Ion Channel Toxins Research Articles

Bioinformatic characterizations and prediction of K+ and Na+ ion channels effector toxins.

BackgroundK+ and Na+ channel toxins constitute a large set of polypeptides, which interact with their ion channel targets. These polypeptides are classified in two different structural groups. Recently a new structural group called birtoxin-like appeared to contain both types of toxins has been described. We hypothesized that peptides of this group may contain two conserved structural motifs in K+ and/or Na+ channels scorpion toxins, allowing these birtoxin-like peptides to be active on K+ and/or Na+ channels.ResultsFour multilevel motifs, overrepresented and specific to each group of K+ and/or Na+ ion channel toxins have been identified, using GIBBS and MEME and based on a training dataset of 79 sequences judged as representative of K+ and Na+ toxins.Unexpectedly birtoxin-like peptides appeared to present a new structural motif distinct from those present in K+ and Na+ channels Toxins. This result, supported by previous experimental data, suggests that birtoxin-like peptides may exert their activity on different sites than those targeted by classic K+ or Na+ toxins.Searching, the nr database with these newly identified motifs using MAST, retrieved several sequences (116 with e-value < 1) from various scorpion species (test dataset). The filtering process left 30 new and highly likely ion channel effectors.Phylogenetic analysis was used to classify the newly found sequences. Alternatively, classification tree analysis, using CART algorithm adjusted with the training dataset, using the motifs and their 2D structure as explanatory variables, provided a model for prediction of the activity of the new sequences.ConclusionThe phylogenetic results were in perfect agreement with those obtained by the CART algorithm.Our results may be used as criteria for a new classification of scorpion toxins based on functional motifs.

Phase Coupling of a Circadian Neuropeptide With Rest/Activity Rhythms Detected Using a Membrane-Tethered Spider Toxin

Drosophila clock neurons are self-sustaining cellular oscillators that rely on negative transcriptional feedback to keep circadian time. Proper regulation of organismal rhythms of physiology and behavior requires coordination of the oscillations of individual clock neurons within the circadian control network. Over the last decade, it has become clear that a key mechanism for intercellular communication in the circadian network is signaling between a subset of clock neurons that secrete the neuropeptide pigment dispersing factor (PDF) and clock neurons that possess its G protein-coupled receptor (PDFR). Furthermore, the specific hypothesis has been proposed that PDF-secreting clock neurons entrain the phase of organismal rhythms, and the cellular oscillations of other clock neurons, via the temporal patterning of secreted PDF signals. In order to test this hypothesis, we have devised a novel technique for altering the phase relationship between circadian transcriptional feedback oscillation and PDF secretion by using an ion channel–directed spider toxin to modify voltage-gated Na+ channel inactivation in vivo. This technique relies on the previously reported “tethered-toxin” technology for cell-autonomous modulation of ionic conductances via heterologous expression of subtype-specific peptide ion channel toxins as chimeric fusion proteins tethered to the plasma membrane with a glycosylphosphatidylinositol (GPI) anchor. We demonstrate for the first time, to our knowledge, the utility of the tethered-toxin technology in a transgenic animal, validating four different tethered spider toxin ion channel modifiers for use in Drosophila. Focusing on one of these toxins, we show that GPI-tethered Australian funnel-web spider toxin δ-ACTX-Hv1a inhibits Drosophila para voltage-gated Na+ channel inactivation when coexpressed in Xenopus oocytes. Transgenic expression of membrane-tethered δ-ACTX-Hv1a in vivo in the PDF-secreting subset of clock neurons induces rhythmic action potential bursts and depolarized plateau potentials. These in vitro and in vivo electrophysiological effects of membrane-tethered δ-ACTX-Hv1a are consistent with the effects of soluble δ-ACTX-Hv1a purified from venom on Na+ channel physiological and biophysical properties in cockroach neurons. Membrane-tethered δ-ACTX-Hv1a expression in the PDF-secreting subset of clock neurons induces an approximately 4-h phase advance of the rhythm of PDF accumulation in their terminals relative to both the phase of the day:night cycle and the phase of the circadian transcriptional feedback loops. As a consequence, the morning anticipatory peak of locomotor activity preceding dawn, which has been shown to be driven by the clocks of the PDF-secreting subset of clock neurons, phase advances coordinately with the phase of the PDF rhythm of the PDF-secreting clock neurons, rather than maintaining its phase relationship with the day:night cycle and circadian transcriptional feedback loops. These results (1) validate the tethered-toxin technology for cell-autonomous modulation of ion channel biophysical properties in vivo in transgenic Drosophila, (2) demonstrate that the kinetics of para Na+ channel inactivation is a key parameter for determining the phase relationship between circadian transcriptional feedback oscillation and PDF secretion, and (3) provide experimental support for the hypothesis that PDF-secreting clock neurons entrain the phase of organismal rhythms via the temporal patterning of secreted PDF signals.

