Neurotoxins

Nervous System
The nervous system functions by conducting an electrical signal or impulse along the length of the nerve and transmitting it across a junction (called the synaptic cleft) to another nerve or to a muscle fiber. When a nerve impulse reaches the terminus of the nerve, an influx of ions promotes the release of vesicles containing a neurotransmitter such as acetylcholine, allowing this messenger molecule to diffuse across the synaptic cleft and bind to specific receptors.

Ion Channels
Many of the important binding sites of the nervous system, such as ion channels, were elucidated using bacterial neurotoxins. Channels for the ions Na+ and Ca2+ play crucial roles in the transmission of a nerve impulse and are found pre- and post-synaptically for sodium and presynaptically only for calcium channels.

Na+ channels are made up of four transmembrane loops with repeating binding sites. The neuronal sodium channels are important during the initial phase of action potential due to the voltage-sensitive production of an inward movement of Na+ and a rapid depolarization from the resting potential continuing to a slight positive overshoot. In addition, Na+ channels play a major role in determining the excitability of central neurons as revealed through the bacterial toxins tetrodotoxin and saxitoxin being selective towards particular channel subtypes. Thus, sodium channels are often described as being tetrodotoxin-sensitive or saxitoxin-sensitive if toxin binding interferes with the nerve impulse. By preventing the agonist-induced conformational change in the receptor ion channel required for the influx of sodium that is essential for membrane depolarization, these toxins inhibit neurotransmitter action and induce paralysis.

Ca2+ channels, while not directly involved in the conductance of a nerve impulse, function to prolong the depolarization through the inward movement of Ca2+ thus causing the release of acetylcholine vesicles. Several calcium channel subtypes (L, N, P/Q, R and T) abound the nerve terminus, being differentiated through their sensitivity to different toxins. The P-type calcium channel, for example, was specifically characterized by its sensitivity towards the spider toxin omega-agatoxin.

Upon mobilization by the influx of Ca2+ through the calcium channels, the acetylcholine containing vesicles fuse with the membrane and exocytotically release their contents into the synaptic cleft and rapidly diffuse. Interference with the release of acetylcholine produces flaccid paralysis while increased release causes severe cramping of muscles. The toxins from the Clostridium bacteria are representative of the type that interferes with the release of acetylcholine. Botulism bacteria specifically causing flaccid paralysis through targeting the cholinergic motor nerve endings while the tetanus bacteria selectively cause spastic paralysis by targeting spinal neurons and causing an increase of acetylcholine release.

Acetylcholine Receptors (AchR)
Acetylcholine has two modes of action, a nicotine-like (nicotinic) or a muscarine-like (muscarinic) action, with the former blocked by curare and the later by atropine. Nicotinic acetylcholine receptors are found primarily at neuromuscular junctions while muscarinic acetylcholine receptors are found primarily in the central nervous system. Functionally the two receptors are also different, nicotinic AChRs are ligand-gated ion channels while muscarinic AChRs are part of a larger class of G-protein coupled receptors. This larger class utilizes the full-power of the intracellular secondary messenger system which involves an increase of intracellular Ca2+ .

Nicotinic Acetylcholine Receptors (nAChR)
Binding by two molecules of acetylcholine to the nicotinic AChR causes a conformational change resulting in the formation of an ion pore. This produces a rapid increase in cellular permeability of Na+ and Ca2+ ions, depolarization and excitation, resulting in muscular contraction. Receptor subunits are either alpha (alpha2 - alpha9) or beta (beta2 - beta5) types, which leads to quite a number of potential combinations but the alpha-subunit is always present in two identical copies as these are the sites to which acetylcholine binds. The alpha-subunits also determine the binding sites through interaction with the other subunits. Neurotoxins targeting this site reversibly block the opening and prevent acetylcholine from forming a pore and allowing cations to pass through.

Neuronal nicotinic acetylcholine receptors (nnAChR) have been classified into two groups based on responses to the snake venom toxin alpha-bungarotoxin (BuTX), being either alpha-BuTX-sensitive or ­insensitive. alpha-BuTX-sensitive receptors are composed of alpha7, alpha8 and/or alpha9 subunits while alpha-BuTX -insensitive receptors are composed of alpha2, alpha3, or alpha4 subunits with beta2, beta4 and/or alpha5 subunits.

Muscarinic Acetylcholine Receptors
Muscarinic receptors are found in the central nervous system synapses rather than at the neuromuscular junction, as is the case with nicotinic acetylcholine receptor specific toxins. Muscarinic receptors are involved in a large number of physiological functions including heart rate and force, contraction of smooth muscles and the release of neurotransmitters. Molecular cloning has determined five subtypes of muscarinic receptors, based on pharmacological activity they have been broken up into M1-M5. All five subtypes are found in the central nervous system while M1-M4 are also scattered widely through a myriad of tissues.

M1, M3 and M5 receptors cause the activation of phospholipase C, generating two secondary messengers (IP3 and DAG) eventually leading to an intracellular increase of Ca2+, while M2 and M4 inhibit adenylate cyclase thus decreasing the production of the second messenger cAMP. Importantly, activation of the M2 receptor in the heart mediates the closing of calcium channels to reduce the force and rate of contraction. Ligand binding to the receptor causes a poorly understood conformational change that mediates the association with and activation of an intracellular G-protein. This G-protein converts GTP to GDP resulting in the disassociation of the activated G-protein allowing this enzyme to catalyze intracellular events.

Competitive binding by the potent venoms of many animals produces interference of the binding of acetylcholine to the receptors resulting in flaccid paralysis.

Sources Of Neurotoxins
Interference with the fundamental communication of the body is a very effective manner of causing envenomation, the venom components selectively targeting important sites of the nervous system in either agonistic or antagonistic manners. In the most dangerous species of venomous snakes and other animals, the most significant action of the venom lies in its effect upon the victim's nervous system, hindering the operation of muscles and causing paralysis leading to death from respiratory failure. As such, these potent molecules are useful for demonstrating the structure-function relationships of toxins and also the tremendous potential venoms have as a source of useful investigational ligands or even as therapeutics. Neurotoxins are found in a wide variety of animals, covering a great diversity from arachnids to amphibians to mollusks to snakes.