Georgina B. Gurrola

Scorpion venom components that affect ion-channels function

The number and types of venom components that affect ion-channel function are reviewed. These are the most important venom components responsible for human intoxication, deserving medical attention, often requiring the use of specific anti-venoms. Special emphasis is given to peptides that recognize Na(+)-, K(+)- and Ca(++)-channels of excitable cells. Knowledge generated by direct isolation of peptides from venom and components deduced from cloned genes, whose amino acid sequences are deposited into databanks are nowadays in the order of 1.5 thousands, out of an estimate biodiversity closed to 300,000. Here the diversity of components is briefly reviewed with mention to specific references. Structural characteristic are discussed with examples taken from published work. The principal mechanisms of action of the three different types of peptides are also reviewed. Na(+)-channel specific venom components usually are modifier of the open and closing kinetic mechanisms of the ion-channels, whereas peptides affecting K(+)-channels are normally pore blocking agents. The Ryanodine Ca(++)-channel specific peptides are known for causing sub-conducting stages of the channels conductance and some were shown to be able to internalize penetrating inside the muscle cells.

A toxin to nervous, cardiac, and endocrine ERG K+ channels isolated from Centruroides noxius scorpion venom

Toxins isolated from a variety of venoms are tools for probing the physiological function and structure of ion channels. The ether‐a‐go‐go‐related genes (erg) codify for the K+ channels (ERG), which are crucial in neurons and are impaired in human long‐QT syndrome and Drosophila ‘seizure’ mutants. We have isolated a peptide from the scorpion Centruroides noxius Hoffmann that has no sequence homologies with other toxins, and demonstrate that it specifically inhibits (IC50 = 16±1 nM) only ERG channels of different species and distinct histogenesis. These results open up the possibility of investigating ERG channel structure‐function relationships and novel pharmacological tools with potential therapeutic efficacy.—Gurrola, G. B., Rosati, B., Rocchetti, M., Pimienta, G., Zaza, A., Arcangeli, A., Olivotto, M., Possani, L. D., Wanke, E. A toxin to nervous, cardiac, and endocrine ERG K+ channels isolated from Centruroides noxius scorpion venom. FASEB J. 13, 953–962 (1999)

Scorpion venom components as potential candidates for drug development

Abstract Scorpions are well known for their dangerous stings that can result in severe consequences for human beings, including death. Neurotoxins present in their venoms are responsible for their toxicity. Due to their medical relevance, toxins have been the driving force in the scorpion natural compounds research field. On the other hand, for thousands of years, scorpions and their venoms have been applied in traditional medicine, mainly in Asia and Africa. With the remarkable growth in the number of characterized scorpion venom components, several drug candidates have been found with the potential to tackle many of the emerging global medical threats. Scorpions have become a valuable source of biologically active molecules, from novel antibiotics to potential anticancer therapeutics. Other venom components have drawn attention as useful scaffolds for the development of drugs. This review summarizes the most promising candidates for drug development that have been isolated from scorpion venoms.

Rate dependency of delayed rectifier currents during the guinea‐pig ventricular action potential

1 The action potential clamp technique was exploited to evaluate the rate dependency of delayed rectifier currents (IKr and IKs) during physiological electrical activity. IKr and IKs were measured in guinea‐pig ventricular myocytes at pacing cycle lengths (CL) of 1000 and 250 ms. 2 A shorter CL, with the attendant changes in action potential shape, was associated with earlier activation and increased magnitude of both IKr and IKs. Nonetheless, the relative contributions of IKr and IKs to total transmembrane current were independent of CL. 3 Shortening of diastolic interval only (constant action potential shape) enhanced IKs, but not IKr. 4 I Kr was increased by a change in the action potential shape only (constant diastolic interval). 5 In ramp clamp experiments, IKr amplitude was directly proportional to repolarization rate at values within the low physiological range (< 1.0 V s−1); at higher repolarization rates proportionality became shallower and finally reversed. 6 When action potential duration (APD) was modulated by constant current injection (I‐clamp), repolarization rates > 1.0 V s−1 were associated with a reduced effect of IKr block on APD. The effect of changes in repolarization rate was independent of CL and occurred in the presence of IKs blockade. 7 In spite of its complexity, the behaviour of IKr was accurately predicted by a numerical model based entirely on known kinetic properties of the current. 8 Both IKr and IKs may be increased at fast heart rates, but this may occur through completely different mechanisms. The mechanisms identified are such as to contribute to abnormal rate dependency of repolarization in prolonged repolarization syndromes.

Activation of ryanodine receptors by imperatoxin A and a peptide segment of the II-III loop of the dihydropyridine receptor.

Excitation-contraction coupling in skeletal muscle is believed to be triggered by direct protein-protein interactions between the sarcolemmal dihydropyridine-sensitive Ca2+ channel and the Ca2+ release channel/ryanodine receptor (RyR) of sarcoplasmic reticulum. A 138-amino acid cytoplasmic loop between repeats II and III of the α1 subunit of the skeletal dihydropyridine receptor (the II-III loop) interacts with a region of the RyR to elicit Ca2+ release. In addition, small segments (10–20 amino acid residues) of the II-III loop retain the capacity to activate Ca2+ release. Imperatoxin A, a 33-amino acid peptide from the scorpion Pandinus imperator, binds directly to the RyR and displays structural and functional homology with an activating segment of the II-III loop (Glu666-Leu690). Mutations in a structural motif composed of a cluster of basic amino acids followed by Ser or Thr dramatically reduce or completely abolish the capacity of the peptides to activate RyRs. Thus, the Imperatoxin A-RyR interaction mimics critical molecular characteristics of the II-III loop-RyR interaction and may be a useful tool to elucidate the molecular mechanism that couples membrane depolarization to sarcoplasmic reticulum Ca2+ release in vivo.