The Cysteine-rich Secretory Protein Domain of Tpx-1 Is Related to Ion Channel Toxins and Regulates Ryanodine Receptor Ca2+ Signaling

The cysteine-rich secretory proteins (Crisp) are predominantly found in the mammalian male reproductive tract as well as in the venom of reptiles. Crisps are two domain proteins with a structurally similar yet evolutionary diverse N-terminal domain and a characteristic cysteine-rich C-terminal domain, which we refer to as the Crisp domain. We presented the NMR solution structure of the Crisp domain of mouse Tpx-1, and we showed that it contains two subdomains, one of which has a similar fold to the ion channel regulators BgK and ShK. Furthermore, we have demonstrated for the first time that the ion channel regulatory activity of Crisp proteins is attributed to the Crisp domain. Specifically, the Tpx-1 Crisp domain inhibited cardiac ryanodine receptor (RyR) 2 with an IC(50) between 0.5 and 1.0 microM and activated the skeletal RyR1 with an AC(50) between 1 and 10 microM when added to the cytoplasmic domain of the receptor. This activity was nonvoltage-dependent and weakly voltage-dependent, respectively. Furthermore, the Tpx-1 Crisp domain activated both RyR forms at negative bilayer potentials and showed no effect at positive bilayer potentials when added to the luminal domain of the receptor. These data show that the Tpx-1 Crisp domain on its own can regulate ion channel activity and provide compelling evidence for a role for Tpx-1 in the regulation of Ca(2+) fluxes observed during sperm capacitation.

Neurotoxic Peptides in the Multicomponent Venom of the Spider Cupiennius Salei Part I. Primary Structure of Neurotoxic Peptides in Relation to their Biological Function

Beside other components, spider venom contains neurotoxically acting peptides which predominantly target a variety of different ion channels within the spider's prey. From the multicomponent venom of Cupiennius salei 13 toxic peptides (CSTX-1 – CSTX-13) were purified by a combination of gel filtration, cation-exchange chromatography, and reverse-phase HPLC. The amino acid sequence of two important neurotoxic peptides, CSTX-1 (74 residues, 8352.6 Da) and CSTX-9 (68 residues, 7530.9 Da), as well as CSTX-2a (61 residues, 6865.7 Da), a truncated form of CSTX-1, were determined by Edman degradation. Sequence comparison of CSTX-1 with CSTX-9 revealed an identity of 53% as well as identical positions of all eight cysteine residues. CSTX-1 and CSTX-9 share the same disulphide bridge pattern linking Cys1–Cys4, Cys2–Cys5, Cys3–Cys8 and Cys6–Cys7. This disulphide bridge pattern is also found in other spider toxins such as agatoxins and curtatoxins. CSTX-1 and CSTX-9 belong to the family of the ion channel toxins containing the inhibitor cystine knot structural motif with the consensus sequence: CX3-7CX3-10CX0-7CX1-8CX4-20C. CSTX-2a and CSTX-9, lacking the lysine-rich C-terminal tail of CSTX-1, exhibit a much lower toxicity for Drosophila melanogaster than CSTX-1, providing good evidence for the direct involvement of this lysine-rich C-terminal tail in the toxicity of CSTX-1.

Ion channel toxins and therapeutics: from cone snail venoms to ciguatera.

