Dendrotoxin

Dendrotoxins are a class of neurotoxins produced by mamba snakes (Dendroapsis) that block particular subtypes of voltage-gated potassium channels in neurons, thereby enhancing the release of acetylcholine at neuromuscular junctions. Because of their high potency and selectivity for potassium channels, dendrotoxins have proven to be extremely useful as pharmacological tools for studying the structure and function of these ion channel proteins.

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Alpha-dendrotoxin is the major facilitatory protein in the venom of the Eastern green mamba, Dendroaspis angusticeps. However, several homologues (beta-, gamma-, delta-dendrotoxins) have also been isolated from the same venom. Two similar proteins, known as dendrotoxin-K and dendrotoxin-I, were isolated from the venom of the black mamba, Dendroaspis polylepis and have been well studied. Other less well known dendrotoxins include dendrotoxin-Dv14 from the Western green mamba, Dendroaspis viridis, and, more recently, the epsilon-dendrotoxins ^[1] isolated from D. angusticeps. Even more recently, another class of toxins, the kalicludines, were isolated from the sea anemone Anemonia sulcata and shown to be structurally homologous to the dendrotoxins.

Discovery and History

In 1979, Barrett and Harvey demonstrated that the venom of the Eastern green mamba snake was reported to increase the twitch height of isolated nerve-muscle preparations by facilitating the release of acetylcholine ^[2]. Separation of the venom into components by Harvey and Karlsson (1980) resulted in the isolation and purification of single polypeptide which they called “dendrotoxin” after the Latin name of the mamba snake. Dendrotoxin was subsequently shown to possess potent and selective potassium channel blocking activity, and its biological activity was demonstrated in several neuronal systems. In 1988, dendrotoxin was renamed alpha-dendrotoxin (alpha-DTX) when several dendrotoxin homologues (subsequently named beta-, gamma-, and delta-dendrotoxin) were isolated from the same venom. Dendrotoxins were also discovered in other mamba snake venoms. Two peptide components of black mamba venom were isolated and named dendrotoxin-I (DTX-I) and dendrotoxin-K (DTX-K). The three-dimensional structure of alpha-dendrotoxin has been determined crystallographically, while the 3D structures of dendrotoxin-K and dendrotoxin-I have been determined based on NMR spectral data.

Functional Effects in the Nervous System

Dendrotoxins have been shown to block particular subtypes of voltage-gated potassium (K⁺) channels in neuronal tissue. In the nervous system, voltage-gated K⁺ channels control the excitability of nerves and muscles by controlling the resting membrane potential and by repolarizing the membrane during action potentials. Dendrotoxin has been shown to bind the nodes of Ranvier of motor neurons^[3] and to block the activity of these potassium channels. In this way, dendrotoxins prolong the duration of action potentials and increase acetylcholine release at the neuromuscular junction, which may result in muscle hyperexcitability and convulsive symptoms.

Binding Sites in the Brain

When injected into the CNS, dendrotoxin induces epileptic activity and, with high enough doses, neuronal damage. With the use of ¹²⁵I-labelled dendrotoxins, the distribution of binding sites in the brain has been well studied. Dense areas of binding in the rat brain are the thalamus, midbrain, and neocortex ^[4]. Other studies with rat brain synaptosomes indicate that alpha- and delta-dendrotoxin preferentially block the rapidly inactivating voltage-gated (“A-type”) K⁺ currents, while beta- and gamma-dendrotoxin block the noninactivating voltage-gated (delayed-rectifier) K⁺ channels ^[5].

Potassium Channel Targets

Despite their structural similarity, there is high variation among dendrotoxins in terms of the potency and selectivity for voltage-gated potassium channel subtypes. Furthermore, voltage-gated potassium channels are one of the most diverse channel types, and include about 40 different channels and 12 distinct subfamilies (K_V1 to K_V12)^[6]. Not surprisingly then, dendrotoxins may preferentially block one or more subtypes of these channels over another. For example, dendrotoxin-I has been reported to block K_V1.1, K_V1.2, and K_V1.6 channels expressed in Xenopus oocytes. The homologous dendrotoxin-K, however, was selective for only K_V1.1. Furthermore, alpha-dendrotoxin also blocks both K_V1.1 and K_V1.2 in the nanomolar range, but has much lower affinity for other cloned channels. More recently, gamma-dendrotoxin has been reported to block a large conductance Ca²⁺-activated potassium channel in neuroblastoma cells ^[7].

