Research profile

Conotoxins as tools for the understanding of the electrical excitability of cells

Our studies are directed towards the identification and characterization of pharmacologically active substances which interact with ion channels. To this end we investigate the pharmacological profile of such substances by electrophysiological measurements in expression systems. The questions we ask are: „What is structurally needed to block a voltage gated ion channel ?“ and „What is crucial for the specificity of a substance for a given ion channel subtype ?“ One focus of our research is the analysis of the interaction of different cone snail toxins with particular ion channels. These studies not only imply the biophysical characterization of the interaction of a conotoxin with its target molecule but also the investigation of the physiological implications of this interaction. The purpose of our studies is to create a basis for a potential pharmacological or even clinical use of these substances. Due to the key role of ion channels in many physiological processes, substances interacting with these proteins may have a great variety of possible clinical implications.
Fig 1: Fish hunting cone snails Middle: Conus geographus, the cone snail most dangerous for humans
Certain conotoxins already undergo clinical trials. What are ion channels? These are proteins which are embedded in the outer membrane of nearly all cells of an organism. They mediate the fast, selective transport of ions through the membrane and they are important for many diverse functions in a cell. Voltage-gated ion channels are key molecules for the generation of electrical signals in muscle cells and neurones. Since the first successful cloning of a voltage activated Na+ channel in 1984 a variety of different genes encoding Na+ K+ or Ca+ channels have been identified. A molecular picture of the structural elements important for the activity of voltage gated ion channels has emerged from intensive studies combining molecular biological and electrophysiological techniques. Thus the pore-forming a-subunit of K+ channels consists of six transmembrane segments and four a-subunits form the functional channel. The a-subunit of Na+- and Ca+ channels consists of four homologous domains with six transmembrane segments each. The transmembrane segment S4 is an important part of the voltage sensor and the linker between S5 and S6 is important for the lining of the ion permeation pathway and is called the “P-loop”. The physiologically active ion channel complex is composed of the a-subunit and auxiliary protein subunits. Due to the molecular identification of the different channel proteins the physiological importance of these molecules can be studied in detail. Accordingly, an increasing number of voltage-activated channels have been identified where mutations of these proteins are the basis for pathological disorders like epilepsy, deafness or heart arrhythmia. What are conotoxins? During evolution different venomous organisms have evolved specific ligands interacting with ion channels. Several toxins affecting voltage ion channels have been identified from marine cone snails. These predatory animals are well known for their nice shells but they also exhibit a very interesting biology
since several members of the species are able to hunt fish (Fig. 1). The venoms of cone snails consist of a huge variety of different peptides, the so called conotoxins. Conotoxins are small, usually cysteine rich peptides which have been grouped into several families according to the molecules they are interacting with. For example, d-conotoxins interact with voltage-activated Na+ channels, w-conotoxins with Ca2+ channels and so forth. The cysteine backbone has been shown to be very important for the function of these peptides (Fig. 2).
Fig 2: Primary structure of conotoxins binding to voltage gated ion channels d-conotoxins interact with Na+ channels; w-conotoxins with Ca2+ channels; k-conotoxins with K+ channels; m-conotoxins bind to Na+ channels. Other conotoxins identified interact with: acetylcholine receptors, NMDA subtypes of glutamate receptors or 5HT3 receptors.

Toxins affecting voltage gated ion channels have been indispensable not only for the investigation of the physiological function but also for the investigation of the structure of these proteins. The pharmacological properties of the conotoxins identified so far show that these peptides usually exhibit the following characteristics: Conotoxins are highly specific, unusually potent and extremely diverse. These features make
conotoxins a very interesting tool for the investigation of the structure and the function of different ion channels. For example, w-conotoxins are able to differentiate between different subtypes of Ca2+ channels which have been subdivided into groups according to their sensitivity to various w-conotoxins. An example with k-conotoxin PVIIA, the first identified conotoxin known to block voltage activated K+ channels, may illustrate our strategy to identify and characterize the interaction of conotoxins with their target molecules. k-PVIIA from the fish-hunting snail Conus purpurascens consists of 27 amino acids. It has the same cysteine backbone as d- and w-conotoxins. The role of k-PVIIA in prey capture has been established: this peptide is a key molecule for the very rapid immobilization of the fish prey by an “excitatory shock”. By using the Xenopus oocyte expression system it has been shown that k-PVIIA blocks Shaker mediated K+ currents with an an IC50 of about 100 nM. The Shaker K+ channel from Drosophila is the first voltage-activated K+ channel which has been cloned. No mammalian channel has been shown to be sensitive to k-PVIIA yet. Residues known to be at the extracellular mouth of the ion channel pore are important for the interaction with the toxin. The binding of PVIIA to the Shaker channel is a bimolecular reaction which is state-dependent. This means that the affinity and the kinetics of the interaction of the toxin with the ion channel are different depending on whether the channel protein is closed or open. Therefore the conformational changes ion channels undergo during their activity can also be of major importance for the binding of a ligand. Furthermore, the affinity of k-PVIIA to the closed state of the channel depends on the extracellular K+ concentration indicating that the extracellular milieu is important for the interaction. These results demonstrate that the interaction of a pharmacologically active substance with a voltage gated K+ channel not only depends on the structure of the molecules but results from an interplay of many parameters.