential targets for pain therapeutics by channel inhibition.

Ratiometry for improving the signal-to-noise with the red-shifted voltage dye di-4ANBDQBS

Voltage-sensitive fluorescent dyes allow non-invasive recording of action potentials in single cells. These experiments often have low signal-to-noise ratios (SNR). To improve SNR, ratiometric fluorescence methods have been utilized. In these studies, we used di-4-ANBDQBS (Cytovolt1), a far-red-shifted voltage-sensitive dye with a stable signal that works through the Stark effect. HEK cells were loaded with CytoVolt1 that was excited at 528 nm using our optical system. Fluorescence emission was measured at 720 nm with a long pass filter and 655 nm with a bandpass filter, corresponding to opposite slope regions of the emission spectrum.

Open-state structure and pore gating mechanism of the cardiac sodium channel

Voltage-gated sodium (Nav) channels open to initiate the action potential in excitable cells, then close within a few milliseconds via fast inactivation. Rapid opening and fast inactivation focus the depolarizing stimulus of the cardiac action potential and support the rapid and efficient conduction needed for a regular heartbeat. Fast inactivation is disrupted by mutations in the DIII/DIV linker of Nav channels, including mutations in the canonical IFM motif (isoleucine/phenylalanine/ methionine) of the “fast inactivation particle” .

Open state of bacterial sodium channel: insights from molecular dynamics simulations

Voltage gated sodium channels are membrane proteins that play an important role in propagating action potentials in excitable cells. In response to changes in the potential difference across the membrane, these channels cycle through three main functional states - resting, open and inactivated state. Thanks to advances in structural biology, the structures of the bacterial sodium channel from Magnetococcus marinus (NavMS) and Arcobacter butzleri (NavAB) have been captured in different functional states.

Tracing the Epileptic Consequences of Neurocysticercosis

Abstract: This Paper aims to provide a detailed overview of some basic and complex topics in the field of Neuroscience. Focusing on the overall occurrence of Action Potentials in Nerve cells, an insightful description of the processes which take place at the cellular and molecular levels is also provided. What follows is a discussion on how Neurocysticercosis, a parasitic infection which adversely affects the Central Nervous System, leads to Epileptic Consequences in the human body, as stated in the title. The explanation of these consequences contains vital information as to how such complex diseases and their consequences are merely errors in Action Potentials at the cellular level. With this, the paper also uses information from previously conducted studies, which found a causal linkage between Neurocysticercosis and Epilepsy. Keywords: Action Potentials; Neurocysticercosis; Epilepsy; Neuroscience; Central Nervous System; Pork Tapeworm; Taenia Solium

Parvalbumin expressing interneurons control spike-phase coupling of hippocampal cells to theta oscillations

Encoding of information by hippocampal neurons is defined by the number and the timing of action potentials generated relative to ongoing network oscillations in the theta (5–14 Hz), gamma (30–80 Hz) and ripple frequency range (150–200 Hz). The exact mechanisms underlying the temporal coupling of action potentials of hippocampal cells to the phase of rhythmic network activity are not fully understood. One critical determinant of action potential timing is synaptic inhibition provided by a complex network of Gamma-amino-hydroxy-butyric acid releasing (GABAergic) interneurons. Among the various GABAergic cell types, particularly Parvalbumin-expressing cells are powerful regulators of neuronal activity. Here we silenced Parvalbumin-expressing interneurons in hippocampal areas CA1 and the dentate gyrus in freely moving mice using the optogenetic silencing tool eNpHR to determine their influence on spike timing in principal cells. During optogenetic inhibition of Parvalbumin-expressing cells, local field potential recordings revealed no change in power or frequency of CA1 or dentate gyrus network oscillations. However, CA1 pyramidal neurons exhibited significantly reduced spike-phase coupling to CA1 theta, but not gamma or ripple oscillations. These data suggest that hippocampal Parvalbumin-expressing interneurons are particularly important for an intact theta-based temporal coding scheme of hippocampal principal cell populations.

The effective use of blebbistatin to study the action potential of cardiac pacemaker cells of zebrafish (Danio rerio) during incremental warming.