Dendrotoxin Structure

  Dendrotoxins are ~7kDa proteins consisting of a single peptide chain of approximately 57-60 amino acids. Several homologues of alpha-dendrotoxin have been isolated, all possessing a slightly different sequence. However, the molecular architecture and folding conformation of these proteins are all very similar. Dendrotoxins possess a very short 3₁₀-helix near the N-terminus of the peptide, while a two turn alpha-helix occurs near the C-terminus. A two-stranded antiparallel β-sheet occupies the central part of the molecular structure. These two β-strands are connected by a distorted β-turn region ^[8] that is thought to be important for the binding activity of the protein. All dendrotoxins are cross-linked by three disulfide bridges, which add stability to the protein and greatly contribute to its structural conformation. The cysteine residues forming these disulfide bonds have been conserved among all members of the dendrotoxin family, and are located at C7-C57, C16-C40, and C32-C53 (numbering according to alpha-dendrotoxin).

The dendrotoxins are structurally homologous to the Kunitz-type serine protease inhibitors, including bovine pancreatic trypsin inhibitor (BPTI). Alpha-dendrotoxin and BPTI have been shown to have 35% sequence identity as well as identical disulfide bonds. Despite the structural homology between these two proteins, dendrotoxins do not appear to exhibit any measurable protease activity like BPTI. This loss of activity appears to result from the absence of key amino acid residues that produce structural differences that hinder the key interactions necessary for the protease activity seen in BPTI.

Dendrotoxins are basic proteins that possess a net positive charge when present in neutral pH. Most of the positively-charged amino acid residues of dendrotoxins are located in the lower part of the structure, creating a cationic domain on one side of the protein. Positive charge results from lysine (Lys) and arginine (Arg) residues that are concentrated in three primary regions of the protein: near the N-terminus (Arg3, Arg4, Lys5), near the C-terminus (Arg54, Arg55) and at the narrow β-turn region (Lys28, Lys29, Lys30)^[9]. It is believed that these positively-charged residues can play a critical role in dendrotoxin binding activity, as they can make potential interactions with the anionic sites (negatively-charged amino acids) in the pore of potassium channels.

Biological Activity

Mode of Action

A single dendrotoxin molecule associates reversibly with a potassim channel in order to exert its inhibitory effect. It is proposed that this interaction is mediated by electrostatic interactions between the positively-charged amino acid residues in the cationic domain of dendrotoxin and the negatively-charged residues in the ion channel pore. Potassium channels, similar to other cation-selective channels, are believed to have a cloud of negative charges that precede the opening to the channel pore that help conduct potassium ions through the permeation pathway. It is generally believed (though not proven) that a dendrotoxin molecules bind to anionic sites near the extracellular surface of the channel and physically occlude the pore, thereby preventing ion conductance. However, Imredy and MacKinnon ^[10] have proposed that delta-dendrotoxin may have an off-center binding site on their target proteins, and may inhibit the channel by altering the structure of the channel, rather than physically blocking the pore.

Biologically Important Residues

Many studies have attempted to identify which amino acid residues are important for binding activity of dendrotoxins to their potassium channel targets. Harvey et al. ^[11]used residue-specific modifications to identify positively-charged residues that were crucial to the blocking activity of dendrotoxin-I. They reported that acetylation of Lys5 near the N-terminal region and Lys29 in the beta-turn region led to substantial decreases in DTX-I binding affinity. Similar results have been shown with dendrotoxin-K using site-directed mutagenesis to substitute positively-charged lysine and arginine residues to neutral alanines. These results, along with many others, have implicated that the positively-charged lysines in the N-terminal half, particularly Lys5 in the 3₁₀-helix, play a very important role in the dendrotoxin binding to their potassium channel targets. The lysine residues in the β-turn region has provided more confounding results, appearing to be biologically critical in some dendrotoxin homologues and not necessary for others. Furthermore, mutation of the entire lysine triplet (K28-K29-K30) to Ala-Ala-Gly in alpha-DTX resulted in very little change in biological activity.

There is a general agreement that the conserved lysine residue near the N-terminus (Lys5 in alpha-DTX) is crucial for the biological activity of all dendrotoxins, while additional residues, such as those in the beta-turn region, might play a role in dendrotoxin specificity by mediating the interactions of individual toxins to their individual target sites. This not only helps explain the stringent specificity of some dendrotoxins for different subtypes of voltage-gated K⁺ channels, but also accounts for differences in the potency of dendrotoxins for common K⁺ channels. For example, Wang et al. ^[12]showed that the interaction of dendrotoxin-K with K_V1.1 is mediated by its lysine residues in both the N-terminus and the β-turn region, while alpha-dendrotoxin appears to interact with its target solely through the N-terminus. This less expansive interactive domain may help explain why alpha-dendrotoxin is less discriminative while dendrotoxin-K is strictly selective for K_V1.1.

