Molecular mechanism of scorpion neurotoxins acting on sodium channels

Scorpion toxins that affect sodium channel gating traditionally are divided into alpha- and beta-classes. They show vast diversity in their selectivity for phyletic- or isoform-specific sodium channels. This article discusses the molecular mechanism of the selectivity. Moreover, a phylogenetic tree of scorpion toxins has been constructed, which, together with the worldwide distribution of toxins and the zoogeographic dispersion of the studied genera, offers an insight into the evolution of diverse scorpion toxins.     

海马脑片抗癫痫药物研究的离体模型

目的：建立离体海马脑片癫痫样放电模型并用于抗癫痫药物研究。方法：在豚鼠海马脑片上灌流青霉素建立颠阗痫样放电的离体模型。并用此模型对抗癫痫药物苯巴比妥钠和苯妥英钠两种药物在不同浓度下对癫痫样放电的对抗作用进行了定量分析,结果：在海马脑片上灌流致痫药物可建立一个较好的离体组织癫痫样放电模型,苯的对抗作用进行了定量分析,结果：在海马脑片上灌流致痫药物可建立一个较好的离体组织癫痫样放电模型。苯巴比妥和苯妥英钠在一定浓度下均有显著对抗癫痫样放电的作用,且与整体实验的结果相一致。结论：本实验建立有离体脑片模型具有实验手段简单,方法灵活,易于建立药物量效关系等优点,可用于抗癫痫药物筛选和研究。     

A model of sodium channels

We have explored the permeation and blockage of ions in sodium channels, relating the channel structure to function using electrostatic profiles and Brownian dynamics simulations. The model used resembles the KcsA potassium channel with an added external vestibule and a shorter selectivity filter. The electrostatic energy landscape seen by permeating ions is determined by solving Poisson's equation. The two charged amino acid rings of Glu-Glu-Asp-Asp (EEDD) and Asp-Glu-Lys-Ala (DEKA) around the selectivity filter region are seen to play a crucial role in making the channel sodium selective, and strongly binding calcium ions such that they block the channel. Our model closely reproduces a range of experimental data including the current-voltage curves, current-concentration curves and blockage of monovalent ions by divalent ions.     

A model of sodium channels

We have explored the permeation and blockage of ions in sodium channels, relating the channel structure to function using electrostatic profiles and Brownian dynamics simulations. The model used resembles the KcsA potassium channel with an added external vestibule and a shorter selectivity filter. The electrostatic energy landscape seen by permeating ions is determined by solving Poisson's equation. The two charged amino acid rings of Glu-Glu-Asp-Asp (EEDD) and Asp-Glu-Lys-Ala (DEKA) around the selectivity filter region are seen to play a crucial role in making the channel sodium selective, and strongly binding calcium ions such that they block the channel. Our model closely reproduces a range of experimental data including the current-voltage curves, current-concentration curves and blockage of monovalent ions by divalent ions.     

Solution structure of toxin 2 from centruroides noxius Hoffmann, a beta-scorpion neurotoxin acting on sodium channels

We have determined the solution structure of Cn2, a beta-toxin extracted from the venom of the New World scorpion Centruroides noxius Hoffmann. Cn2 belongs to the family of scorpion toxins that affect the sodium channel activity, and is very toxic to mammals (LD50=0.4 microg/20 g mouse mass). The three-dimensional structure was determined using 1H-1H two-dimensional NMR spectroscopy, torsion angle dynamics, and restrained energy minimization. The final set of 15 structures was calculated from 876 experimental distance constraints and 58 angle constraints. The structures have a global r. m.s.d. of 1.38 A for backbone atoms and 2.21 A for all heavy atoms. The overall fold is similar to that found in the other scorpion toxins acting on sodium channels. It is made of a triple-stranded antiparallel beta-sheet and an alpha-helix, and is stabilized by four disulfide bridges. A cis-proline residue at position 59 induces a kink of the polypeptide chain in the C-terminal region. The hydrophobic core of the protein is made up of residues L5, V6, L51, A55, and by the eight cysteine residues. A hydrophobic patch is defined by the aromatic residues Y4, Y40, Y42, W47 and by V57 on the side of the beta-sheet facing the solvent. A positively charged patch is formed by K8 and K63 on one edge of the molecule in the C-terminal region. Another positively charged spot is represented by the highly exposed K35. The structure of Cn2 is compared with those of other scorpion toxins acting on sodium channels, in particular Aah II and CsE-v3. This is the first structural report of an anti-mammal beta-scorpion toxin and it provides the necessary information for the design of recombinant mutants that can be used to probe structure-function relationships in scorpion toxins affecting sodium channel activity.     

