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Among the variety of toxins that bind and kill the cells, regardless of the origin, structure, size, and complexity, all have the cell plasma membrane as the target. This has been expressed by Rappuoli and Cesare (3). The toxins range from simple proteins peptides to large and sophisticated oligomeric proteins. Some toxins will have a lytic effect. These are defined as direct lytic factors. Others release enzymes that chemically degrade the cell membrane. Different animals produce a wide range of toxins, whose mode of action on the target cell membranes is varied. Examples of such animals are snakes, mollusks, scorpions, and sea anemones. However, all have one similarity of being able to permeate the cells.
According to Rochat, (124), physiologically useful high-affinity peptone oxides have been isolated from various animals such as scorpions, mollusks, snakes, and spiders. The toxins have a high degree of potency and thus act in picomolar to nanomolar concentrations range by altering the normal channel gating. The toxins may either block or cause a physical structural change of the channels they target. Massaro (4) has expressed that these toxins can be divided into four categories. One of those is the alpha neurotoxins which bind with high affinity to muscular acetylcholine receptors only. Snakes produce a variety of toxins such as alpha neurotoxins or curamimetic toxins which act on nicotinic acetylcholine receptors. Dendrotoxins are found in the alpha neurotoxins class isolated from snake venom of mamba snakes. They are a family of homologous proteins, as expressed by Rochat (4). This paper looks into the biochemistry of the dendrotoxins, taking into account their different types, structure, constituent amino acids, mode of action, their effects, and their relevance in research. It also discusses the active amino acid residues at the active site responsible for the proteins binding activity on target membranes. The dendrotoxins discussed in this report are from the black mamba snake.
Types of Dendrotoxins
According to Massaro, (4), one of the types is alpha neurotoxins. He classifies the neurotoxins on the basis of their length, number of fingers, source, and specificity. One class of alpha neurotoxins has a high affinity and is specific to the muscular receptors. They consist of a large family, as expressed by Massaro, (4), with short three-fingered toxins and are from the elapids and hydrophiids, (collectively known as Elapidae). This type of neurotoxin is also found in the Waglerins.
Another group is the alpha neurotoxins, that bind with high affinity to both muscular and some receptors on the neurons. These are the alpha 7, 8, and 9. They belong to a family of long-chain and three-finger toxins found in venoms from the Elapidae.
The third category consists of neurotoxins that bind to the neuronal receptors only and with a high affinity. Only four toxins in this category have been discovered yet. This has been expressed by Massaro (4). All are long-chain three-fingered toxins from the elapids. The last category includes the nonconventional neurotoxins, which have additional disulfide bonds in their first loop. They are also called weak neurotoxins and interact with low affinities on muscular types of acetylcholine receptors.
Effects on the Nervous Structure
The dendrotoxins act on the nicotinic acetylcholine receptors. These receptors are important in neural transmission. They control the transformation of chemical messages into an electric signal, binding the neurotransmitter acetylcholine, which generates postsynaptic depolarization. The chemical message is elicited by stimuli, and carried by the nerves to the brain for interpretation and response. At the site of the stimuli, the message is picked by cellular receptors. The re information is relayed by neurons which form part of the nervous system. The nerves link through the synaptic cleft. The membrane preceding the cleft is the pre-synaptic membrane, while the one after is referred to as the postsynaptic membrane. A chemical neurotransmitter substance known as acetylcholine is released, polarizing the presynaptic and postsynaptic membrane to facilitate the transmission of the signal during action potential. During resting potential, the acetylcholine should be in its inactive form. The sensory neurons possess potassium channels on their surface and nodes of Ranvier. Any toxic effect on these receptors will therefore have an effect on the transmission of nerve impulses in the organism. The extend of the resulting effect on the nervous system will be depended on the quantities of the toxins that have been able to bind to the target cell membranes. The alpha dendrotoxins are however highly potent.
Dendrotoxins have been shown to block particular subtypes of voltage-gated potassium channels in neuronal tissue. These channels control both presynaptic and postsynaptic membrane activities during resting and active potential ( Nelson 460) Dendrotoxins irreversibly bind on to nodes of Ranvier blocking the channels. Nelson (460) argues that in this way, they prolong the duration of action potential and increase acetylcholine release at the neuromuscular junction, which may result in muscle hyperexcitability and convulsive symptoms.
The Chemical Structure
According to Nelson, (467), dendrotoxins are proteins that weigh approximately 7 Kilo Daltons and consist of a single peptide chain of approximately 57-60 amino acids. The alpha dendrotoxins are homologous and have a crystal structure. The molecular structure and folding of the homologs are similar. This view has been supported by Nelson. (467) The two most identical homologs are I and K. Dendrotoxins poses a very short 310-helix near the N terminus of the peptide, and a two-turn alpha-helix occurs near the C- terminus. A two-stranded antiparallel ²-sheet is found at the central part of the molecular structure. The two strands are separated by a region for binding action. According to Nelson (470), this region also greatly contributes to the structural conformation. The intramolecular disulfide bonds are formed from cysteine residues and are present in all homologs. They are located at C7-C57, C16-C40, and C32-C53, in which the numbering is according to alpha dendrotoxin. They possess three intramolecular disulfide bonds.
