Single Sodium Channels Research Articles

СОЕДИНЕНИЕ N-АЦЕТИЛ-6-АМИНОГЕКСАНОВОЙ КИСЛОТЫ ПОДАВЛЯЕТ НЕРВНУЮ ПРОВОДИМОСТЬ

Local anesthetic effect of N-acetyl-6-aminohexanoate salt (compound LHT-9-17) in the form of a substance with a purity of 99.8% was studied and also assessed the effect of the substance on the conductivity of single sodium channels and the conductivity of the nerve conductor at normal pH values and against the background of acidosis. Experimental ligature periodontitis was modeled in 20 laboratories male Wistar rats weighing 200-220 g. Local anesthetic effects of 2% LHT-9-17 and 2% lidocaine hydrochloride solution induced by infraorbital administration was assessed at day 12 of the pathology development. Conduction of single Na-channels was assessed by patch-clamp method on isolated giant neurons of Limnea stagnalis. Registration of the action potential of Rana radibunda isolated sciatic nerve was carried out in perfusion chamber contained 0.00001 to 0.01 μM LCT-9-17. Experiments included increasing the force of stimulating incentives until the maximum response. Compound LHT-9-17 has local anesthetic activity, including during the inflammatory process. The analgesic effect is due to the suppression of the conductivity of Na-channels of nerve conductors. The activity kept under inflammatory conditions may be due to the action of substances against the inflammatory state.

Single-compartment model of a pyramidal neuron, fitted to recordings with current and conductance injection.

For single neuron models, reproducing characteristics of neuronal activity such as the firing rate, amplitude of spikes, and threshold potentials as functions of both synaptic current and conductance is a challenging task. In the present work, we measure these characteristics of regular spiking cortical neurons using the dynamic patch-clamp technique, compare the data with predictions from the standard Hodgkin-Huxley and Izhikevich models, and propose a relatively simple five-dimensional dynamical system model, based on threshold criteria. The model contains a single sodium channel with slow inactivation, fast activation and moderate deactivation, as well as, two fast repolarizing and slow shunting potassium channels. The model quantitatively reproduces characteristics of steady-state activity that are typical for a cortical pyramidal neuron, namely firing rate not exceeding 30Hz; critical values of the stimulating current and conductance which induce the depolarization block not exceeding 80mV and 3, respectively (both values are scaled by the resting input conductance); extremum of hyperpolarization close to the midpoint between spikes. The analysis of the model reveals that the spiking regime appears through a saddle-node-on-invariant-circle bifurcation, and the depolarization block is reached through a saddle-node bifurcation of cycles. The model can be used for realistic network simulations, and it can also be implemented within the so-called mean-field, refractory density framework.

Sodium Channels Involved in the Initiation of Action Potentials in Invertebrate and Mammalian Neurons

Living organisms react to external stimuli to adapt their activity to the environment for survival. Acquired information is encoded by neurons by action potentials (APs) in a series of discrete electrical events. Rapid initiation of the AP is critical for fast reactions and strongly relies on voltage-activated Na+-selective channels (NaVs), which are widely expressed by both invertebrate and vertebrate neurons. Intuitively, NaVs of higher mammals should be activated faster than those of any other species. In addition to improved NaV channel structure, central mammalian neurons also demonstrate a patterned distribution of specific types of NaV1 channels at and near the site of AP initiation within the axonal initial segment (AIS). The AIS has different types of fast Nav1 channels and is thought to provide the biological basis for efficient frequency coding of information. In the present work, we review data related to the channels underlying fast initiation of action potentials in vertebrates and invertebrates, along with their evolution, distribution, and known specific roles. Current research has established that all mammalian NaV1 (1.1–1.9) channels share a similar structure, with 4 conservative transmembrane D-domains with a highly homologous sequence, but significant differences in the length of the functional cytoplasmic linkers. Similarly, the structure of NaV1 channels in invertebrates is generally similar to that of mammals, but it shows high variability across the evolutionary tree in the length of the linkers. AP initiation in mammalian cortical neurons is mediated by NaV1.2 and NaV1.6 channels, whereas interneurons mostly rely on NaV1.1 channels in their firing. Although invertebrate NaV1 channels normally display relatively slow kinetics, their activation is fast enough to produce APs, even in simple animals such as Placozoa. Remarkably, fast sodium-based excitability is not limited to animals. Recently, a photosynthetic prokaryote has been found to show rapidly activated sodium currents provided by their independently evolved single D-domain EuKatB sodium channels.