Ion channels are intimately linked to all neurotransmission and neurotransmitter release processes, but in disease states often contribute adversely to disease pathology. The diversity and distribution of ion channel types and subtypes being uncovered through the use of molecular biology and toxin probes present an exciting opportunity for the discovery of new, more selective drugs. Among ion channels targeted by cone shell venom peptides (conotoxins) are the voltage-sensitive sodium, calcium, and potassium channels which open and then close (inactivate) in response to membrane depolarization, and thus regulate neurotransmission and the neurotransmitter release process. Conotoxins also target ligand-gated ion channels, including the NMDA-glutamate channel and the nicotinic acetylcholine receptor channel. The diversity of subtypes, especially those subtypes upregulated in disease states, makes ion channels a rapidly expanding therapeutic area. Conotoxins represent some of the most selective inhibitors of ion channel subtypes and have often been used as the defining ligand. In this overview, the structures and therapeutic potential of conotoxins active at ion channels are highlighted. The activity and structures are then contrasted with ciguatoxins, which are responsible for the food poisoning known as ciguatera. A universal liquid chromatography/mass spectrometry approach to the detection of these classes of toxins is briefly discussed.

Pyrethroid Insecticides: Poisoning Syndromes, Synergies, and Therapy

Pyrethroid insecticides are widely used, but there have been relatively few reports of systemic poisoning. These reports have, however, shown that pharmacotherapy is difficult and that the duration of poisoning can be unexpectedly long. Pyrethroids are ion channel toxins prolonging neuronal excitation, but are not directly cytotoxic. Two basic poisoning syndromes are seen. Type I pyrethroids produce reflex hyperexcitability and fine tremor. Type II pyrethroids produce salivation, hyperexcitability, choreoathetosis, and seizures. Both produce potent sympathetic activation. Local effects are also seen: skin contamination producing paresthesia and ingestion producing gastrointestinal irritation. The slow absorption of pyrethroids across the skin usually prevents systemic poisoning, although a significant reservoir of pyrethroid may remain bound to the epidermis. Carboxyesterase inhibitors can enhance pyrethroid toxicity in high-dose experimental studies. Hence, the unauthorized pyrethroid/organophosphate mixtures marketed in some developing countries may precipitate human poisoning. Pyrethroid paresthesia can be treated by decontamination of the skin, but systemic poisoning is difficult to control with anticonvulsants. Pentobarbitone, however, is surprisingly effective as therapy against systemic type II pyrethroid poisoning in rats, probably due to its dual action as a chloride channel agonist and a membrane stabilizer.

Spider toxins : A new group of potassium channel modulators

Spider toxins that target potassium channels constitute a new class of pharmacological tools that can be used to probe the structure and function of these channels at the molecular level. The limited studies performed to date indicate that these peptide toxins may facilitate the analysis of K+ channels that have proved insensitive to peptide inhibitors isolated from other animal sources. Thus far, two classes of K+ channel-selective spider toxins have been isolated, sequenced, and pharmacologically characterised – the hanatoxins (HaTx) from Grammastola spatulata and heteropodatoxins (HpTx) from Heteropoda venatoria. The hanatoxins block Kv2.1 and Kv4.2 voltage-gated K+ channels. In Kv2.1 K+ channels this occurs as a consequence of a depolarising shift in the voltage dependence of activation and not by occlusion of the channel pore. These toxins show minimal sequence homology with other peptide inhibitors of K+ channels, but they do share some homology with other ion channel toxins from spiders, particularly with regard to the spacing between cysteine residues. We have recently isolated three K+ channel antagonists from the venom of the Australian funnel-web spider Hadronyche versuta; at least two of these toxins are likely to constitute a new class of spider toxins active on K+ channels as they are approximately twice as large as HaTx and HpTx.

