Analysis of the selectivity filter of the voltage-gated sodium channel NavRh

NaChBac is a bacterial voltage-gated sodium (Na_v) channel that shows sequence similarity to voltage-gated calcium channels. To understand the ion-permeation mechanism of Na_v channels, we combined molecular dynamics simulation, structural biology and electrophysiological approaches to investigate the recently determined structure of Na_vRh, a marine bacterial NaChBac ortholog. Two Na⁺ binding sites are identified in the selectivity filter (SF) in our simulations: The extracellular Na⁺ ion first approaches site 1 constituted by the side groups of Ser181 and Glu183, and then spontaneously arrives at the energetically more favorable site 2 formed by the carbonyl oxygens of Leu179 and Thr178. In contrast, Ca²⁺ ions are prone to being trapped by Glu183 at site 1, which then blocks the entrance of both Na⁺ and Ca²⁺ to the vestibule of the SF. In addition, Na⁺ permeates through the selective filter in an asymmetrical manner, a feature that resembles that of the mammalian Na_v orthologs. The study reported here provides insights into the mechanism of ion selectivity on Na⁺ over Ca²⁺ in mammalian Na_v channels.     

Thermal and chemical unfolding and refolding of a eukaryotic sodium channel

Voltage-gated sodium channels are dynamic membrane proteins essential for signaling in nervous and muscular systems. They undergo substantial conformational changes associated with the closed, open and inactivated states. However, little information is available regarding their conformational stability. In this study circular dichroism spectroscopy was used to investigate the changes in secondary structure accompanying chemical and thermal denaturation of detergent-solubilised sodium channels isolated from Electrophorus electricus electroplax. The proteins appear to be remarkably resistant to either type of treatment, with “denatured” channels, retaining significant helical secondary structure even at 77 °C or in 10% SDS. Further retention of helical secondary structure at high temperature was observed in the presence of the channel-blocking tetrodotoxin. It was possible to refold the thermally-denatured (but not chemically-denatured) channels in vitro. The correctly refolded channels were capable of undergoing the toxin-induced conformational change indicative of ligand binding. In addition, flux measurements in liposomes showed that the thermally-denatured (but not chemically-denatured) proteins were able to re-adopt native, active conformations. These studies suggest that whilst sodium channels must be sufficiently flexible to undergo major conformational changes during their functional cycle, the proteins are highly resistant to unfolding, a feature that is important for maintaining structural integrity during dynamic processes.     

The cation selectivity filter of the bacterial sodium channel,NaChBac

The Bacillus halodurans voltage-gated sodium-selective channel (NaChBac) (Ren, D., B. Navarro, H. Xu, L. Yue, Q. Shi, and D.E. Clapham. 2001b. SCIENCE: 294:2372-2375), is an ideal candidate for high resolution structural studies because it can be expressed in mammalian cells and its functional properties studied in detail. It has the added advantage of being a single six transmembrane (6TM) orthologue of a single repeat of mammalian voltage-gated Ca(2+) (Ca(V)) and Na(+) (Na(V)) channels. Here we report that six amino acids in the pore domain (LESWAS) participate in the selectivity filter. Replacing the amino acid residues adjacent to glutamatic acid (E) by a negatively charged aspartate (D; LEDWAS) converted the Na(+)-selective NaChBac to a Ca(2+)- and Na(+)-permeant channel. When additional aspartates were incorporated (LDDWAD), the mutant channel resulted in a highly expressing voltage-gated Ca(2+)-selective conductance.     

Cardiac sodium channel Na(v)1.5 interacts with and is regulated by the protein tyrosine phosphatase PTPH1

In order to identify proteins interacting with the cardiac voltage-gated sodium channel Na(v)1.5, we used the last 66 amino acids of the C-terminus of the channel as bait to screen a human cardiac cDNA library. We identified the protein tyrosine phosphatase PTPH1 as an interacting protein. Pull-down experiments confirmed the interaction, and indicated that it depends on the PDZ-domain binding motif of Na(v)1.5. Co-expression experiments in HEK293 cells showed that PTPH1 shifts the Na(v)1.5 availability relationship toward hyperpolarized potentials, whereas an inactive PTPH1 or the tyrosine kinase Fyn does the opposite. The results of this study suggest that tyrosine phosphorylation destabilizes the inactivated state of Na(v)1.5.     