Blebbistatin potently inhibits actin-myosin interaction, preventing contractile activity of excitable cells including cardiac myocytes, despite electrical excitation of an action potential (AP). We collected intracellular microelectrode recordings of pacemaker cells located in the sinoatrial region (SAR) of the zebrafish heart at room temperature and during acute warming to investigate whether or not blebbistatin inhibition of contraction significantly alters pacemaker cell electrophysiology. Changes were evaluated based on 16 variables that characterized the AP waveform. None of these AP variables nor the spontaneous heart rate were significantly modified with the application of 10 μM blebbistatin when recordings were made at room temperature. Compared with the control group, the blebbistatin-treated group showed minor changes in the rate of spontaneous diastolic depolarization (P = 0.027) and the 50% and 80% repolarization (P = 0.008 and 0.010, respectively) in the 26°C–29°C temperature bin, but not at higher temperatures. These findings suggest that blebbistatin is an effective excitation-contraction uncoupler that does not appreciably affect APs generated in pacemaking cells of the SAR and can, therefore, be used in zebrafish cardiac studies.

QT interval prolongation, torsades de pointes and psychotropic medication

QT interval reflects the duration of action potential of myocardial cells. Its prolongation in electrocardiogram is related to risk of torsades de pointes (TdP), a form of polymorphous ventricular tachycardia, and sudden cardiac death, which is one of the main causes of early mortality in psychiatric patients. The fact that only a proportion of patients with drug-induced QT prolongation manifest TdP or die shows that QT prolongation alone is not the perfect biological predictive marker of arrhythmia manifestation. Yet, it continues to be useful but needs to be combined with other known risk factors. This paper is a narrative review based on search in reference books and in PubMed and ScienceDirect databases using the terms [Torsades de pointes OR QT prolongation] AND [Psychotropic drugs OR antipsychotics OR antidepressants]. Its aim is to present basic knowledge about the pathophysiology of TdP arrhythmia and QT prolongation and their relationship with psychotropic drugs. TdP is a relatively slow-rate tachycardia and, in some cases, may stop abruptly without manifestation of clinical symptoms. The diagnosis is based on electrocardiographic findings, the symptoms are similar to those of any tachyarrhythmia and they are related to heart rate and its effects on arterial pressure and cardiac work. QT interval prolongation and TdP result from structural and functional disorders of ion channels and related proteins which are implicated in the process of ventricular repolarization. Psychotropic drugs are commonly used not only in psychiatric patients but in many patients with somatic disease as well. They can affect transmission of electrical impulses from sinus node to ventricular myocardium in various ways and most of its classes prolong QT interval in therapeutic doses or in cases of intoxication. Thus, their careful administration and monitoring of patients, especially hospitalized ones, are of vital importance. More data is available concerning the effects of antipsychotic and antidepressant medication. Among the first group, first generation, classical antipsychotics, especially the lower potency drugs, are considered to carry greater risk. Caution should be exercised when administering thioridazine, haloperidol, pimozide, sertindole and ziprasidone. Among antidepressants, caution is warranted when administering tricyclic antidepressants, citalopram and escitalopram. Among the other psychotropics, much information is available about methadone.

Mechanisms and physiological implications of cooperative gating of clustered ion channels.

Ion channels play a central role in the regulation of nearly every cellular process. Dating back to the classic 1952 Hodgkin-Huxley model of the generation of the action potential, ion channels have always been thought of as independent agents. A myriad of recent experimental findings exploiting advances in electrophysiology, structural biology, and imaging techniques, however, have posed a serious challenge to this long-held axiom, as several classes of ion channels appear to open and close in a coordinated, cooperative manner. Ion channel cooperativity ranges from variable-sized oligomeric cooperative gating in voltage-gated, dihydropyridine-sensitive CaV1.2 and CaV1.3 channels to obligatory dimeric assembly and gating of voltage-gated NaV1.5 channels. Potassium channels, transient receptor potential channels, hyperpolarization cyclic nucleotide-activated channels, ryanodine receptors (RyRs), and inositol trisphosphate receptors (IP3Rs) have also been shown to gate cooperatively. The implications of cooperative gating of these ion channels range from fine-tuning excitation-contraction coupling in muscle cells to regulating cardiac function and vascular tone, to modulation of action potential and conduction velocity in neurons and cardiac cells, and to control of pacemaking activity in the heart. In this review, we discuss the mechanisms leading to cooperative gating of ion channels, their physiological consequences, and how alterations in cooperative gating of ion channels may induce a range of clinically significant pathologies.