The neuromuscular junction contains the distal nerve terminal, Schwann cell, synaptic cleft, and muscle end plate, which together provide an array of receptors and substrates for drug action. Neuromuscular transmission is predominantly dependent on acetylcholine as the natural transmitter. Acetylcholine, when released from the prejunctional nerve terminal, binds to acetylcholine receptors (AChRs) that are present either prejunctionally or postjunctionally within the neuromuscular junction (synapse) and, depending on their structural composition, are classified into the usual muscle subtype AChRs or a variety of neuronal subtype AChRs.
Muscle relaxants have multiple sites of action. Although the major actions occur by mechanisms and at sites described as agonistic and antagonistic actions at postjunctional receptors for depolarizing and nondepolarizing muscle relaxants (NDMRs), this description of neuromuscular drug action is a simplistic one. Neuromuscular transmission is impeded by NDMRs because they prevent access of acetylcholine to its preferred recognition site on the postjunctional nicotinic AChRs.
If the concentration of NDMR is increased, another, noncompetitive action—block of the ion channel—is superimposed. The postjunctional paralytic effects of muscle relaxants can be enhanced by the actions of the relaxant on prejunctional AChRs, which modulate the release of acetylcholine. The latter can be documented as fade that occurs with increased frequency of stimulation. Fade can also be seen when the postjunctional AChRs alone are functionally blocked (e.g., by bungarotoxin) or when the number of AChRs (e.g., myasthenia gravis) is decreased. Hence, the neuromuscular junction is a complex and dynamic system in which the phenomena produced by drugs are composites of actions that vary with the drug, dose, activity at the nerve terminal and muscle, time after administration, presence of anesthetics or other drugs, and age and condition of the patient.
Inhibition of acetylcholinesterase enzyme by anticholinesterases (e.g., neostigmine) increases the concentration of acetylcholine in the synaptic cleft, which can compete with and displace the NDMRS and thus reverse the paralysis. These anticholinesterase drugs (e.g., neostigmine) also have other effects, including the nerve terminal and the receptor, by an allosteric mechanism. Acute bolus or prolonged administration of anticholinesterases can have deleterious effects on neuromuscular function in otherwise healthy patients. The modified cyclodextrin, sugammadex, is a novel and innovative class of compound that reverses paralysis of only steroidal muscle relaxants by encapsulation of this series of compounds.
Depolarizing compounds (e.g., succinylcholine) initially react with the acetylcholine recognition site and, like the transmitter, open AChR ion channels during depolarization of the end-plate membrane. Unlike the transmitter, they are not subject to hydrolysis by acetylcholinesterase and therefore remain in the junction. Soon after the administration of succinylcholine, some receptors are desensitized, and, although occupied by the agonist, they do not open to allow current to flow to depolarize the muscle membrane area.
If the depolarizing relaxant is applied in higher-than-usual concentrations or is allowed to remain at the junction for a long time, then other neuromuscular effects occur; depolarizing relaxants in higher concentrations have effects on prejunctional structures, and the combination of prejunctional and postjunctional effects plus secondary ones on muscle and nerve homeostasis results in the complicated phenomenon known as phase II block. Some of the other drugs used clinically (e.g., Botox) have effects on the motor nerve and therefore indirectly on muscle. Systemic infection with clostridial toxins ( Clostridium botulinum , gas gangrene) can lead to systemic paralysis as a result of decreased release of acetylcholine from the nerve terminal. NDMRs administered for 24 hours or longer can have effects on the postsynaptic receptor and simulate denervation state (chemical denervation) manifested by upregulated postsynaptic AChRs. Magnesium given to preeclamptic mothers decreases the release of acetylcholine with a potential for muscle weakness in the mother or newborn. In recognizing these sites and mechanisms, we begin to bring our theoretical knowledge closer to explaining the phenomena observed when these drugs are exposed to living humans.