电压门控钠通道NaV1.7与痛信号的产生

电压门控钠通道NaV1.7选择性高表达在伤害感受性脊髓背根神经节的感觉神经元上,在疼痛电信号的产生、传导和调控中具有重要的生理功能。伤害性感受器上的NaV1.7亦在慢性神经痛和炎症痛的病理生理过程中发挥关键作用。近年来的研究发现,人类遗传性疼痛症(如红斑性肢痛病)与NaV1.7钠离子通道基因SCN9A的某些功能增强型突变相关。最近Cox等首次报道了SCN9A突变将导致人先天痛觉完全丧失,而无痛症患者机体其它功能正常,提示NaV1.7将可能成为有效治疗疼痛而无副作用的一个新靶标。     

Subconductance states of single sodium channels modified by chloramine-T and sea anemone toxin in neuroblastoma cells

Single channel currents of chloramine-T (Chl-T) and sea anemone toxin (ATX-II) modified sodium channels were studied in neuroblastoma cells. With both substances similar subconductance states have been observed. The conductances of the sublevels were multiples of the unit step which was about onefourth of the most frequently occurring main conductance. Thus, the current levels observed were one fourth, half and five-fourths of the main current size. Both substances caused a slower decay of the averaged current compared to the current of the native channels. The main single-channel conductance was 15.2 pS (T=16°C) for the Chl-T and 10.8 pS (T=12°C) for the ATX-II modified channels. The channel open time was doubled by ATX-II, but was not increased significantly by Chl-T. The existence of the subconductance states suggests that the native channels may also have multiple open conformations.     

Protonation state of the selectivity filter of bacterial voltage-gated sodium channels is modulated by ions

The selectivity filter (SF) of bacterial voltage-gated sodium channels consists of four glutamate residues arranged in a C₄ symmetry. The protonation state population of this tetrad is unclear. To address this question, we simulate the pore domain of bacterial voltage-gated sodium channel of Magnetococcus sp. (Na_vMs) through constant pH methodology in explicit solvent and free energy perturbation calculations. We find that at physiological pH the fully deprotonated as well as singly and doubly protonated states of the SF appear feasible, and that the calculated pKa decreases with each additional bound ion, suggesting that a decrease in the number of ions in the pore can lead to protonation of the SF. Previous molecular dynamics simulations have suggested that protonation can lead to a decrease in the conductance, but no pKa calculations were performed. We confirm a decreased ionic population of the pore with protonation, and also observe structural symmetry breaking triggered by protonation; the SF of the deprotonated channel is closest to the C₄ symmetry observed in crystal structures of the open state, while the SF of protonated states display greater levels of asymmetry which could lead to transition to the inactivated state which possesses a C₂ symmetry in the crystal structure. We speculate that the decrease in the number of ions near the mouth of the channel, due to either random fluctuations or ion depletion due to conduction, could be a self-regulatory mechanism resulting in a nonconducting state that functionally resembles inactivated states.     

Purification, sequence, and pharmacological properties of sea anemone toxins from Radianthus paumotensis. A new class of sea anemone toxins acting on the sodium channel

Four new toxins have been isolated from the sea anemone Radianthus paumotensis: RpI, RpII, RpIII, and RpIV. They are polypeptides comprised of 48 or 49 amino acids; the sequence of RpII has been determined. Toxicities of these toxins in mice and crabs are similar to those of the other known sea anemone toxins, but they fall into a different immunochemically defined class. The sequence of RpII shows close similarities with the N-terminal end (up to residue 20) of the previously sequenced long sea anemone toxins, but most of the remaining part of the molecule is completely different. Like the other sea anemone toxins, Radianthus toxins are active on sodium channels; they slow down the inactivation process. Through their Na+ channel action, Radianthus toxins stimulate Na+ influx into tetrodotoxin-sensitive neuroblastoma cells and tetrodotoxin-resistant rat skeletal myoblasts. The efficiency of the toxins is similar in the two cellular systems. In that respect, Radianthus toxins behave much more like scorpion neurotoxins than sea anemone toxins from Anemonia sulcata or Anthopleura xanthogrammica. In binding experiments to synaptosomal Na+ channels, Radianthus toxins compete with toxin II from the scorpion Androctonus australis but not with toxins II and V from Anemonia sulcata.     