According to Massaro, (6), these three-dimensional basic polypeptides have 6 cysteine amino acid residues that form three disulfide bonds. All the toxins show significant sequence homology to Kunitz protease inhibitors such as bovine pancreatic trypsin inhibitors. Most potent neurotoxicity shows negligible inhibitory activity. Site-directed mutagenesis has shown that the functional site of the dendrotoxins includes six major binding residues, all on the N-terminus, with Lys5 and Leu9 being the most important. They do not have nerve activity and do not destroy the nerve terminus.
Rochat (4) expresses that the dendrotoxins are structurally homologous to the Kuntz-type serine protease inhibitors, including bovine pancreatic trypsin inhibitor (BPTI). The disulfide bonds found in Alpha dendrotoxins and BPTI are identical. (Tipton, 40). However, dendrotoxins have lower protease inhibitory ability as compared to BPTI. This view has been expressed by Tipton. (40). This has been due to the absence of key amino acids causing significant structural differences (Nelson 456).
Dendrotoxins are basic proteins in nature and possess a net positive electrostatic charge at neutral pH. A bigger percentage of residues with an overall net positive charge form the lower cationic part of the protein. This has been expressed by Tipton (45). The cations responsible for these acidic effects are located in lysine and arginine. These are mainly found in three primary regions of the protein: near the N-terminus (Arg3, Arg4, Lys5), near the C-terminus (Arg54, Arg55), and at the narrow ²-turn region (Lys28, Lys29, Lys30). These cations are able to electrostatically interact with the anionic ends in the channel pores. The interaction results from overall charge differences. They work in virtually an irreversible way.
Biologically Important Residues
Studies have been done to determine the specific residues involved in dendrotoxin binding to target channels. According to Rappuli (65), Harvey is one of the scholars that have been involved in such studies. Harvey employed a method of modifying specific amino acids. He aimed at identifying the cations that could hinder the activity of one of the homologs, dendrotoxin-1. He acetylated lysine residue at the N-terminal at the fifth position, and lysine 29. He observed this dendrotoxin homolog had a great percentage decline in its potassium channel binding affinity. Site-directed mutagenesis was performed on a different homolog k. In this; cationic lysine and arginine were substituted with alanine which is neutral. A significant decrease in binding affinity was also noted. He, therefore, concluded that the cationic lysine residues at the N terminal, especially lysine 5 was key in the binding activity.lysine residues occupy three subsequent positions in the sequence. A mutation of the lysine codon (K28-K29-K30) to Ala-Ala-Gly resulted in an insignificant change in the toxins binding activity (Rappuli 69).
TiptoTipton0) states that there is a general agreement among different scholars that the conserved lysine residue near the N-terminus (Lys5 in alpha-DTX) is important for the biological activity of all dendrotoxins. The additional residues, such as those in the beta-turn region, play a role in dendrotoxin specificity. This they do by mediating the interactions of individual toxins to their specific target sites. This explains the stringent specificity of some dendrotoxins for different subtypes of voltage-gated K+ channels. It also accounts for differences in the potency of dendrotoxins for common K+ channels. According to Tipton, (150), another scholar, Wang, showed that the interaction of dendrotoxin-K with KV1.1 is mediated by its lysine residues in both the N-terminus and the ²-turn region. In this study, as expressed by Tipton (150), Wang concluded that alpha dendrotoxins can only interact with target channels through N-terminus only. This less expansive interactive domain explains why alpha-dendrotoxin is less specific while dendrotoxin-K is strictly selective for KV1.1.
A study was conducted on the effect of lysine acetylation. In this study, as expressed by Harvey (1263-1273), acetylating of dendrotoxin was done with acetic anhydride. Monoacetyl derivatives of the seven lysine residues and a wider derivative isolated through chromatography. The derivative acetyl Lys 29 and the derivative of Tyr 24 and Eys 28 were found to have more than 1000 lower toxicity than the native toxin. Lys 29 is part of a lysine triplet, Lys 28, 29, 30 are not found in the functional site. The Association of an acetyl group to Lys 29 resulted in structural changes that rendered the dendrotoxin inactive. Acetylating of Lys 28 only produces little effect on the toxin. However, the modification of both Lys 28 and Tyr 24 rendered the toxin almost inactive. Lysine 5 was found to have a protruding side chain that does not interact with any other group in the toxin. Harvey, therefore, concluded that this is the part of the functional site that is specific for the binding of the toxin on the potassium channels.
