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.