Reduced sodium channel Na(v)1.1 levels in BACE1-null mice

The Alzheimer BACE1 enzyme cleaves numerous substrates, with largely unknown physiological consequences. We have previously identified the contribution of elevated BACE1 activity to voltage-gated sodium channel Na(v)1.1 density and neuronal function. Here, we analyzed physiological changes in sodium channel metabolism in BACE1-null mice. Mechanistically, we first confirmed that endogenous BACE1 requires its substrate, the β-subunit Na(v)β(2), to regulate levels of the pore-forming α-subunit Na(v)1.1 in cultured primary neurons. Next, we analyzed sodium channel α-subunit levels in brains of BACE1-null mice at 1 and 3 months of age. At both ages, we found that Na(v)1.1 protein levels were significantly decreased in BACE1-null versus wild-type mouse brains, remaining unchanged in BACE1-heterozygous mouse brains. Interestingly, levels of Na(v)1.2 and Na(v)1.6 α-subunits also decreased in 1-month-old BACE1-null mice. In the hippocampus of BACE1-null mice, we found a robust 57% decrease of Na(v)1.1 levels. Next, we performed surface biotinylation studies in acutely dissociated hippocampal slices from BACE1-null mice. Hippocampal surface Na(v)1.1 levels were significantly decreased, but Na(v)1.2 surface levels were increased in BACE1-null mice perhaps as a compensatory mechanism for reduced surface Na(v)1.1. We also found that Na(v)β(2) processing and Na(v)1.1 mRNA levels were significantly decreased in brains of BACE1-null mice. This suggests a mechanism consistent with BACE1 activity regulating mRNA levels of the α-subunit Na(v)1.1 via cleavage of cell-surface Na(v)β(2). Together, our data show that endogenous BACE1 activity regulates total and surface levels of voltage-gated sodium channels in mouse brains. Both decreased Na(v)1.1 and elevated surface Na(v)1.2 may result in a seizure phenotype. Our data caution that therapeutic BACE1 activity inhibition in Alzheimer disease patients may affect Na(v)1 metabolism and alter neuronal membrane excitability in Alzheimer disease patients.     

Cumulative hydropathic topology of a voltage-gated sodium channel at atomic resolution

Voltage-gated sodium channels (NavChs) are biological pores that control the flow of sodium ions through the cell membrane. In humans, mutations in genes encoding NavChs can disrupt physiological cellular activity thus leading to a wide spectrum of diseases. Here, we present a topological connection between the functional architecture of a NavAb bacterial channel and accumulation of atomic hydropathicity around its pore. This connection is established via a scaling analysis methodology that elucidates how intrachannel hydropathic density variations translate into hydropathic dipole field configurations along the pore. Our findings suggest the existence of a nonrandom cumulative hydropathic topology that is organized parallel to the membrane surface so that pore's stability, as well as, gating behavior are guaranteed. Given the biophysical significance of the hydropathic effect, our study seeks to provide a computational framework for studying cumulative hydropathic topological properties of NavChs and pore-forming proteins in general.     

The voltage-dependent Na+ channel of insect nervous system identified by receptor sites for tetrodotoxin, and scorpion and sea anemone toxins

Receptor sites for some of the most important toxins known to be specific for voltage-sensitive Na+ channel in the mammalian nervous system have been identified in a purified membrane preparation of house fly brain. Very high affinities have been found for the association of tetrodotoxin or tetrodotoxin derivatives with the insect Na+ channel (Kd = 0.03 - 0.08 nM). The gamma toxin from the Brazilian scorpion Tityus serrulatus forms a complex with the Na+ channel having a Kd of 6.1 pM. The Kd value for toxin II from the sea anemone Anemonia sulcata is 0.12 microM. These results show a high degree of conservation of the pharmacological properties of the brain Na+ channels between insects and mammals.     

Role of auxiliary beta1-, beta2-, and beta3-subunits and their interaction with Na(v)1.8 voltage-gated sodium channel

The nociceptive C-fibers of the dorsal root ganglion express several sodium channel isoforms that associate with one or more regulatory beta-subunits (beta1-beta4). To determine the effects of individual and combinations of the beta-subunit isoforms, we co-expressed Nav1.8 in combination with these beta-subunits in Xenopus oocytes. Whole-cell inward sodium currents were recorded using the two-microelectrode voltage clamp method. Our studies revealed that the co-expression beta1 alone or in combination with other beta-subunits enhanced current amplitudes, accelerated current decay kinetics, and negatively shifted the steady-state curves. In contrast, beta2 alone and in combination with beta1 altered steady-state inactivation of Nav1.8 to more depolarized potentials. Co-expression of beta3 shifted steady-state inactivation to more depolarized potentials; however, combined beta1beta3 expression caused no shift in channel availability. The results in this study suggest that the functional behavior of Nav1.8 will vary depending on the type of beta-subunit that expressed under normal and disease states.     