Mode of Action
The different steps that guide cytotoxicity also apply in dendrotoxins. As expressed by Rappuoli and Cesare, (3), they are able to interact selectively with the thick glycosylated layer found in ithenost cells. They quickly bind to selected membrane proteins with high affinity, resulting in an increased concentration of toxins on the membrane. This is followed by a conformational change According to Marshall and Harvey (70), dendrotoxins inhibit some proteinases, but this may be unrelated to their ability to block potassium channels. They have been shown to block some types of potassium channels and not others. They are highly potent hence act in nanomolar to picomolar rangHigh-affinity binding sites have been detected in the brain. They are not very toxic on systemic administration, but their toxicity increases by 10,000 fold on direct b injection to the brain. They induce repetitive firing of neurons and also enhance transmitter release.
According to Marshall and Harvey (83), they only block some specific neuronal potassium channels. Dendrotoxins once on the target channels reversibly inhibit them. This interaction is thought to be mediated by interactions between the cationic and anionic charges of the amino residues. Potassium channels have negative charges on their extracellular surface. Dendrortoxins are believed to bind to these sites, inhibiting ion conductance. The delta dendrotoxins however bind on the target channels, causing their structural alteration. They do not cause physical blockage of the pore, unlike alpha dendrotoxins.
Dendrotoxins cause little overall change to perineural potassium currents, though they still induce the repetitive firing of the motor nerve. According to the findings of Rochat, they induce an increase in the quantal content of endplate potentials and repetitive firing of the endplate potential. According to Munday et al (163), dendrotoxins are highly potent blockers of KV1.1, KV1.2, and KV1.6 potassium channels, inhibiting the neurotransmission. It can cause localized impairment of nerve function after mucous membrane exposure.
Dendrotoxins Use in Research
According to Massora, (9) snake neurotoxins, especially the short-chained three-fingered types are regarded as remarkable tools by toxicologists, pharmacologists, and biochemists. This is because they bear an imprint of the region of acetylcholine receptors which is likely to be close to or even overlapping the area where the acetylcholine neurotransmitter binds. Identification of this region would be crucial in understanding how the neurotransmitter works.
Dendrotoxins also come in handy in research studies because they are among the simplest proteins usable to address essential questions about proteins such as protein-protein interaction.
The three-fingered neurotoxin is not exotic but is found in many other functionally unrelated proteins. According to Massora (12), this offers a basis for the study of molecular Biology and evolutionary relationships for the protein folds. In regard to these three reasons, dendrotoxins are research tools.
Rochat (130) argues that dendrotoxins are tools valuable tools for the study of potassium-dependent channels in neurons. Experimentally, they have greater effects in mammalian preparations at body temperature, as expressed by Marshall and Harvey (73), Potassium channels in vertebrate neurons display a high degree of diversity. The channels are important in cellular signal transduction through the regulation of biological membranes ionic flux. The channels are thus targets for various toxins of biological origin.
Different peptide toxins have sn experimentally isolated successfully. Dendrotoxins are quite selective in their mode of action. This attribute has made them find their application in studies to determine the channels structures. This would be useful in classifying them since they are diverse (Munday 165).
There has been advancement in this area with the availability of radiolabelled dendrotoxins. Their radioactivity has been used in the identification of new toxins. Kalicludine toxins derived from sea anemone have been successfully radiolabelled and used in such identifications. Information on the channels structures provides clues that are medically useful in the synthesis of therapeutic products. (Nelson 247)
Dendrotoxins can be used as tools to localize potassium channels. They can also be used as tools to probe potassium channel subunits and oligomeric structures in the brain. These two views have been expressed by Massaro (462). This has been successfully done in the rat brain. He further expresses that they can also be exploited in studies of neurodegeneration. He attributes this to their potent convulsing effect in both systemic and intracerebral administrations. In the recent past, dendrotoxin homologs have been successfully been used in such studies in intact animals, opening new avenues of research towards the development of novel therapeutic strategies for neurons protection.
References
Massaro. J. Edward. Handbook of Neurotoxicology. New Jersey: Humana Publishers. 2002. Web.
Munday, S.T. et al. Dendrotoxin Poisoning in a Neurobiochemist. Clinical Toxicology, 2003: pg. 163-165. Web
Nelson, David and Michael. M. Lehninger Principles of Biochemistry. 4th Ed. New York: W H Freeman &CO. Publishers. 2004. Web.
Rapulli, R, and Cesare Monteaco. Guide to Protein toxins ands their use in cell Biology. Oxford: Oxford University press publishers. 2000. Web.
Rochat, Herve, and Marie France. Animal Toxins: Facts and protocols. Berlin: Birkhauser publishers. 2000. Web.
Tiptong, Keith, and Federico Dajas. Neurotoxins in neurobiology: Their actions and applications. West Sussex: Ellis Horwood Publishers. 1994. Web.
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