Contemporary research work has focused on the control of AChR expression on the postjunctional membrane in normal and diseased states. The presence or absence of the conventional mature and immature isoforms seems to complicate matters further. In certain pathologic states (e.g., denervation, stroke, sepsis, burns, immobilization, chronic use of NDMRs), AChRs are upregulated, with increased expression of the immature isoform and de-novo expression of the nicotinic α7 acetylcholine receptors (α7 AChRs). Altered functional and pharmacologic characteristics of the immature (fetal or γ-subunit–containing receptors) and α7 AChRs expressed in pathologic muscle wasting conditions result in increased sensitivity to succinylcholine with hyperkalemia and resistance (insensitivity) to NDMRs.
An area of increasing attention is control of the expression of mature versus the other two receptor isoforms (immature γ- and α7 AChRs on the synapse). Re-expression of the immature γ- and α7 AChRs is probably related to aberrant growth factor signaling.
Genetic mutations in the AChRs, that result in prolonged or fast open-channel time, can lead to a myasthenia-like state, even in the presence of normal receptor numbers. The weakness is usually related to ineffective depolarization or to the altered open-channel time, or to both.
Although cholinergic neurotransmission at the neuromuscular junction is the most widely studied synapse within the nervous system, complete knowledge of its workings has not yet been achieved. The physiology of neuromuscular transmission could be analyzed and understood at the simplest level by using the classic model of nerve signaling to muscle through the acetylcholine receptor (AChR). The mammalian neuromuscular junction and the nicotinic AChRs are the prototypical and most extensively studied synapse and receptor, respectively. Research has provided more detailed information on processes that, within the classic scheme, can modify neurotransmission and response to drugs. One example is the role of qualitative or quantitative changes in AChRs that modify neurotransmission and the response to drugs. In myasthenia gravis, for example, the decrease in AChRs results in decreased efficiency of neurotransmission (and therefore muscle weakness) and altered sensitivity to neuromuscular relaxants. Another example is the importance of nerve-related (prejunctional) changes that alter neurotransmission and the response to muscle relaxants. Yet, muscle relaxants act in ways that are not encompassed by the classic scheme of a unitary site of action. The observation that muscle relaxants can have prejunctional effects or that some muscle relaxants can also have agonist-like stimulatory actions on the receptor, whereas others have effects not explainable by purely postsynaptic actions on muscle, has provided new insight into some previously unexplained observations. Although muscle relaxants are known to have effects on the presynaptic and postsynaptic receptors of the neuromuscular junction, recent evidence indicates that they can react with nicotinic and muscarinic AChRs other than those in muscle, including receptors on the carotid body, on the vagus to the heart, and on bronchial smooth muscle. Although this multifaceted action-response scheme makes the physiologic and pharmacologic neurotransmission more complex, these added insights also bring experimentally derived knowledge much closer to clinical observations. This review dwells on the basic physiology and anesthesia-related pharmacology of the neuromuscular junction. Several reviews that provide more detailed insight into the physiological and pathological processes that alter function and pharmacology of the neuromuscular junction are available for the initiated reader.
Neuromuscular transmission occurs by a fairly simple and straightforward mechanism. The nerve synthesizes acetylcholine and stores it in small, uniformly sized packages called vesicles . Stimulation of the nerve causes these vesicles to migrate to the surface of the nerve, rupture, and discharge acetylcholine into the cleft separating the nerve from muscle. AChRs in the end plate of the muscle respond by opening their channels for influx of sodium ions into the muscle to depolarize the muscle. The end-plate potential created is continued along the muscle membrane by the opening of sodium channels present throughout the muscle membrane to initiate a muscle contraction. The acetylcholine immediately detaches from the receptor and is destroyed by the enzyme, acetylcholinesterase, which is also present in the cleft. Exogenous drugs that activate the nicotinic AChR, that is, agonists such as depolarizing muscle relaxants (e.g., succinylcholine or nicotine), can also act on these receptors and mimic the effect of acetylcholine and cause depolarization of the end plate. Nondepolarizing muscle relaxants (NDMRs) also act on the receptors, but they prevent acetylcholine from binding to the receptor and thus prevent depolarization by agonists. Because these NDMRs prevent the action of agonists (e.g., acetylcholine, carbachol, succinylcholine), they belong to the class of compounds known as antagonists at the muscle AChRs. Other compounds, frequently called reversal drugs or antagonists of neuromuscular paralysis (e.g., neostigmine, prostigmine), inhibit acetylcholinesterase and therefore impair the hydrolysis of acetylcholine. The increased accumulation of undegraded acetylcholine can effectively compete with NDMRs and thereby displace the latter from the receptor (i.e., law of mass action) and antagonize the effects of NDMRs.
