TOXINS AND DRUGS AFFECTING SYNAPTIC TRANSMISSION

Much of our knowledge of the synaptic physiology of the neuromuscular junction and the identities of its
various molecular components have been derived from experiments using specific pharmacologic agents
and toxins that permit functional dissection of the system. Figure 8-17 illustrates the relative synaptic
location and corresponding pharmacology of AChE, as well as several ion channels and proteins involved
in exocytosis.

Guanidinium Neurotoxins Such as Tetrodotoxin Prevent Depolarization of the Nerve Terminal, Whereas
Dendrotoxins Inhibit Repolarization
The action potential is the first step in transmission: a nerve action potential arriving at the terminal initiates
the entire process. As discussed in Chapter 7, the depolarizing phase of the action potential is mediated by
voltage-dependent Na+ channels that are specifically blocked by nanomolar concentrations of the small
guanidinium neurotoxins tetrodotoxin (TTX) and saxitoxin (STX) (see Fig. 7-5C).

The mamba snake toxin dendrotoxin (p. 194) has an effect that is precisely opposite that of TTX: it
facilitates the release of ACh that is evoked by nerve stimulation. Dendrotoxins are a family of
approximately 59-residue proteins with three disulfide bonds that block certain iso-forms of voltage-gated
K+ channels by binding to an extracellular site in the P-region domain with high affinity. These toxins reveal
the important role of K+ channels in terminating the process of transmitter release. Blockade of presynaptic
K+ channels by dendrotoxin inhibits repolarization of the presynaptic membrane, thereby prolonging the
duration of the action potential and facilitating the release of transmitter in response to the entry of extra
Ca2+ into the nerve terminal.

By Inhibiting Ca2+ Channels in the Active Zone of the Nerve Terminal, Molluscan Toxins Such as
ω-Conotoxin Prevent the Triggering of Exocytosis by Intracellular Ca 2+
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Table 8-2. NEUROTOXINS THAT BLOCK FUSION OF SYNAPTIC VESICLES

TOXIN TARGET
Tetanus Synaptobrevin
Botulinum B, D, F, G Synaptobrevin
Botulinum A/E SNAP-25
Botulinum C1 Syntaxin

SNAP-25, synaptosome-associated protein-25 kDa.

The exocytotic fusion of mature synaptic vesicles positioned at presynaptic active zones and the
subsequent release of ACh require the entry of Ca2+ into the nerve terminal. Ca2+ enters the presynaptic
terminal through voltage-gated Ca2+ channels that are activated by the depolarization of an incoming action
potential. One type of voltage-gated Ca2+ channel, the N-type isoform, has been localized to the region of
the active zone of the frog neuromuscular junction. Voltage-clamp experiments demonstrate that a class of
molluscan peptide toxins called ω-conotoxins (p. 191) block N-type Ca2+ currents in a practically
irreversible fashion. Exposure of a frog nerve-muscle preparation to ω-conotoxin thus inhibits the release of
neurotransmitter. This effect is manifested as an abolition of muscle EPP when the preparation is
stimulated via the nerve. The ω-conotoxins are 24 to 29 residues long and contain three disulfide bonds.
Imaging with confocal laser scanning microscopy has shown that ω-conotoxin binds at highest density to
voltage-dependent Ca2+ channels in the presynaptic nerve terminal, directly across the synaptic cleft from
AChR channels. This observation implies that Ca2+ channels are located precisely at the active zones of
synaptic vesicle fusion. This arrangement provides for focal entry and short-range diffusion of Ca2+
entering the nerve terminal to the exact sites involved in promoting Ca2+-dependent transmitter release.

By Cleaving Proteins Involved in Exocytosis, Bacterial Toxins Such as Tetanus and Botulinum Toxins
Prevent Fusion of the Synaptic Vesicles
Another class of neurotoxins that specifically inhibits neurotransmitter release includes the tetanus and
botulinum toxins. These large protein toxins (∼150 kDa) are respectively produced by the bacteria
Clostridium tetani and C. botulinum (see the box titled Clostridial Catastrophes). C. tetani is the causative
agent of tetanus ("lockjaw"), which is characterized by a general increase in muscle tension and muscle
rigidity, beginning most often with the muscles of mastication. The reason for this paradoxical enhancement
of muscle action is that the toxins have their greatest effect on inhibition of synaptic transmission by
inhibitory neurons in the spinal cord, neurons that would normally inhibit muscle contraction. C. botulinum
causes botulism, which is characterized by weakness and paralysis of skeletal muscle, as well as a variety
of symptoms that are related to inhibition of cholinergic nerve endings in the autonomic nervous system.