CYP450 biosensors based on gold chips for antiepileptic drugs determination

Three-electrode configuration chips containing a Pt, Au and a screen-printed Ag/AgCl as counter, working and reference electrode, respectively, have been developed. Selective determination of Phenobarbital (PB) has been carried out by Cytochrome P450 2B4 (CYP450) immobilization into a polypyrrole matrix onto the gold working electrode. Chronoamperometric experiments show a PB diffusion coefficient of 2.42 × 10⁻⁶ cm² s⁻¹, a reproducibility and repeatability in terms of residual standard deviation (RSD) of 13% and 5.51%, respectively, and a limit of detection (LOD) of 0.289 μmol dm⁻³ ( = β = 0.05) for the developed CYP450-biosensor chip. Its performance has been showed by the determination of PB in pharmaceutical drugs. HPLC has been used as reference technique.     

A Drosophila systems model of pentylenetetrazole induced locomotor plasticity responsive to antiepileptic drugs

Background  

Rodent kindling induced by PTZ is a widely used model of epileptogenesis and AED testing. Overlapping pathophysiological mechanisms may underlie epileptogenesis and other neuropsychiatric conditions. Besides epilepsy, AEDs are widely used in treating various neuropsychiatric disorders. Mechanisms of AEDs' long term action in these disorders are poorly understood. We describe here a Drosophila systems model of PTZ induced locomotor plasticity that is responsive to AEDs.     

Differential effects of antiepileptic drugs on human bone cells

Antiepileptic drugs (AED) have been associated to in vivo deleterious consequences in bone tissue. The present work aimed to characterize the cellular and molecular effects of five different AED on human osteoclastogenesis and osteblastogenesis. It was observed that the different drugs had the ability to differentially modulate both processes, in a way dependent on the identity and dose of the AED. Shortly, valproic acid stimulated either osteoclastogenesis and osteoblastogenesis, whereas carbamazepine, gabapentin, and lamotrigine revealed an opposite behavior; topiramate elicited a decrease of osteoclast development and an increase in osteoblast differentiation. This is the first report describing the direct effects of different AED on human primary bone cells, which is a very important issue, because these drugs are usually consumed in long-term therapeutics, with acknowledged in vivo effects in bone tissue.     

An asymmetrical kinetic model for veratridine interactions with sodium channels in molluscan neurons

Veratridine alkaloid induces bi-stability or saw-tooth-shaped long potential waves in molluscan neurons. Voltage clamp experiments reveal the production of a slow sodium current whose changes are described by an asymmetric kinetic diagram relating the states of the sodium channels. Methods of the qualitative theory of differential equations were used to determine the condition necessary for such a model to have either an oscillatory solution or a bi-stable behavior. The kinetic diagram was modified to account for the frequency dependence of the slow sodium current production upon repeated short depolarizations. The modified kinetic diagram suggests that open and inactivated sodium channels are turned into channels with slow kinetic parameters; the transition from open channels would be fast, irreversible and restricted to part of the open channels, whereas that from inactivated channels would be slow and fully reversible upon repolarization.     

Voltage-gated sodium channels

Epilepsy is a brain disorder characterized by seizures and convulsions. The basis of epilepsy is an increase in neuronal excitability that, in some cases, may be caused by functional defects in neuronal voltage gated sodium channels, Nav1.1 and Nav1.2. The effects of antiepileptic drugs (AEDs) as effective therapies for epilepsy have been characterized by extensive research. Most of the classic AEDs targeting Nav share a common mechanism of action by stabilizing the channel’s fast-inactivated state. In contrast, novel AEDs, such as lacosamide, stabilize the slow-inactivated state in neuronal Nav1.1 and Nav1.7 isoforms. This paper reviews the different mechanisms by which this stabilization occurs to determine new methods for treatment.     