The neuromuscular junction is specialized on both the nerve side and on the muscle side to transmit and receive chemical messages. Each motor neuron runs without interruption from the ventral horn of the spinal cord or medulla to the neuromuscular junction as a large, myelinated axon ( Fig. 12.1 A ). As the motor neuron approaches the muscle, the neuron repeatedly branches to contact many muscle cells and gather them into a functional group known as a motor unit (see Fig. 12.1 B ). The architecture of the nerve terminal is quite different from that of the rest of the axon. As the terminal reaches the muscle fiber, it loses its myelin, forms a spray of terminal branches against the muscle surface, and is covered by Schwann cells. This arrangement conforms to the architecture on the synaptic area of the muscle membrane (see Fig. 12.1 C ). The nerve is separated from the surface of the muscle by a gap of approximately 50 nm, called the junctional cleft or synaptic cleft . The nerve and muscle are held in tight alignment by protein filaments called basal lamina that span the cleft between the nerve and end plate. The muscle surface is heavily corrugated, with deep invaginations of the junctional cleft—the primary and secondary clefts—between the folds in the muscle membrane; thus, the end plate’s total surface area is very large. The depths of the folds also vary between muscle types and species. Human neuromuscular junctions, relative to muscle size, are smaller than those of the mouse, although the junctions are located on muscle fibers that are much larger. Human junctions have longer junctional foldings and deeper gutters. The sodium channels, which propagate the wave of depolarization, are located in the depths of the folds (see Fig. 12.1 D ). The shoulders of the folds are densely populated with AChRs, approximately 5 million of them in each junction. AChRs are sparse in the depths between the folds.
The trophic function of the nerve is vital for the development and maintenance of adequate neuromuscular function. Before birth, each muscle cell commonly has contacts with several nerves and has several neuromuscular junctions. At birth, all but one of the nerves retract, and a single end plate remains (see section on “Neuromuscular Junction at the Extremes of Age”). Once formed, the nerve-muscle contact, especially the end plate, is durable. Even if the original nerve dies, the one replacing it innervates exactly the same region of the muscle. The nerve endings on fast muscles are larger and more complicated than those on slow muscles. The reason for this is unclear. These differences in the nerve endings on muscle surfaces may play a role in the difference in response of fast- and slow-twitch muscle fibers to muscle relaxants.
Because all the muscle cells in a unit are excited by a single neuron, stimulation of the nerve electrically or by an action potential originating from the ventral horn or by any agonist, including depolarizing relaxants (e.g., succinylcholine), causes all muscle cells in the motor unit to contract synchronously. Synchronous contraction of the cells in a motor unit is called fasciculation and is often vigorous enough to be observed through the skin. Although most adult human muscles have only one neuromuscular junction per cell, an important exception is some of the cells in extraocular muscles. The extraocular muscles are tonic muscles, and, unlike other mammalian striated muscles, they are multiply innervated with several neuromuscular junctions strung along the surface of each muscle fiber. Quite in contrast to other muscles, even the adult ocular muscle contains mature and immature fetal receptors (see section on “Biology of Prejunctional and Postjunctional Nicotinic Acetylcholine Receptors”) segregated into distinct synapses on different fibers. The ocular muscles slowly contract and relax rather than quickly as do other striated muscles; they can maintain a steady contraction, or contracture, the strength of which is proportional to the stimulus received. Physiologically, this specialization apparently holds the eye steadily in position. Ocular muscles are important to an anesthesiologist because depolarizing muscle relaxants (e.g., succinylcholine) affect them differently than they do on most skeletal muscles. Instead of causing a brief contraction, followed by paralysis, the depolarizing drug causes a long-lasting contracture response that pulls the eye against the orbit and could contribute to an increase in intraocular fluid pressure. The clinical significance of the succinylcholine-induced increase in intraocular pressure has been questioned. Although many textbooks invoke the reported extrusion of intraocular contents with succinylcholine, the basis for this effect seems to be anecdotal. Clinical studies, however, have indicated that succinylcholine-induced contractions of the extraocular muscles can last as long as 1 to 2 minutes and isometric tensions larger than 12 g can develop for each extraocular muscle. Thus, succinylcholine probably should not be given to patients with open eye injuries.