Uses in Research

Potassium channels of vertebrate neurons display a high degree of diversity that allows neurons to precisely tune their electrical signaling properties by expression of different combinations of potassium channel subunits. Furthermore, because they regulate ionic flux across biological membranes, they are important in many aspects of cellular regulation and signal transduction of different cell types. Therefore, voltage-gated potassium channels are targets for a wide range of potent biological toxins from such organisms as snakes, scorpions, sea anemones, and cone snails. Thus, venom purification has led to the isolation of peptide toxins such as the dendrotoxins, which have become useful pharmacological tools for the study of potassium channels. Because of their potency and selectivity for different subtypes of potassium channels, dendrotoxins have become useful as molecular probes for the structural and functional study of these proteins. This may help improve our understanding of the roles played by individual channel types, as well as assist in the pharmacological classification of these diverse channel types^[13]. Furthermore, the availability of radiolabelled dendrotoxins provides a tool for the screening of other sources in a search for new potassium channel toxins, such as the kalicludine class of potassium channel toxins in sea anemones. Lastly, the structural information provided by dendrotoxins may provide clues to the synthesis of therapeutic compounds that may target particular classes of potassium channels.

References

1. ^ [Tytgat J, Vandenberghe I, Ulens C, Van Beeumen J (2001). New polypeptide components purified from mamba venom. PEBS Letters 491:217-221]
2. ^ [Harvey AL (2001). Twenty years of dendrotoxins. Toxicon 39:15-26]
3. ^ [Gasparini S, Danse J-M, Licoq A, Pinkasfeld S, Zinn-Justin S, Young LC, C.L. de Medeiros C, Rowan EG, Harvey AL, and Me’nez A (1998). Delineation of the Functional Site of alpha-dendrotoxin: The functional topographies of dendrotoxins are different but share a conserved core with those of other K_V1 potassium channel-blocking toxins. J Biol Chem 273:25393-25403]
4. ^ [Haghdoust H, Janahmadi M, Behzadi G (2007). Physiological role of dendrotoxin sensitive K+ channels in the rat cerebellar Purkinje neurons. Physiol Res 56:6]
5. ^ [Tricaud N, Marchot P, Martin-Eauclaire MF (2001). On the kaliotoxin and dendrotoxin binding sites on rat brain synaptosomes. Toxicon 38:1749-1758 ]
6. ^ [Catterall WA, Cestèle S, Yarov-Yarovoy V, Yu FH, Konoki K, Scheuer T (2007). Voltage-gated ion channels and gating modifier toxins. Toxicon 49, 124-141]
7. ^ [Inglis V, Karpinski E, Benishin C (2003). Gamma-dendrotoxin blocks large conductance Ca²⁺-activated K⁺ channels in neuroblastoma cells. Life Sciences 73, 2291-2305]
8. ^ [Katoh E, Nishio H, Inui T, Nishiuchi Y, Kimura T, Sakakibara S, Yamazaki T (2000). Structural Basis for the Biological Activity of Dendrotoxin-I, a Potent Potassium Channel Blocker. Biopolymers 54:44-57 ]
9. ^ [Swaminathan P, Hariharan M, Murali R, Singh CU (1996). Molecular Structure, Conformational Analysis, and Structure-Activity Studies of Dendrotoxin and Its Homologues Using Molecular Mechanics and Molecular Dynamics Techniques. J Med Chem. 39:2141-2155]
10. ^ [Imredy JP, and MacKinnon R (2000). Energetic and Structural Interactions between delta-Dendrotoxin and a Voltage-gated Potassium Channel. J. Mol. Biol. 296:1283-1294]
11. ^ [Harvey AL, Rowan EG, Vatanpour H, Engstrom A, Westerlund B, Karlsson E (1997). Changes to biological activity following acetylation of dendrotoxin I from Dendroaspis polylepis (black mamba). Toxicon 35:1263-1273]
12. ^ [Wang FC, Bell N, Reid P, Smith LA, McIntosh P, Robertson B, and Dolly JO (1999). Identification of residues in dendrotoxin K responsible for its discrimination between neuronal K⁺ channels containing K_V1.1 and 1.2 alpha subunits. Eur J Biochem 263:222-229]
13. ^ [Yoshida S and Matsumoto S (2005). Effects of alpha-dendrotoxin on K⁺ currents and action potentials in tetrodotoxin-resistant adult rat trigeminal ganglion neurons. J Pharmacol Exp Ther 314:437-445]