Resurgent Current and Voltage Sensor Trapping Enhanced Activation by a β-Scorpion Toxin Solely in Nav1.6 Channel SIGNIFICANCE IN MICE PURKINJE NEURONS

Resurgent currents are functionally crucial in sustaining the high frequency firing of cerebellar Purkinje neurons expressing Nav1.6 channels. β-Scorpion toxins, such as CssIV, induce a left shift in the voltage-dependent activation of Nav1.2 channels by “trapping” the IIS4 voltage sensor segment. We found that the dangerous Cn2 β-scorpion peptide induces both the left shift voltage-dependent activation and a transient resurgent current only in human Nav1.6 channels (among 1.1-1.7), whereas CssIV did not induce the resurgent current. Cn2 also produced both actions in mouse Purkinje cells. These findings suggest that only distinct β-toxins produce resurgent currents. We suggest that the novel and unique selectivity of Cn2 could make it a model drug to replace deep brain stimulation of the subthalamic nucleus in patients with Parkinson disease.

Primary structure and synthesis of Imperatoxin A (IpTxa), a peptide activator of Ca2+ release channels/ryanodine receptors

We present the complete amino acid sequence of Imperatoxin A (IpTxa), a 33‐amino‐acid peptide from the venom of the scorpion P. imperator which activates Ca2+ release channels/ryanodine receptors (RyR) of sarcoplasmic reticulum (SR). The amino acid sequence of IpTxa shows no homology to any scorpion toxin so far described, but shares some homology to the amino acid sequence of Tx2‐9 and agelenin, two spider toxins that target neuronal P‐type Ca2+ channels. We also describe the total synthesis of IpTxa and demonstrate that it efficiently activates RyRs with potency and affinity identical to those of native IpTxa. The use of synthetic IpTxa should help in the identification of the structural motifs of RyR critical for channel gating.

Structure and function of scorpion toxins affecting K+-channels

This chapter reviews current literature dealing with peptides isolated from the venom of scorpions. Only peptides that recognize K+-channels are reported. They are called K+-channel-ligands or simply peptides, because the actual toxicity effects of all these peptides has not been demonstrated. The primary structures of 35 peptides are reviewed, and a general nomenclature has been proposed to define 9 distinct sub-families of related sequences. Partial sequences were not included in this classification. They are 29 to 39 amino acid residues long peptides with a common structural motif composed of an α-helix segment and three anti-parallel β-sheet strands stabilized by three or four disulfide bridges. Binding and/or electrophysiological experiments showed that the affinity of these peptides for the various sub-types of K+-channels varies from micromolar to picomolar concentrations. Some data on the three-dimensional structures and on specific functions of the various peptides on voltage-dependent K+-channels, high-conductance and small-conductance, Ca2+-dependent K+-channels are also briefly reviewed.

Isolation and physiological characterization of taicatoxin, a complex toxin with specific effects on calcium channels

Taicatoxin is a new complex oligomeric toxin that was isolated from the venom of the Australian taipan snake Oxyuranus scutellatus scutellatus. It is composed of three different molecular entities: an alpha-neurotoxin-like peptide of mol. wt 8000, a neurotoxic phospholipase of mol. wt of 16,000 and a serine protease inhibitor of mol. wt 7000, linked by non-covalent bonds, at an approximate stoichiometry of 1:1:4. The most active form of the complex was isolated by ion exchange chromatography through DE-Cellulose followed by two steps of CM-Cellulose chromatography at pH 4.7 and pH 6.0, respectively. At this stage the complex migrates as a single component in beta-alanine-acetate-urea gel electrophoresis and is very toxic to mice (1 or 2 micrograms of the complex protein kills a mouse of 20 g within 2 hr). It blocks the high threshold calcium channel current of excitable membranes in heart and does not affect the low threshold calcium channel current. The block occurs at a site that is accessible extracellularly but not intracellularly. The block is selective for calcium channels, reversible, does not affect single channel conductance but only changes channel gating, and is voltage dependent with higher affinity for inactivated channels. The phospholipase activity of the complex toxin can be separated by affinity-chromatography using a phospholipid analog (PC-Sepharose). The resulting complex contains only alpha-neurotoxin and protease inhibitor and is still capable of blocking calcium channels, although with less potency than the native oligomeric form. Sephadex G-50 gel filtration chromatography in the presence of high salt (1M NaCl) at alkaline pH (8.2), separates the alpha-neurotoxin-like peptide from the protease inhibitor, but at this stage the resulting peptides lose physiological activity towards the calcium channels. The amino acid sequence of the protease inhibitor was determined by automatic Edman degradation. The alpha-neurotoxin-like peptide and two isosubunits displaying phospholipase activity were sequenced at the N-terminal part of the molecule.

A large number of novel Ergtoxin-like genes and ERG K+-channels blocking peptides from scorpions of the genus Centruroides

Twenty‐three novel sequences similar to Ergtoxin (ErgTx) were obtained by direct sequencing of peptides or deduced from gene cloned using cDNAs of venomous glands of Centruroides (C.) elegans, C. exilicauda, C. gracilis, C. limpidus limpidus, C. noxius and C. sculpturatus. These peptides have from 42 to 47 amino acid residues cross‐linked by four disulfide bridges. They share sequence similarities (60–98% compared with ErgTx1) and were shown to block ERG K+‐channels of F‐11 clone (N18TG‐2×rat DRG) cultured cells. An unrooted phylogenetic tree analysis of these peptides showed that they conform at least five different subfamilies, of which three are novel subfamilies.