Numerical analysis of the Sodium–Water Reaction in a minichannel to evaluate the safety of a Printed Circuit Steam Generator

ABSTRACT A printed circuit heat exchanger is expected to improve the safety of a sodium-cooled Fast reactor when it is used as the steam generator. To use such a Printed Circuit Steam Generator (PCSG), the Sodium–Water Reaction (SWR) should be evaluated first. Because the diameter and length of a sodium channel in the PCSG are 4 mm and 2 m, respectively, a numerical approach is useful to understand the SWR characteristics in a minichannel. CFD modeling is conducted using commercial code and the SWR in a single sodium channel is analyzed under operating conditions. H2O at 16.8 MPa and 683 K is injected into the sodium channel through the cracks of different sizes (0.1–1.0 mm). Although the SWR is extreme in the early stage, most of the Na(l) is pushed towards the inlet and outlet of the channel within 15.3–35.3 ms as H2O inflowed. The peak temperature is around 1200–1600 K in the early stage of the SWR and the temperature reaches around 683 K. Moreover, the calculated depth of target wastage is around 0.62–3.34 μm. Therefore, the effect of the SWR is negligible in the PCSG core under operating conditions.

The conformational cycle of a prototypical voltage-gated sodium channel.

Electrical signaling was a dramatic development in evolution, allowing complex single-cell organisms like Paramecium to coordinate movement and early metazoans like worms and jellyfish to send regulatory signals rapidly over increasing distances. But how are electrical signals generated in biology? In fact, voltage-gated sodium channels conduct sodium currents that initiate electrical signals in all kingdoms of life, from bacteria to man. They are responsible for generating the action potential in vertebrate nerve and muscle, neuroendocrine cells, and other cell types1,2. Because of the high level of conservation of their core structure, it is likely that their fundamental mechanisms of action are conserved as well. Here we describe the complete cycle of conformational changes that a bacterial sodium channel undergoes as it transitions from resting to activated/open and inactivated/closed states, based on high-resolution structural studies of a single sodium channel. We further relate this conformational cycle of the ancestral sodium channel to the function of its vertebrate orthologs. The strong conservation of amino acid sequence and three-dimensional structure suggests that this model, at a fundamental level, is relevant for both prokaryotic and eukaryotic sodium channels, as well as voltage-gated calcium and potassium channels.

Resting-State Structure and Gating Mechanism of a Voltage-Gated Sodium Channel

Voltage-gated sodium (NaV) channels initiate action potentials in nerve, muscle, and other electrically excitable cells. The structural basis of voltage gating is uncertain because the resting state exists only at deeply negative membrane potentials. To stabilize the resting conformation, we inserted voltage-shifting mutations and introduced a disulfide crosslink in the VS of the ancestral bacterial sodium channel NaVAb. Here, we present a cryo-EM structure of the resting state and a complete voltage-dependent gating mechanism. The S4 segment of the VS is drawn intracellularly, with three gating charges passing through the transmembrane electric field. This movement forms an elbow connecting S4 to the S4-S5 linker, tightens the collar around the S6 activation gate, and prevents its opening. Our structure supports the classical "sliding helix" mechanism of voltage sensing and provides a complete gating mechanism for voltage sensor function, pore opening, and activation-gate closure based on high-resolution structures of a single sodium channel protein.

