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Abstract:

Voltage-gated sodium channels (VGSCs) and voltage-gated potassium channels (VGPCs) play a crucial role in many physiological processes in both excitable and non-excitable cells. Considered the importance of VGSCs and VGPCs it is not surprising that many poisonous and venomous organisms have developed potent neurotoxins targeting these transmembrane proteins. Designed by nature to kill, these toxins can be useful tools in the design of for instance new pesticides. Furthermore, with the high association of certain diseases with VGSCs and VGPCs, they may as well be applied as lead compounds in the development of new medicines. The research presented here focuses on the purification of novel potential ligands of VGSCs and VGPCs and the evaluation of their potency and selectivity through electrophysiology. Furthermore, by structure-function studies, the crucial points for toxin-channel interaction are highlighted. Finally, where applicable, an explanation for intoxication in humans is given based on the obtained results. The research has been concentrating on amphibians and scorpions since their poison/venom is highly diverse in neurotoxins.Certain amphibians have developed an efficient defense system by accumulating toxic compounds from prey into their skin. Examples of suchalkaloids are pumiliotoxins (PTXs). In general, PTXs are known as positive modulators of VGSCs. However, PTX 251D does not share these characteristics and mice and insect studies showed that PTX 251D is highly toxic. In the first chapter, we searched for the possible target of PTX 251D.The toxin was therefore made synthetically and tested on four VGSCs (mammalian Nav1.2/β1, Nav1.4/β1, Nav1.5/β1 and insect Para/tipE) and five VGPCs (mammalian Kv1.1-1.2, Kv1.3, Kv11.1 (hERG) and insect Shaker IR). PTX 251D not only inhibited the Na+ influx through the mammalian VGSCs but also affected the steady-state activation and inactivation. Interestingly, in the insect ortholog, the inactivation process wasdramatically affected. Additionally, PTX 251D inhibited the K+ efflux through all five tested VGPCs and slowed down the deactivation kinetics of the mammalian VGPCs. Kv1.3 was the most sensitive channel, with an IC50 value 10.8 ± 0.5 µM. To the best of our knowledge this is the first report of a PTX affecting VGPCs. The results of PTX 251D on VGPCs may, in analogy with the known convulsant 4-aminopyridine, explain the toxic effect in mice and insects. In chapter 2, the venom of the Iranian scorpion Odonthobuthus doriae (Buthidae) was studied and a VGPC toxin (OdK2) was purified, sequenced and characterized physiologically. Based on multiple sequence alignments, OdK2 was classified as α-KTx3.11. The pharmacological effects of OdK2 were studied on a panel of eight different cloned VGPCs (vertebrate Kv1.1-Kv1.6, hERG and insect Shaker IR). Interestingly, OdK2 selectively inhibits the currents through Kv1.3 channels with an IC50 value of 7.2 ± 2.7 nM.Scorpion β-toxins represent a particular pharmacological group of voltage-gated sodium channel(VGSC) neurotoxins. They typically shift the voltage dependence of activation to more hyperpolarizing potentials and reduce the peak current amplitude by binding to receptor-site 4. In chapter 3, the purification and functional characterization of the first voltage-gated sodium channel toxins, CeII8 and CeII9, isolated from the scorpion Centruroides elegans (Buthidae) are reported. Both toxins were electrophysiologically characterized on four mammalian VGSCs (Nav1.2/β1, Nav1.4/β1, Nav1.5/β1 and Nav1.7 /β1). Although CeII8 has the highest sequence similarity with scorpion α-toxins, inhibiting the inactivation of VGSCs, 300 nM toxin had a clear β-toxin effect and was selective towards Nav1.7/β1, involved in short-term and inflammatory pain. To the best of our knowledge, CeII8 is the first β-toxin active on Nav1.7/β1. CeII9, a typical anti-mammalian β-toxin, selectively modulated Nav1.4/β1 at a concentration of 700 nM. Through these effects, the high lethality to mice could be explained. Interestingly, both toxins, despite their differences in amino acid sequence, only altered the biophysical properties of a fraction of the expressed sodium channels. Since these effects have also been reported for the β-toxin CssIV, the bioactive surfaces of the toxins have been compared to each other.Given their medical importance, most attention has been paid towards the venom compositionof scorpions of the Buthidae family. Nevertheless, research has shown that the venom of scorpions of other families is as well a remarkable source of unique peptidyl toxins. In the last chapter, a peptide, HelaTx1, with unique primary sequence was isolated from the telson of the scorpion Heterometrus laoticus (Scorpionidae). Based on the amino acid sequence, the peptide could be cloned and the cDNA sequence revealed. HelaTx1 was chemically synthesized and functionally characterized on VGPCs of the Shaker-related, Shab-related, Shaw-related and Shal-related subfamilies.Furthermore, the toxin was also tested on small- and intermediate conductance Ca2+ activated K+ channels. From the channels studied, Kv1.1 and Kv1.6 were found to be the most sensitive (Kv1.1 EC50 = 9.9 ± 1.6 µM). The toxin did not alter the activation of the channels. Competition experiments with TEA showed that the toxin is an external pore blocker. Through mutational studies residues in the pore crucial for bindingof the toxin could be highlighted. Given the amino acid sequence, the predicted secondary structure and the biological activity on VGPCs, HelaTx1 should be included in the κ-KTx family. Based on a phylogenetic study, a revision of this VGPC blocker family was done. A rearrangement into five subfamilies is suggested, from which the κ-KTx5subfamily is formed by HelaTx1.