Molecular cloning of an epidermal growth factor-like toxin and two sodium channel toxins from the sea anemone Stichodactyla gigantea

An epidermal growth factor (EGF)-like toxin (gigantoxin I) and two sodium channel toxins (gigantoxins II and III), previously isolated from the sea anemone Stichodactyla gigantea, were cloned for their cDNAs. The precursor protein of gigantoxin I is composed of a signal peptide, propart and mature peptide, similar to those of gigantoxins II and III, and is much simpler in structure than those of mammalian EGFs. In addition, gigantoxin I as well as gigantoxins II and III was demonstrated to be contained in nematocysts, suggesting that gigantoxin I functions as a toxin in S. gigantea.     

Mechanisms and models of cardiac sodium channel inactivation

Shortly after cardiac Na⁺ channels activate and initiate the action potential, inactivation ensues within milliseconds, attenuating the peak Na⁺ current, I_Na, and allowing the cell membrane to repolarize. A very limited number of Na⁺ channels that do not inactivate carry a persistent I_Na, or late I_Na. While late I_Na is only a small fraction of peak magnitude, it significantly prolongs ventricular action potential duration, which predisposes patients to arrhythmia. Here, we review our current understanding of inactivation mechanisms, their regulation, and how they have been modeled computationally. Based on this body of work, we conclude that inactivation and its connection to late I_Na would be best modeled with a “feet-on-the-door” approach where multiple channel components participate in determining inactivation and late I_Na. This model reflects experimental findings showing that perturbation of many channel locations can destabilize inactivation and cause pathological late I_Na.     

Phylogenetic characterization of the epithelial Na+ channel (ENaC) family

Summary

Twenty-one sequenced protein members of the epithelial Na⁺ channel (ENaC) family have been identified and characterized in terms of their sizes, hydropathy profiles, sequence similarities and phylogenies. These proteins derive from mammals, the frog Xenopus laevis and the worm Caenorhabditis elegans. The eleven sequenced vertebrate proteins fall into four subfamilies designated α, β, γ, and δ. The 10 C. elegans proteins do not cluster with the vertebrate proteins, and they all proved to be distantly related to each other. Nonetheless, the 21 ENaC proteins exhibit the same apparent topology, each with two transmembrane spanning segments separated by a large extracellular loop. All but two ENaC proteins possess highly conserved extracellular domains containing numerous conserved cysteine residues as well as adjacent C-terminal amphipathic transmembrane spanning segments, postulated to contribute to the formation of the hydrophilic pores of these oligomeric channel protein complexes. It is proposed that the well-conserved extracellular domains serve as receptors to control the activities of the channels. A topological model for the ENaC family proteins is presented.     

小剂量芬太尼联合咪达唑仑对大鼠皮质神经元钠离子通道电流的影响

目的：观察小剂量芬太尼联合咪达唑仑对大鼠大脑皮层神经元细胞膜电压门控性钠离子通道电流的影响。方法：用膜片钳全细胞记录方式观察小剂量芬太尼联合咪达唑仑对原代培养的新生SD大鼠大脑皮层神经元钠离子通道电流的影响。实验分为空白组,即未用药组;芬太尼5μg/L（F5）组和芬太尼5μg/L＋咪达唑仑200μg/L（F5＋M200）组。结果：F5＋M200组平均最大电流密度为（-213.98±91.68）pA/pF,明显低于空白组（-267±115.36）pA/pF（n=5,P〈0.05）和F5组（-231.90±97.16）pA/pF（n=5,P〈0.05）。结论：小剂量芬太尼联合咪达唑仑对皮质神经元钠离子通道电流的抑制作用较单一芬太尼组具有增强效应,这可能是临床两种药物合用后镇静镇痛作用增强原因之一。     

Activity-dependent modulation of neuronal KV channels by retinoic acid enhances CaV channel activity