Structures of closed and open states of a voltage-gated sodium channel

Bacterial voltage-gated sodium channels (BacNavs) serve as models of their vertebrate counterparts. BacNavs contain conserved voltage-sensing and pore-forming domains, but they are homotetramers of four identical subunits, rather than pseudotetramers of four homologous domains. Here, we present structures of two NaVAb mutants that capture tightly closed and open states at a resolution of 2.8-3.2 Å. Introduction of two humanizing mutations in the S6 segment (NaVAb/FY: T206F and V213Y) generates a persistently closed form of the activation gate in which the intracellular ends of the four S6 segments are drawn tightly together to block ion permeation completely. This construct also revealed the complete structure of the four-helix bundle that forms the C-terminal domain. In contrast, truncation of the C-terminal 40 residues in NavAb/1-226 captures the activation gate in an open conformation, revealing the open state of a BacNav with intact voltage sensors. Comparing these structures illustrates the full range of motion of the activation gate, from closed with its orifice fully occluded to open with an orifice of ∼10 Å. Molecular dynamics and free-energy simulations confirm designation of NaVAb/1-226 as an open state that allows permeation of hydrated Na+, and these results also support a hydrophobic gating mechanism for control of ion permeation. These two structures allow completion of a closed-open-inactivated conformational cycle in a single voltage-gated sodium channel and give insight into the structural basis for state-dependent binding of sodium channel-blocking drugs.

Initial comparison of energy measures for neural stimulation in a single conductance channel.

This paper considers the utility of several alternative energy measures to reduce the energy required by a stimulation current source to charge a neuron membrane capacitance to a prescribed value in the case of a single sodium channel. For a simple case, minimizing the energy of the nonlinear channel conductance provides improved efficiency in terms of stimulator energy as compared to minimizing a squared-integral measure of the stimulation current. This work lays the foundation for expanding this investigation to a full conductance-based Hodgkin-Huxley model.

A 3 T sodium and proton composite array breast coil

The objective of this study was to determine whether a sodium phased array would improve sodium breast MRI at 3 T. The secondary objective was to create acceptable proton images with the sodium phased array in place. A novel composite array for combined proton/sodium 3 T breast MRI is compared with a coil with a single proton and sodium channel. The composite array consists of a 7-channel sodium receive array, a larger sodium transmit coil, and a 4-channel proton transceive array. The new composite array design utilizes smaller sodium receive loops than typically used in sodium imaging, uses novel decoupling methods between the receive loops and transmit loops, and uses a novel multichannel proton transceive coil. The proton transceive coil reduces coupling between proton and sodium elements by intersecting the constituent loops to reduce their mutual inductance. The coil used for comparison consists of a concentric sodium and proton loop with passive decoupling traps. The composite array coil demonstrates a 2-5× improvement in signal-to-noise ratio for sodium imaging and similar signal-to-noise ratio for proton imaging when compared with a simple single-loop dual resonant design. The improved signal-to-noise ratio of the composite array gives breast sodium images of unprecedented quality in reasonable scan times.

The response of NaV1.3 sodium channels to ramp stimuli: multiple components and mechanisms

Na(V)1.3 voltage-gated sodium channels have been shown to be expressed at increased levels within axotomized dorsal root ganglion neurons and within injured axons within neuromas and have been implicated in neuropathic pain. Like a number of other sodium channel isoforms, Na(V)1.3 channels produce a robust response to slow ramplike stimuli. Here we show that the response of Na(V)1.3 to ramp stimuli consists of two components: an early component, dependent upon ramp rate, that corresponds to a window current that is dependent upon closed-state inactivation; and a second component at more depolarized potentials that is correlated with persistent current which is detected for many tens of milliseconds after the start of a depolarizing pulse. We also assessed the K354Q Na(V)1.3 epilepsy-associated mutant channel, which is known to display an enhanced persistent current and demonstrate a strong correlation with the second component of the ramp response. Our results show that a single sodium channel isoform can produce a ramp response with multiple components, reflecting multiple mechanisms, and suggest that the upregulated expression of Na(V)1.3 in axotomized dorsal root ganglion neurons and enhanced ramp current in K354Q mutant channels can contribute in several ways to hyperexcitability and abnormal spontaneous firing that contribute to hyperexcitability disorders, such as epilepsy and neuropathic pain.

Visualizing Individual Sodium Channels on the Move

Visualization of voltage-gated sodium channels at work is an important requirement for the understanding of rapid electrical signaling in nerve cells. In this issue of Chemistry & Biology, Ondrus and colleagues have mastered this challenge by chemical synthesis of a fluorescent antagonist and by monitoring single sodium channels in living cells with unprecedented optical resolution.