The cystine knot structure of ion channel toxins and related polypeptides

An increasing number of ion channel toxins and related polypeptides have been found to adopt a common structural motif designated the inhibitor cystine knot motif (Pallaghy P. K., Nielsen, K. J., Craik, D. J., Norton, R. S. (1994) A common structural motif incorporating a cystine knot and triple-stranded β-sheet in toxic and inhibitory polypeptides. Protein Science 3, 1833–1839). These globular, disulfide-stabilized molecules come from phylogenetically diverse sources, including spiders, cone shells, plants and fungi, and have various functions, although many target voltage-gated ion-channels. The common motif consists of a cystine knot and a triple-stranded, anti-parallel β-sheet. Examples of ion-channel toxins known to adopt this structure are the ω-conotoxins and ω-agatoxins, and, more recently, robustoxin, versutoxin and protein 5 from spiders, as well as κ-conotoxin PVIIA and conotoxin GS from cone shells. The variations on the motif structure exemplified by these structures are described here. We also consider the sequences of several polypeptides that might adopt this fold, including SNX-325 from a spider, δ-conotoxin PVIA and the μO-conotoxins from cone shells, and various plant and fungal polypeptides. The interesting case of the two- and three-disulfide bridged binding domains of the cellobiohydrolases from the fungus Trichoderma reesei is also discussed. The compact and robust nature of this motif makes it an excellent scaffold for the design and engineering of novel polypeptides with enhanced activity against existing targets, or with activity against novel targets.

Structure and function of ion channel toxins

Structure and function of ion channel toxins

The pharmacology of potassium ion channel toxins: A. L. Harvey (Department of Physiology and Pharmacology, Strathclyde Institute for Drug Research, Strathclyde University, Glasgow G1 1XW, U.K.)

The pharmacology of potassium ion channel toxins: A. L. Harvey (Department of Physiology and Pharmacology, Strathclyde Institute for Drug Research, Strathclyde University, Glasgow G1 1XW, U.K.)

Omega-Aga-I: a presynaptic calcium channel antagonist from venom of the funnel web spider, Agelenopsis aperta.

Spider venoms are proving to be important sources of specific ion channel toxins. Venom of Agelenopsis aperta, a funnel web spider, contains a class of polypeptide toxins which blocks neuromuscular synapses at nanomolar concentrations. Detailed physiological analyses of block caused by one of these toxins, omega-Aga-I, show that it suppresses transmitter release at insect and frog neuromuscular junctions and blocks calcium spikes in insect neuronal cell bodies. omega-Aga-I may define a binding site on neuronal calcium channels which is common to both vertebrates and invertebrates.

Enhancement of brain calcium antagonist binding by phencyclidine and related compounds.

The abilities of compounds structurally or pharmacologically related to phencyclidine to increase the apparent affinity of the [3H]dihydropyridine calcium channel antagonist [3H]nitrendipine were examined in lysed synaptosomal membrane preparations of rat brain. The p-bromo analog of phencyclidine (1-(1-(4-bromophenyl)cyclohexyl)piperidine) was the most efficacious compound tested in enhancing the apparent affinity of [3H]nitrendipine. The efficacy of this compound was approximately two-fold greater than PCP. The stereoisomers of PCMP (1-(1-phenylcyclohexyl-3-methylpiperidine) were also more efficacious than phencyclidine, although only a small degree of stereoselectivity was observed. Levoxadrol, dexoxadrol and the enantiomers of ketamine did not potentiate [3H]nitrendipine binding. The enantiomers of SKF 10047 (n-allylormetazocine), dextrorphan, levorphanol and the ion channel toxins histrionicotoxin and pumiliotoxin-B also increased the apparent affinity of [3H]nitrendipine, while several local anesthetics and mu-opiate receptor ligands were without effect. These studies suggest that the ability of phencyclidine and structurally related compounds to increase the apparent affinity of [3H]nitrendipine is not mediated through an interaction with phencyclidine receptors, but may represent a unique site for allosteric modulation of neuronal dihydropyridine calcium channel antagonist binding sites.

Effects of ion channel toxins and specific neurotoxins on the cyclic nucleotide content of cerebellar slices, primary brain cultures and neural cell lines

cAMP and cGMP were measured in mouse cerebellar slices, neural cell lines and primary nerve cell cultures from rats after treatment with different neurotoxins and high potassium.