The perijunctional zone is the area of muscle immediately beyond the junctional area and is critical to the function of the neuromuscular junction. The perijunctional zone contains a mixture of receptors, including a smaller density of AChRs and a high density of sodium channels (see Fig. 12.1 D ). The admixture enhances the capacity of the perijunctional zone to respond to the depolarization (i.e., end-plate potential) produced by the AChRs and to transduce it into the wave of depolarization that travels along the muscle to initiate muscle contraction. The density of sodium channels in the perijunctional area is richer than in more distal parts of the muscle membrane. The perijunctional zone is close enough to the nerve ending to be influenced by transmitter released from it. Moreover, special variants (i.e., isoforms) of receptors and sodium channels can appear in this area at different stages of life and in response to abnormal decreases in nerve activity (see section on “Biology of Prejunctional and Postjunctional Nicotinic Acetylcholine Receptors”). Congenital abnormalities in the AChRs or in the sodium and calcium channels (i.e., mutations) are also known. These variabilities seem to contribute to the differences in response to relaxants that are observed in patients with different pathologic conditions and ages.
The contents of a nerve ending are not homogeneous. As illustrated in Figs. 12.1 C and 12.2 , vesicles are congregated in the portion toward the junctional surface, whereas microtubules, mitochondria, and other support structures are located toward the opposite side. The vesicles containing transmitter are ordered in repeating clusters alongside small, thickened, electron-dense patches of membrane referred to as active zones or release sites . This thickened area is a cross section of a band running across the width of the synaptic surface of the nerve ending that is believed to be the structure to which vesicles attach (active zones) before they rupture into the junctional cleft (see section on “Process of Exocytosis”). High-resolution scanning electron micrographs reveal small protein particles arranged alongside the active zone between vesicles. These particles are believed to be special channels—voltage-gated calcium channels—that allow calcium to enter the nerve and cause the release of vesicles. The rapidity with which the neurotransmitter is released (200 μs) suggests that voltage-gated calcium channels are close to the release sites. Proteomic studies suggest that at least 26 genes encode presynaptic proteins, and mutations in 12 of them cause defects in presynaptic structure that can lead to decreased acetylcholine release and muscle weakness. These defects can be related to exocytosis, endocytosis, formation of active and periactive zones, vesicle transport, and neuropeptide modulation.
When observing the electrophysiologic activity of a skeletal muscle, small, spontaneous depolarizing potentials at neuromuscular junctions can be seen. These potentials have only one hundredth the amplitude of the evoked end-plate potential produced when the motor nerve is stimulated. Except for amplitude, these potentials resemble the end-plate potential in the time course and manner they are affected by drugs. These small-amplitude potentials are called miniature end-plate potentials (MEPPs). Statistical analysis led to the conclusion that they are unitary responses; that is, there is a minimum size for the MEPP, and the sizes of all MEPPs are equal to or multiples of this minimum size. Because MEPPs are too large to be produced by a single molecule of acetylcholine, it was deduced that they are produced by uniformly sized packages, or quanta, of transmitter released from the nerve (in the absence of stimulation). The stimulus-evoked end-plate potential is the additive depolarization produced by the synchronous discharge of quanta from several hundred vesicles. The action potential that is propagated to the nerve ending allows the entry of calcium into the nerve through voltage-gated calcium channels, which causes vesicles to migrate to the active zone, fuse with the neural membrane at release sites, and discharge their acetylcholine into the junctional cleft. Because the release sites are located immediately opposite the receptors on the postjunctional surface, little transmitter is wasted, and the response of the muscle is coupled directly with the signal from the nerve.
