Title: Novel potassium channel toxins from scorpion venom: from elucidation to potential application
Other Titles: Nieuwe kalium kanaal toxines uit schorpioenvenoom: van opheldering tot potentiële toepassing
Authors: Abdel-Mottaleb, Yousra; M0319928
Issue Date: 6-Sep-2007
Abstract: Summary K+ channels are a ubiquitous family of membrane proteins that play a critical role in a wide variety of physiological processes, including the regulation of heart rate, muscle contraction, neurotransmitter release, neuronal excitability, insulin secretion, epithelial electrolyte transport, cell volume regulation and cell proliferation. Based on that critical role, K+ channels have been recognized as potential targets for therapeutic drugs for many years. Much of our knowledge on K+ channels was elucidated using specific peptide ligands isolated from a number of venomous organisms. Recently, this field received a strong support and increased interest due to the solution of the three-dimensional structure of a couple of K+ channels. At the same time, several new subfamilies of specific toxins for K+ channels were isolated from scorpion venoms, enhancingthe availability and diversity of such useful molecular tools. It opened new lines of research for the better understanding of K+ channel biophysics and pharmacology. In this study, we tackled 3 points:Our first objective was to search for novel, potent and selective K+ channel toxins from scarcely studied scorpion species such as the Argentinean scorpion Tityus trivittatus and the Iranian scorpion Odonthobuthus doriae. In chapter 1, we isolated and characterized the first example of a new sub-family of toxins (α-KTx20.1) from the scorpion Tityus trivittatus . Its effects were verified using 7 different cloned K+ channels (vertebrate Kv1.1-1.5, Shaker IR and hERG) expressed in Xenopus leavis oocytes. The toxin-induced effects show large differences among the different K+ channels and a preference towards Kv1.3 (IC50 = 7.9±1.4 nM). Interestingly, the toxin lacks a typical dyad that might be responsible for its relatively high potency on Kv1.3 channels.In chapter 2, the very first member of K+ channels toxins from the venom of the Iranian scorpion Odonthobuthus doriae (OdK1) was purified, sequenced and characterized physiologically. OdK1 was classified as α-KTX 8.5. The pharmacological effects of OdK1 were studied on six different cloned K+ channels (vertebrate Kv1.1-Kv1.5 and Shaker IR) expressed in Xenopus laevis oocytes. Interestingly, OdK1 selectively inhibited the currents through Kv1.2 channels with an IC50 value of 183 ±3 nM but did not affect any of the other channels. This unique pharmacologic profile offers a tool for the study of conditions such as multiple sclerosis and spinal chord injury where the specific block of Kv1.2 channels might prove beneficial.In chapter 3, from the same Iranian scorpion, Odonthobuthus doriae , we have been able to isolate a third toxin, this time selective to Kv1.3channels. The amino acid sequence of OdK2 bears a typical dyad but the potency of OdK2 is less than other members of the same subfamily α-KTx3. Nevertheless, OdK2 shows a unique selectivity towards Kv1.3 (5000 fold the IC50 value of Kv1.3 failed to inhibit seven other Kv channels: Kv1.1, Kv1.2, Kv1.4, Kv1.5, Kv1.6, Shaker IR or hERG channels). To determine the footprint of OdK2 on Kv1.3, we made use of an existing Kv1.1mutant which was made to match His399 of Kv1.3 channels. Qualitatively,we proved that the mutant is affected by OdK2 but quantitatively, the effect was not equal to Kv1.3. An explanation could be that the toxin interacts with a His399 causing the partial block and that alternative contact points are required to block the channel. The precise footprint of OdK2 on Kv1.3 channels remains to be elucidated, ideally via co-crystallization, knowing that the subtle difference in the amino acid sequence ofthe pore region causes dramatic effects on the selectivity of the toxinto the channels. Our second objective was to carry out structure-function studies which are important for understanding the underlying molecular determinants ofthe potency and selectivity of the toxins to K+ channels and ultimately, the design of tailor-made peptides targeting specific channels.In chapter 4, we have isolated a new 23-residue peptide from the venom of Heterometrus spinifer that shares ~60% identity with κ-hefutoxin 1 and based on sequence similarity, was identified as the third member of the κ-KTx subfamily and hence designated as κ-KTx1.3. However, despite the presence of the putative functional dyad (Y5 and K19)in identical positions in its sequence, κ-KTx1.3 failed to reproduce the blocking activity of κ-hefutoxin 1 on Kv1.2 and Kv1.3 channels. On analyzing the primary structure of κ-KTx1.3, it was found that the critical lysine (K19) was flanked by another lysine (K20) and it was postulated that this additional positive charge may hinder critical electrostatic interactions reported to occur between the dyad lysine extremity and the carbonyl oxygen atoms of conserved residues in the Kv1 channel selectivity filter. We have therefore, chemically synthesized mutants of κ-KTx1.3 substituting the flanking lysine with a neutral amino acid (K20A) or a negatively charged glutamic acid (K20E) as found in κ-hefutoxin 1. Interestingly, by these single-residue substitutions, we were able to assign K+ channel blocking activity to a scorpion venom-derived peptide that was otherwise inactive on Kv channels. Our third objective was to explore the scarcely-studied peptides from the venom of scorpions. Some of those peptides have antimicrobial effectscombined with K+ channels blockade.In chapter 5, we report the functional analyses of HgeScplp1, HgeβKTx and TstβKTx, including tests for K+ channel blockade, as well as for cytolytic and antimicrobial activities. More deep analyses of a naturally-occurring variant and a chemically-obtained fragment of HgeScplp1 indicate that the C-terminal part of β-KTxs and scorpine-like peptides are responsible for K+ channel blockade. The topology of the genesencoding for HgeScplp1 and TstβKTx, also are reported, supporting the monophyletic origin of β-KTxs and scorpine-like peptides. Finally, sequence analyses and robust phylogenetic reconstruction are used todiscuss the evolutive history of this kind of peptides and to highlightthe versatility of the CS-α/β as a permissive scaffold for K+channel recognition and blockade.
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Toxicology and Pharmacology

Files in This Item:

There are no files associated with this item.

Request a copy


All items in Lirias are protected by copyright, with all rights reserved.