Can Quantum Entanglement Between Ion Transition States Effect Action Potential Initiation?

The involvement of atomic determinants in molecular models underlying ion-conducting proteins suggests a revisitation of classical concepts that are based on rate-theory models (e.g. ‘gating’ particles) and bulk solvation concepts. Here, we investigate possible effects of a quantum correlation regime within ion-conducting molecules (voltage gated ion channels) on the onset dynamics of propagating voltage pulses (action potentials, APs). In particular, we focus on the initiation characteristics of action potentials, (API). We model the classical onset parameters of the sodium current in the Hodgkin–Huxley equation as three similar but independent probabilistic mechanisms that can become quantum correlated. The underlying physics is general and can involve entanglement between various degrees of freedom underlying ion transition states or ‘gating states’ during conduction, for example, Na+ ions in different channel locations, or different coordination states of ions with atoms lining sub-regions of the protein (‘filter-states’). We find that the resulting semi-classical version of the Hodgkin–Huxley equation, incorporating entangled sodium channel system states, can either enhance or slow down the rise in membrane potentials at the time of signal initiation. As in principle a single sodium channel can drive the membrane to an AP threshold, we suggest that the observed effects of a semi quantum-classical signal description point to a self-amplification of Na+ channels and may be due to quantum interferences within the atomic environment of channel atoms. If inserted into canonical generators of AP signals, the suggested quantum term can further enhance signal onset-rapidness, an aspect that has recently been observed in real cortical neurons and that seems to be inevitable for the encoding of high-frequency input.

Acetato de eslicarbazepina: una nueva alternativa en el tratamiento de las crisis focales

Epilepsy is one of the most common neurological diseases. In recent years an important number of drugs have been added to the therapeutic options we have available to us. With the aim of offering an optimal clinical effectiveness, the mechanisms of action or chemical structures of the antiepileptic drugs recently introduced onto the market have been modified with respect to the first, so-called classical or conventional, antiepileptics. Eslicarbazepine acetate belongs to this group of recently incorporated pharmaceuticals and is a novel single daily dose voltage-gated sodium channel blocker, which acts selectively in groups of rapid-activation neurons. It has been approved for indication in associated therapy in adults with partial onset seizures, with or without secondary generalisation. It is widely metabolised to eslicarbazepine and, to a lesser extent, to R-licarbazepine and oxcarbazepine. In 800 mg and 1200 mg doses it has been shown to bring about a significant reduction in a high percentage of patients with refractory epilepsy in simultaneous treatment with up to three antiepileptic drugs, and this effectiveness is maintained in open follow-up studies lasting up to a year. It is generally speaking well-tolerated; most of the adverse side-effects range in intensity from mild to moderate, and the percentage of patients who withdraw from treatment for this reason is low. Eslicarbazepine acetate is an alternative treatment in associated therapy in patients with partial epilepsy who do not respond adequately to treatment in monotherapy.

The Roles of Sodium Channels in Nociception: Implications for Mechanisms of Neuropathic Pain

Animal models have provided useful insights into the development and treatment of neuropathic pain. New genetic data from both human studies and transgenic mouse models suggest that specific voltage-gated sodium channel subtypes are associated with specific types of pain and, as such, may be useful analgesic drug targets for a variety of pain types including neuropathic pain. Global voltage-gated sodium channel blockers such as lidocaine have proven efficacy in treating pain but can be limited by adverse effects when administered systemically. Selective sodium channel blockers targeting channels at the periphery (Nav1.7, Nav1.8, and Nav1.9) could potentially reduce the side effect profile. Individual isoforms of voltage-gated sodium channels have been linked to particular types of pain. Nav1.7 is a useful target for ameliorating acute mechanical pain and inflammatory pain, and strong evidence also suggests that Nav1.9 could be targeted for treating inflammatory pain. Selective blockers of Nav1.8 could also have clinical benefit for visceral pain. Although there is no association between a single sodium channel isoform and neuropathic pain, combined blockade of peripherally expressed isoforms Nav1.7, Nav1.8, and Nav1.9 may prove useful.

