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Title: Purification, structural and functional characterization of novel voltage-gated ion channel ligands from amphibians and scorpions.
Other Titles: Opzuivering, structurele en functionele karakterisatie van nieuwe liganden voor spanningsafhankelijke ionenkanalen uit het gif van amfibieën en schorpioenen.
Authors: Vandendriessche, Thomas
Issue Date: 19-Dec-2011
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 such alkaloids 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/ß1, Nav1.4/ß1, Nav1.5/ß1 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 was dramatically 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 &plusmn; 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 &#945;-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 &plusmn; 2.7 nM.Scorpion ß-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/ß1, Nav1.4/ß1, Nav1.5/ß1 and Nav1.7 /ß1). Although CeII8 has the highest sequence similarity with scorpion &#945;-toxins, inhibiting the inactivation of VGSCs, 300 nM toxin had a clear ß-toxin effect and was selective towards Nav1.7/ß1, involved in short-term and inflammatory pain. To the best of our knowledge, CeII8 is the first ß-toxin active on Nav1.7/ß1. CeII9, a typical anti-mammalian ß-toxin, selectively modulated Nav1.4/ß1 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 ß-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 composition of 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 &plusmn; 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 binding of 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 &#954;-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 &#954;-KTx5 subfamily is formed by HelaTx1.<w:latentstyles deflockedstate="false" defunhidewhenused="true"  <w:lsdexception="" locked="false" priority="0" semihidden="false"  <object
Table of Contents: Table of Contents

INTRODUCTION - VOLTAGE-GATED ION CHANNELS, AMPHIBIANS AND SCORPIONS: WHAT IS THE LINK? 1
1.1. The electrochemical basis of action potentials 2
1.2. Voltage-gated sodium channels 4
1.2.1. General structure 4
1.2.2. Molecular basis of functioning 5
1.2.2.1. The selectivity filter 5
1.2.2.2. Voltage dependent activation 6
1.2.2.3. The inactivation process 7
1.2.3. Phylogeny and nomenclature 9
1.2.4. Channelopathies 11
1.3. Voltage-gated potassium channels 13
1.3.1. General structure 14
1.3.2. Molecular basis of functioning 15
1.3.2.1. The selectivity filter 15
1.3.2.2. Voltage dependent activation 18
1.3.2.3. The inactivation process 18
1.3.3. Phylogeny and nomenclature 19
1.3.4. Channelopathies 21
1.4. Amphibian toxins 23
1.4.1. Lipophilic alkaloids isolated from amphibian skin 25
1.5. Scorpion toxins 28
1.5.1. Scorpion toxins acting on voltage-gated sodium channels 30
1.5.2. Scorpion toxins acting on voltage-gated potassium channels 34
1.6. References 37

EXPERIMENTAL PROCEDURES 43
2.1. Rapid Amplification of cDNA Ends 44
2.2. Two-electrode voltage-clamp recordings in Xenopus laevis oocytes 45
2.3. Other techniques 47

RATIONALE AND OBJECTIVES 49
3.1. Rationale 50
3.2. Objectives 51

PART I: AMPHIBIANS: A REMARKABLE SOURCE OF BIOLOGICALLY ACTIVE ALKALOIDS 53
MODULATION OF VOLTAGE-GATED NA+ AND K+ CHANNELS BY PUMILIOTOXIN 251D: A “JOINT VENTURE” ALKALOID FROM ARTHROPODS AND AMPHIBIANS 55
4.1. Introduction 56
4.2. Materials and methods 58
4.2.1. Toxin 58
4.2.2. Heterologous expression of voltage-gated ion channels in Xenopus laevis oocytes 58
4.2.3. Electrophysiological studies on cloned voltage-gated ion channels 59
4.3. Results 62
4.3.1. Effects of PTX 251D on voltage-gated sodium channels 62
4.3.2. Effects of PTX 251D on voltage-gated potassium channels 64
4.4. Discussion 66
4.5. Acknowledgements 69
4.6. References 70