The metabolite of vitamin A, retinoic acid (RA), is known to affect synaptic plasticity in the nervous system and to play an important role in learning and memory. A ubiquitous mechanism by which neuronal plasticity develops in the nervous system is through modulation of voltage-gated Ca²⁺ (Ca_V) and voltage-gated K⁺ channels. However, how retinoids might regulate the activity of these channels has not been determined. Here, we show that RA modulates neuronal firing by inducing spike broadening and complex spiking in a dose-dependent manner in peptidergic and dopaminergic cell types. Using patch-clamp electrophysiology, we show that RA-induced complex spiking is activity dependent and involves enhanced inactivation of delayed rectifier voltage-gated K⁺ channels. The prolonged depolarizations observed during RA-modulated spiking lead to an increase in Ca²⁺ influx through Ca_V channels, though we also show an opposing effect of RA on the same neurons to inhibit Ca²⁺ influx. At physiological levels of Ca²⁺, this inhibition is specific to Ca_V2 (not Ca_V1) channels. Examining the interaction between the spike-modulating effects of RA and its inhibition of Ca_V channels, we found that inhibition of Ca_V2 channels limits the Ca²⁺ influx resulting from spike modulation. Our data thus provide novel evidence to suggest that retinoid signaling affects both delayed rectifier K⁺ channels and Ca_V channels to fine-tune Ca²⁺ influx through Ca_V2 channels. As these channels play important roles in synaptic function, we propose that these modulatory effects of retinoids likely contribute to synaptic plasticity in the nervous system.     

Sensitivity of cloned muscle,heart and neuronal voltage-gated sodium channels to block by polyamines: A possible basis for modulation of excitability in vivo

Spermidine and spermine, are endogenous polyamines (PAs) that regulate cell growth and modulate the activity of numerous ion channel proteins. In particular, intracellular PAs are potent blockers of many different cation channels and are responsible for strong suppression of outward K⁺ current, a phenomenon known as inward rectification characteristic of a major class of K_IR K⁺ channels. We previously described block of heterologously expressed voltage-gated Na⁺ channels (Na_V) of rat muscle by intracellular PAs and PAs have recently been found to modulate excitability of brain neocortical neurons by blocking neuronal Na_V channels. In this study, we compared the sensitivity of four different cloned mammalian Na_V isoforms to PAs to investigate whether PA block is a common feature of Na_V channel pharmacology. We find that outward Na⁺ current of muscle (Na_V1.4), heart (Na_V1.5), and neuronal (Na_V1.2, Na_V1.7) Na_V isoforms is blocked by PAs, suggesting that PA metabolism may be linked to modulation of action potential firing in numerous excitable tissues. Interestingly, the cardiac Na_V1.5 channel is more sensitive to PA block than other isoforms. Our results also indicate that rapid binding of PAs to blocking sites in the Na_V1.4 channel is restricted to access from the cytoplasmic side of the channel, but plasma membrane transport pathways for PA uptake may contribute to long-term Na_V channel modulation. PAs may also play a role in drug interactions since spermine attenuates the use-dependent effect of the lidocaine, a typical local anesthetic and anti-arrhythmic drug.     

Design and synthesis of (5-amino-1, 2, 4-triazin-6-yl)(2-(benzo[d] isoxazol-3-yl) pyrrolidin-1-yl)methanone derivatives as sodium channel blocker and anticonvulsant agents

Abstract

A series of novel (5-amino-3-substituted-1, 2, 4-triazin-6-yl) (2-(6-halo-substituted benzo[d]isoxazol-3-yl) pyrrolidin-1-yl) methanone 5a–5r was synthesized. Their anticonvulsant activities were evaluated by the maximal electroshock (MES) test and neurotoxicity was evaluated by the rotorod test. The MES test showed that (5-amino-3-phenyl-1, 2, 4-triazin-6-yl)(2-(6-fluorobenzo[d]isoxazol-3-yl) pyrrolidin-1-yl) methanone 5c was found to be the most potent compound with ED₅₀ value of 6.20?mg/kg (oral/rat) and a protective index (PI?=?ED₅₀/TD₅₀) value of >48.38, which was much higher than the PI of the reference drug phenytoin. To explain the possible mechanism of action of selected derivatives 5b, 5c, 5i and 5o, their influence on sodium channel was evaluated in vitro.