Tetrodotoxin-resistant sodium channels

Summary 1. Tetrodotoxin (TTX) has been widely used as a chemical tool for blocking Na⁺ channels. However, reports are accumulating that some Na⁺ channels are resistant to TTX in various tissues and in different animal species. Studying the sensitivity of Na⁺ channels to TTX may provide us with an insight into the evolution of Na⁺ channels.2. Na⁺ channels present in TTX-carrying animals such as pufferfish and some types of shellfish, frogs, salamanders, octopuses, etc., are resistant to TTX.3. Denervation converts TTX-sensitive Na⁺ channels to TTX-resistant ones in skeletal muscle cells, i.e., reverting-back phenomenon. Also, undifferentiated skeletal muscle cells contain TTX-resistant Na⁺ channels. Cardiac muscle cells and some types of smooth muscle cells are considerably insensitive to TTX.4. TTX-resistant Na⁺ channels have been found in cell bodies of many peripheral nervous system (PNS) neurons in both immature and mature animals. However, TTX-resistant Na⁺ channels have been reported in only a few types of central nervous system (CNS). Axons of PNS and CNS neurons are sensitive to TTX. However, some glial cells have TTX-resistant Na⁺ channels.5. Properties of TTX-sensitive and TTX-resistant Na⁺ channels are different. Like Ca²⁺ channels, TTX-resistant Na⁺ channels can be blocked by inorganic (Co²⁺, Mn²⁺, Ni²⁺, Cd²⁺, Zn²⁺, La³⁺) and organic (D-600) Ca²⁺ channel blockers. Usually, TTX-resistant Na⁺ channels show smaller single-channel conductance, slower kinetics, and a more positive current-voltage relation than TTX-sensitive ones.6. Molecular aspects of the TTX-resistant Na⁺ channel have been described. The structure of the channel has been revealed, and changing its amino acid(s) alters the sensitivity of the Na⁺ channel to TTX.7. TTX-sensitive Na⁺ channels seem to be used preferentially in differentiated cells and in higher animals instead of TTX-resistant Na⁺ channels for rapid and effective processing of information.8. Possible evolution courses for Na⁺ and Ca²⁺ channels are discussed with regard to ontogenesis and phylogenesis.     

Determination of antiepileptic drugs in biological material

Current analytical methodologies applied to the determination of antiepileptic drugs in biological material are reviewed. The role of chromatographic techniques is emphasized. Special attention is focused on new chemical entities as well as current trends such as high-speed liquid chromatographic techniques, hyphenated techniques and electrochromatography techniques. A review with 542 references.     

Inhibition of aquaporin 4 by antiepileptic drugs

The potential of antiepileptic drugs (AEDs) to inhibit the water transport properties of aquaporin 4 (AQP4) was investigated using a combination of in silico and in vitro screening methods. Virtual docking studies on 14 AEDs indicated a range of docking energies that spanned approximately 40 kcal/mol, where the most stabilized energies were consistent with that of the previously identified AQP4 inhibitor acetazolamide. Nine AEDs and one bio-active metabolite were further investigated in a functional assay using AQP4 expressing Xenopus oocytes. Seven of the assayed compounds were found to inhibit AQP4 function, while three did not. A linear correlation was indicated between the in silico docking energies and the in vitro AQP4 inhibitory activity at 20 microM.     

Excitability constraints on voltage-gated sodium channels

We study how functional constraints bound and shape evolution through an analysis of mammalian voltage-gated sodium channels. The primary function of sodium channels is to allow the propagation of action potentials. Since Hodgkin and Huxley, mathematical models have suggested that sodium channel properties need to be tightly constrained for an action potential to propagate. There are nine mammalian genes encoding voltage-gated sodium channels, many of which are more than approximately 90% identical by sequence. This sequence similarity presumably corresponds to similarity of function, consistent with the idea that these properties must be tightly constrained. However, the multiplicity of genes encoding sodium channels raises the question: why are there so many? We demonstrate that the simplest theoretical constraints bounding sodium channel diversity--the requirements of membrane excitability and the uniqueness of the resting potential--act directly on constraining sodium channel properties. We compare the predicted constraints with functional data on mammalian sodium channel properties collected from the literature, including 172 different sets of measurements from 40 publications, wild-type and mutant, under a variety of conditions. The data from all channel types, including mutants, obeys the excitability constraint; on the other hand, channels expressed in muscle tend to obey the constraint of a unique resting potential, while channels expressed in neuronal tissue do not. The excitability properties alone distinguish the nine sodium channels into four different groups that are consistent with phylogenetic analysis. Our calculations suggest interpretations for the functional differences between these groups.