A Single Channel Description of an Inactivation Deficient Sodium Channel Mutant

A characteristic of many voltage-gated ion channels including sodium and potassium channels is that macroscopic currents elicited with a voltage step subsequently decline, or inactivate. Because entry into inactivated states tends to limit the activity of single sodium channels to a small number of openings, identifying the statistical properties of other less stable gating processes can be difficult. Here, we examined the single channel characteristics of a mutant skeletal muscle sodium channel (rat Nav1.4 L435W/L437C/A438W) that exhibits very little macroscopic inactivation (Wang et al., 2003) in response to voltage steps to −60 and −40 mV. Although open dwell time distributions could be more or less described by a single exponential component, both deviations from the single exponential fit and time constants from bi-exponential fits were consistent across patches, suggesting the existence of two open states with time constants of approximately 0.5-1 ms and 2-4 ms. Bursts of openings separated by closures of less than 2 ms were observed frequently, with the number of bursts in a given sweep increasing at more depolarized voltages. The number of opening within each burst was also voltage dependent, increasing from about 3 to 5 openings at −60 and −40 mV, respectively. These data will be presented with a preliminary kinetic description of sodium channel activity in the absence of inactivation. This work was funded by NIH grant GM084140.

Differential Evolution of Voltage-Gated Sodium Channels in Tetrapods and Teleost Fishes

The voltage-gated sodium channel (SCN) alpha subunits are large proteins with central roles in the generation of action potentials. They consist of approximately 2,000 amino acids encoded by 24-27 exons. Previous evolutionary studies have been unable to reconcile the proposed gene duplication schemes with the species distribution and molecular phylogeny of the genes. We have carefully annotated the complete SCN gene sequences, correcting numerous database errors, for a broad range of vertebrate species and analyzed their phylogenetic relationships. We have also compared the chromosomal positions of the SCN genes relative to adjacent gene families. Our studies show that the ancestor of the vertebrates probably had a single sodium channel gene with two characteristic AT-AC introns, the second of which is unique to vertebrate SCN genes. This ancestral gene, located close to a HOX gene cluster, was quadrupled along with HOX in the two rounds of basal vertebrate tetraploidizations to generate the ancestors of the four channels SCN1A, SCN4A, SCN5A, and SCN8A. The third tetraploidization in the teleost fish ancestor doubled this set of genes and all eight are still present in at least three of four investigated teleost fish genomes. In tetrapods, the gene family expanded by local duplications before the radiation of amniotes, generating the cluster SCN5A, SCN10A, and SCN11A on one chromosome and the cluster SCN1A, SCN2A, SCN3A, and SCN9A on a different chromosome. In eutherian mammals, a tenth gene, SCN7A, arose in a local duplication in the SCN1A gene cluster. The SCN7A gene has undergone rapid evolution and has lost the ability to cause action potentials-instead, it functions as a sodium sensor. The three genes in the SCN5A cluster were translocated from the HOX-bearing chromosome in a mammalian ancestor along with several adjacent genes. This evolutionary scenario is supported by the adjacent TGF-β receptor superfamily (comprised of five distinct families) and the cysteine-serine-rich nuclear protein gene family as well as the HOX clusters. The independent expansions of the SCN repertoires in tetrapods and teleosts suggest that the functional diversification may differ between the two lineages.

The Tetrodotoxin Receptor of Voltage-Gated Sodium Channels—Perspectives from Interactions with μ-Conotoxins

Neurotoxin receptor site 1, in the outer vestibule of the conducting pore of voltage-gated sodium channels (VGSCs), was first functionally defined by its ability to bind the guanidinium-containing agents, tetrodotoxin (TTX) and saxitoxin (STX). Subsequent studies showed that peptide μ-conotoxins competed for binding at site 1. All of these natural inhibitors block single sodium channels in an all-or-none manner on binding. With the discovery of an increasing variety of μ-conotoxins, and the synthesis of numerous derivatives, observed interactions between the channel and these different ligands have become more complex. Certain μ-conotoxin derivatives block single-channel currents partially, rather than completely, thus enabling the demonstration of interactions between the bound toxin and the channel’s voltage sensor. Most recently, the relatively small μ-conotoxin KIIIA (16 amino acids) and its variants have been shown to bind simultaneously with TTX and exhibit both synergistic and antagonistic interactions with TTX. These interactions raise new pharmacological possibilities and place new constraints on the possible structures of the bound complexes of VGSCs with these toxins.