In humans, infection by these bacteria can lead to death because the toxins that they synthesize are potent
inhibitors of neurotransmitter release. This inhibition occurs because both tetanus and botulinum toxin
proteins have zinc-dependent endoproteinase activity (Table 8-2). These toxins enter nerve terminals and
specifically cleave three different proteins required for synaptic vesicle exocytosis. Tetanus toxin and
botulinum toxins B, D, F, and G cleave synaptobrevin, an integral membrane protein of the synaptic
vesicle membrane. Botulinum toxins C1 and A/E respectively cleave syntaxin and SNAP-25, two proteins
associated with the presynaptic membrane. These neurotoxins can have useful medical applications. For
example, botulinum toxin is used to treat certain disorders characterized by muscle spasms. Injection of a
small amount of botulinum toxin into the eye muscles of a patient with strabismus (a condition in which both
eyes cannot focus on the same object because of abnormal hyperactivity of particular eye muscles) is able
to suppress aberrant muscle spasms and restore normal vision.

Both Agonists and Antagonists of the Nicotinic Acetylcholine Receptor Can Prevent Synaptic
Transmission
The ionotropic (nicotinic) AChR channel located in the postsynaptic muscle membrane (see Fig. 8-17) also
has a rich and diverse pharmacology that can be exploited for clinical applications, as well as for elucidating
many functional aspects of the neuromuscular junction. Figure 8-18 shows the chemical structures of two
classes of agents that act on the nicotinic AChR. These agents are classified as agonists or antagonists
according to whether they activate opening of the channel or prevent its activation. Many agonists have a
structure similar to that of the natural neurotransmitter ACh. In general, such agonists activate the opening
of AChR channels with the same unitary conductance as those activated by ACh, but with different kinetics
of channel opening and closing. The synthetic drugs carbamylcholine (or carbachol) and succinylcholine
contain the choline moiety of ACh that is required for receptor activation. Carbamylcholine is a carbamyl
ester of choline, whereas succinylcholine (or succinyldicholine) is dimer of ACh linked together via the
acetyl methyl group. Both of these agents are resistant to hydrolysis by muscle AChE, but succinylcholine is
susceptible to hydrolysis by plasma and liver esterases. This property allows for prolonged activation of
AChRs.
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CLOSTRIDIAL CATASTROPHES
Botulism, although hardly one of the most common causes of food poisoning
today, is still the illness that many people think of when food-borne disorders are
discussed. The neurotoxin of Clostridium botulinum is very potent, and only a small
amount of contamination can lead to death. The most common source of botulism is
homemade foods. The spores of this organism can survive boiling temperatures for
a number of hours, and if the cooked food is allowed to stand at room temperature
for more than 16 hours, the clostridial spores can germinate and produce toxin.
Symptoms of the illness may appear from several hours to more than a week after
ingestion, although most cases occur within 18 to 36 hours. Patients begin to
complain of symptoms attributable to inhibition of synaptic vesicle release in the
autonomic nervous system (see Chapter 15), such as dry mouth, double vision, and
difficulty swallowing and speaking, and later begin to experience gastrointestinal
complications, including vomiting, pain, and diarrhea. Symptoms attributable to
inhibition of synaptic vesicle release at the neuromuscular junction such as
weakness and paralysis of the limbs may soon follow; ultimately, paralysis of the
respiratory muscles (see Chapter 26) can be fatal. Prompt intervention with
mechanical ventilation has reduced the mortality from botulism dramatically, and the
figure today stands at about 20%. Almost all deaths occur among the first victims of
a contaminated ingestion because the disease is not quickly recognized; those who
 fall victim later, when the diagnosis is much easier, do much better.
Vaccination has reduced the number of cases of tetanus reported in the United
States to only about 100 each year, almost all occurring in inadequately vaccinated
individuals. The disease is caused by a neurotoxin (tetanospasmin) produced by
Clostridium tetani. The organism gains entry to its host through a cut or puncture
wound. The toxin then travels along the peripheral nerves to the spinal cord, the
major site of its attack. There, the toxin inhibits synaptic-vesicle release by
interneurons that normally inhibit firing of the motor neurons that, in turn, activate
skeletal muscle. Thus, because the toxin suppresses inhibition of the normal reflex
arc, muscular contraction leads to profound spasms, most characteristically of the
jaw muscles but potentially affecting any muscle in the body. Symptoms can
commence on the day of the injury or as long as 2 months later. Complications
include respiratory arrest, aspiration pneumonia, rib fractures caused by the severe
spasms, and a host of other pulmonary and cardiac manifestations.