PART II: BUTHID SCORPIONS: BEYOND THE SECRETS OF SCORPIONS OF MEDICAL IMPORTANCE 73
ODK2, A KV1.3 CHANNEL-SELECTIVE TOXIN FROM THE VENOM OF THE IRANIAN SCORPION ODONTHOBUTHUS DORIAE 75
5.1. Introduction 76
5.2. Materials and methods 78
5.2.1. Purification and sequence determination of OdK2 78
5.2.2. Sequence comparison analysis 79
5.2.3. Expression in oocytes 80
5.2.4. Electrophysiological measurements 80
5.3. Results and discussion 80
5.3.1. Purification and primary sequence determination 80
5.3.2. Comparative sequence analysis 82
5.3.3. Electrophysiological studies 83
5.4. Acknowledgements 86
5.5. References 86

ISOLATION AND CHARACTERIZATION OF TWO NOVEL SCORPION TOXINS: THE α-TOXIN-LIKE CEII8, SPECIFIC FOR NAV1.7 AND THE CLASSICAL ANTI-MAMMALIAN CEII9, SPECIFIC FOR NAV1.4 CHANNELS 89
6.1. Introduction 90
6.2. Materials and methods 92
6.2.1. Source of venom 92
6.2.2. Purification protocols 93
6.2.3. Amino acid sequence determination and mass spectrometry analysis 93
6.2.4. Sequence comparison and molecular modeling 94
6.2.5. Lethality assay in mice 94
6.2.6. VGSC expression in Xenopus oocytes 95
6.2.7. Electrophysiological measurements 95
6.3. Results 96
6.3.1. Purification, amino acid sequence determination and mass spectrometry analysis 96
6.3.2. Sequence comparison and molecular modeling 99
6.3.3. Lethality assay in mice 102
6.3.4. Electrophysiological measurements 102
6.4. Discussion 105
6.5. Acknowledgements 110
6.6. References 110

PART III: UNRAVELING THE VENOM OF NON-BUTHID SCORPIONS: WHAT DO ‘HARMLESS’ SCORPIONS HAVE TO OFFER US? 115
PURIFICATION, MOLECULAR CLONING AND FUNCTIONAL CHARACTERIZATION OF HELATX1 (HETEROMETRUS LAOTICUS): THE FIRST MEMBER OF A NEW Κ-KTX SUBFAMILY 117
7.1. Introduction 118
7.2. Materials and methods 120
7.2.1. Venom source 120
7.2.2. Purification of HelaTx1 120
7.2.3. Mass spectrometry analysis and amino acid sequencing 120
7.2.4. Preparation of total RNA 121
7.2.5. 3’RACE 121
7.2.6. 5’RACE 122
7.2.7. Bioinformatics 122
7.2.8. Two-electrode voltage-clamp experiments on Kv1.3 in Xenopus laevis oocytes 123
7.2.9. Patch clamp experiments on Kv1.x channels expressed in mammalian cells 124
7.2.10. Patch clamp experiments on KCa3.1, KCa2.3, Kv2.1, Kv3.1 and Kv4.2 expressed in mammalian cells 124
7.2.11. Data analysis 125
7.3. Results 126
7.3.1. Purification, primary sequence determination and secondary structure prediction 126
7.3.2. cDNA sequence 128
7.3.3. Sequence comparison and phylogenetic analysis of the κ-KTx family 129
7.3.4. Electrophysiological characterization of HelaTx1 129
7.4. Discussion 134
7.5. Acknowledgements 143
7.6. References 143

CONCLUDING DISCUSSION 147
8.1. Exploration of unique pharmacological treasures in amphibians 148
8.2. Isolation and characterization of novel ligands affecting VGSCs and VGPCs from lethal and ‘harmless’ scorpions 150
8.3. Intoxication mechanism 154
8.4. Future perspectives 156
8.5. References 158

SUMMARY 161

SAMENVATTING 165
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Division of Mechatronics, Biostatistics and Sensors (MeBioS)
Toxicology and Pharmacology
Animal Physiology and Neurobiology Section - miscellaneous
Division of Crop Biotechnics

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