Molecular evolution of Na+ channels in teleost fishes

Voltage-dependent sodium channels are critical for electrical excitability. Invertebrates possess a single sodium channel gene; two rounds of genome duplication early in vertebrates increased the number to four. Since the teleost-tetrapod split, independent gene duplications in each lineage have further increased the number of sodium channel genes to 10 in tetrapods and 8 in teleosts. Here we review how the occurrence of multiple sodium channel paralogs has influenced the evolutionary history of three groups of fishes: pufferfish, gymnotiform and mormyriform electric fish. Pufferfish (tetraodontidae) produce a neurotoxin, tetrodotoxin, that binds to and blocks the pore of sodium channels. Pufferfish evolved resistance to their own toxins by amino acid substitutions in the pore of their sodium channels. These substitutions had to occur in parallel across multiple paralogs for organismal resistance to evolve. Gymnotiform and mormyriform fishes independently evolved electric organs to generate electricity for communication and object localization. Two sodium channel genes are expressed in muscle in most fishes. In both groups of weakly electric fishes, one gene lost its expression in muscle and became compartmentalized in the evolutionary novel electric organ, which is a muscle derivative. This gene then evolved at elevated rates, whereas the gene that is still expressed in muscle does not show elevated rates of evolution. In the electric organ-expressing gene, amino acid substitutions occur in parts of the channel involved in determining how long the channel will be open or closed. The enhanced rate of sequence evolution of this gene likely underlies the species-level variations in the electric signal.

Expression, Purification and Biophysical Characterization of a Superfamily of Prokaryotic Voltage-gated Sodium Channels

Eukaryotic voltage-gated sodium channels are monomeric membrane proteins comprised of four pseudo-repeats of domains containing six transmembrane segments, and have a molecular weight of >200 KDa. Their size and complexity makes them an arduous target for production in heterologous expression systems, a necessary step in acquiring the amounts of protein needed for biophysical and structural characterization. The simplified, single domain bacterial sodium channel containing six transmembrane segments isolated from Bacillus halodurans, NaChBac, can be expressed in E. coli in yields suitable for biophysical characterization and may enable successful crystallization and 3-D structure determination. Upon purification in 0.1% DDM, NaChBac is functional and associates to form a stable homotetramer (Nurani et al. (2008) Biochemistry 31:8114-8121). Seven different bacterial sodium channels with significant homology to NaChBac have been expressed in E. coli, purified in high-yield and characterized for secondary structure, thermal stability and drug binding. The experimental ease of obtaining a pure and homogeneous sample varies amongst the superfamily of sodium channels studied. The bacterial sodium channels are extremely thermal stable but individual members differ in their long-term stability when stored at room temperature, 4°C and −80°C, and their ability to bind the drug mibefradil. These channels also differ in their ability to form stable tetramers upon purification in different detergents. The differences and similarities found in this superfamily of sodium channels may prove valuable for determining general structural features important for specific voltage-gated sodium channel functions. The ability to express, purify and reconstitute multiple active bacterial sodium channels in membrane-mimic environments provides an arsenal of resources for elucidating structural features and identifying residues important for sustaining function in voltage-gated sodium channels. (Supported by a grants from the BBSRC to the MPSI consortium and BAW)

Alcohol-induced Ventricular Fibrillation in Brugada Syndrome

A 37‐year‐old man lost consciousness suddenly due to ventricular fibrillation (VF). After cardioversion, twelve‐lead ECG showed a pattern characteristic of type 1 Brugada. An implantable cardioverter defibrillator (ICD) was implanted for Brugada syndrome. In the following three years, VF occurred eight times after consumption of alcohol. Association between the Brugada syndrome and alcohol consumption has rarely been reported. Recently, it was reported that alcohol has inhibitory effect on single cardiac sodium channel gating and it may be that alcohol acted as a sodium channel blocker in this patient. Here we report a case of alcohol‐induced VF in Brugada syndrome.