Succinylcholine is used to produce sustained muscle relaxation or "flaccid paralysis," which is useful in
certain types of surgery in which it is important to prevent excitation and contraction of skeletal muscles.
This paralytic action occurs because succinylcholine prolongs the opening of AChR channels and thereby
depolarizes the muscle membrane in the vicinity of the end plate. Such depolarization results in initial
repetitive muscle excitation and tremors, followed by relaxation secondary to inactivation of Na+ channels
in the vicinity of the end plate. This latter effect prevents the spread of muscle action potentials beyond the
end-plate region. On a longer time scale, such agents also lead to desensitization of the AChR to agonist,
which further inhibits neuromuscular transmission.

Another important agent acting on AChRs is nicotine, a natural constituent of tobacco that is responsible
for the stimulant action and at least some of the addictive effects of smoking. The selective ability of
nicotine to activate AChR channels is the basis of the classification scheme of "nicotinic" AChRs versus
"muscarinic" AChRs (see Fig. 8-3). Nicotine is not an agonist of the muscarinic or G protein - linked
receptors, which instead are activated by the mushroom alkaloid muscarine. Although nicotine is able to
activate the AChR at the neuromuscular junction, the physiological effects of smoking are primarily
manifested in the CNS and autonomic ganglia, where other neuronal isoforms of nicotinic AChRs are
located.

A classic example of a nicotinic AChR antagonist is d-tubocurarine (see Fig. 8-18), the active ingredient of
curare, a poison extracted from plants of the genus Strychnos. The indigenous tribes of the Amazon region
used curare to poison arrows for hunting. d-Tubocurarine is a competitive inhibitor of ACh binding to two
activation sites on the α subunits of the AChR. This action leads to flaccid paralysis of skeletal muscle from
inhibition of the nicotinic AChR. However, curare does not cause depolarization. A hallmark of the action of
d-tubocurarine is that it can be reversed by an increase in concentration of the natural agonist ACh by
binding competition. A large increase in local ACh concentration can be produced indirectly by an inhibitor
of AChE such as neostigmine (see later).

Figure 8-18 also shows the structure of pancuronium, which is a synthetic bis-quaternary ammonium
steroid derivative. This drug is also useful for the production of neuromuscular blockade in surgery, and it is
actually a more potent, selective competitive antagonist of the muscle nicotinic AChR than D-tubocurarine
is.

Another class of nicotinic AChR inhibitors is a family of approximately 8-kDa proteins present in the venom
of Elapidae snakes (e.g., cobras). These toxins include α-bungarotoxin (α-Bgt) and homologous α toxins,
which bind very strongly to nicotinic receptors. The specific binding of α-Bgt to the nicotinic AChR of
skeletal muscle is virtually irreversible. When α-Bgt binds to the nicotinic AChR, it obstructs the
agonist-binding site and prevents activation of the receptor by ACh. The radioiodinated derivative
125
I-labeled α-Bgt has been widely used as a ligand for purifying the nicotinic AChR from various tissues.
Fluorescent derivatives of α-Bgt can also be used as specific labels for localizing AChRs at the muscle end
plate. The same snake venom (Bungarus multicinctus) that contains α-Bgt also contains a homologous
protein toxin called κ-bungarotoxin (κ-Bgt). This toxin has little effect on nicotinic AChR channels at the
neuromuscular junction, but it does inhibit AChR channels in neuronal tissue. The differential effect of α-Bgt
and κ-Bgt on muscle and neuronal currents activated by both ACh and nicotine led to the recognition that
different classes of nicotinic receptors exist in the CNS versus skeletal muscle. The basis for these
isoforms is the differential expression of multiple genes for homologous nicotinic AChR subunits.

Inhibitors of Acetylcholinesterase Prolong and Magnify the End-Plate Potential

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Figure 8-18 Agonists and antagonists of the nicotinic acetylcholine receptor (AChR).

A variety of specific inhibitors of anticholinesterase have been helpful in defining the contribution of AChE to
responses at the muscle end plate. Inhibition of AChE generally increases the amplitude and prolongs the
duration of the postsynaptic response to ACh; thus, the enzyme plays an important role in limiting the
excitatory action of ACh under normal physiological conditions. In the absence of ACh breakdown by AChE,
the prolonged decay of the EPP reflects the underlying kinetics of activated receptors and slow depletion of
the agonist in the vicinity of the junctional folds by diffusion of ACh.

The plant alkaloid physostigmine (also known as eserine) is the prototypic anticholinesterase (Fig. 8-19).
Neo-stigmine (also called prostigmine), a synthetic anti-AChE drug that is partially analogous to
physostigmine, is used to treat myasthenia gravis. As discussed in the box on p. 224, this disease is
caused by the autoimmune destruction and loss of nicotinic AChRs at the muscle end plate. Because ACh
is an acetyl ester, the catalytic cycle of AChE results in the formation of an intermediate in which a serine
group of the AChE is acetylated. Physostigmine and neostigmine, on the other hand, have a carbamyl ester
linkage that produces a carbamoylated intermediate form of AChE. However, slow hydrolysis of the
carbamoylated enzyme results in an approximately 4-hour duration of esterase inhibition.

Another important class of synthetic AChE inhibitors consists of organophosphorus compounds, which
are irreversible inhibitors. This group is typified by diisopropyl-fluorophosphate (DFP) (see Fig. 8-19). These
compounds react with the aforementioned serine residue of AChE and form an essentially irreversible
covalent modification of the enzyme. Such agents rank highly among the most potent and lethal of toxic
chemicals. Their devastating effect is caused by excessive enhancement of cholinergic neurotransmission,
mediated by both muscarinic and nicotinic receptor pathways throughout the body. For example, exposure
to toxic organophosphorus agents results in the flaccid paralysis of respiratory muscles because of initial
muscle stimulation followed by depolarization blockade. The devastating action of these compounds
dramatically underlines the essential role of AChE in terminating cholinergic neurotransmission. A tragic
application of various volatile forms of these compounds is their use as chemical warfare agents (i.e.,
"nerve gas" such as sarin). Related compounds, such as malathion (see Fig.8-19), which are selectively
toxic to insects, are widely used as agricultural insecticides. Depolarization Blockade
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Figure 8-19 Structures of acetylcholinesterase (AChE) inhibitors.

A natural organophosphorus neurotoxin is produced by Anaebena flosaquae, a toxic cyanobacterium

(blue-green alga). Known as anatoxina(s), this toxin is a potent inhibitor of AChE and is responsible for the
poisoning of dogs and farm animals that drink from contaminated ponds. Another interesting class of
natural inhibitors includes the fasciculins, a family of small protein toxins present in mamba snake venom
that inhibit AChE with very high affinity and specificity.

By Inhibiting Muscle Na+ Channels, Certain Neurotoxins Block Spread of the Postsynaptic Action Potential
and Muscle Contraction
An action potential is not only the first but also the last step in transmission at the neuromuscular junction:
the production of an action potential in the muscle fiber membrane signals the successful completion of
synaptic transmission. As discussed earlier, action potentials, including those in muscle, can be blocked by
TTX and STX. Selective blockade of the muscle action potential can be achieved with a unique toxin called
µ-conotoxin (p. 187), which is obtained from a marine snail (Conus geographus). µ-Conotoxin is a
22-residue, basic peptide with a discoidal, star-like three-dimensional structure that is stabilized by three
disulfide bonds. It is an especially potent blocker of the particular isoform of the voltage-dependent Na+
channel that is present in adult mammalian skeletal muscle, but it has little effect on the Na+ channel
isoforms of nerve or heart. If an intact nerve-muscle preparation is exposed to µ-conotoxin, stimulation of
the nerve still evokes the release of ACh, but the muscle action potential is completely eliminated. Effects
of µ-Conotoxin

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