Alignment of the presynaptic receptor site is achieved by adhesion molecules or specific cell-surface proteins located on both sides of the synapse that grip each other across the synaptic cleft and hold together the prejunctional and postjunctional synaptic apparatuses. One such protein implicated in synapse adhesion is neurexin, which binds to neuroligins on the postsynaptic membrane. The amount of acetylcholine released by each nerve impulse is large, at least 200 quanta of approximately 5000 molecules each, and the number of AChRs activated by transmitter released by a nerve impulse is also large, approximately 500,000 molecules. The ions (mostly sodium and some calcium) that flow through the channels of activated (open) AChRs cause maximum depolarization of the end plate, which results in an end-plate potential that is greater than the threshold for stimulation of the muscle. This system is extremely vigorous. The signal is carried by more molecules of transmitter than are needed, and they evoke a response that is larger than needed. At the same time, only a small fraction of the available vesicles and receptors or channels are used to send each signal. Consequently, transmission has a substantial margin of safety, and, at the same time, the system has substantial capacity in reserve.
Formation of Neurotransmitter at Motor Nerve Endings
The axon of the motor nerve carries electrical signals from the spinal cord to muscles and has all the biochemical apparatus needed to transform the electrical signal into a chemical one. All the ion channels, enzymes, other proteins, macromolecules, and membrane components needed by the nerve ending to synthesize, store, and release acetylcholine and other trophic factors are made in the cell body and transmitted to the nerve ending by axonal transport (see Fig. 12.2 ). The simple molecules, choline and acetate, are obtained from the environment of the nerve ending, where choline is transported by a special system from extracellular fluid to the cytoplasm and acetate in the form of acetyl coenzyme A from mitochondria. The enzyme choline acetyltransferase brings about the reaction of choline and acetate to form acetylcholine. After synthesis, acetylcholine is stored in cytoplasm until it is transported and incorporated into vesicles, which are better positioned for release when an action potential reaches the nerve terminal.
Nerve Action Potential
During a nerve action potential, sodium from outside flows across the membrane, and the resulting depolarizing voltage opens the calcium channels, which allows entry of calcium ions into the nerve and causes acetylcholine to be released. A nerve action potential is the normal activator that releases the transmitter acetylcholine. The number of quanta released by a stimulated nerve is greatly influenced by the concentration of ionized calcium in extracellular fluid. If calcium is not present, then depolarization of the nerve, even by electrical stimulation, will not produce the release of transmitter. Doubling the extracellular calcium results in a 16-fold increase in the quantal content of an end-plate potential. The calcium current persists until the membrane potential is returned to normal by outward fluxes of potassium from inside the nerve cell. Along with calcium channels on the nerve terminal are potassium channels, including the voltage-gated and calcium-activated potassium channels, whose function is to limit entry of calcium into the nerve and therefore depolarization. The calcium current can be prolonged by potassium channel blockers (e.g., 4-aminopyridine, tetraethylammonium), which slow or prevent the efflux of potassium out of the nerve. The increase in quantal content produced in this way can reach astounding proportions. An effect of increasing calcium in the nerve ending is also clinically observed as the so-called posttetanic potentiation (PTP), which occurs after a nerve of a patient paralyzed with an NDMR is stimulated at high, tetanic frequencies. Calcium enters the nerve with every stimulus, but it accumulates during the tetanic period because it cannot be excreted as quickly as the nerve is stimulated. Because the nerve ending contains more than the normal amount of calcium for some time after the tetanus, a stimulus applied to the nerve during this time causes the release of more than the normal amount of acetylcholine. The abnormally large amount of acetylcholine antagonizes the relaxant (temporarily) and causes the characteristic increase in the size of the twitch (i.e., post tetanic facilitation).
Calcium enters the nerve through specialized proteins called calcium channels . Of the several types of calcium channels, two seem to be important for the release of transmitter: P channels and the slower L channels. P channels, probably the type responsible for the normal release of transmitter, are found only in nerve terminals. In motor nerve endings, the calcium channels are located immediately adjacent to the active zones (see Fig 12.2 ). They are voltage-dependent and are opened and closed by changes in membrane voltage caused by the nerve action potential. In addition to calcium channels, several forms of potassium channels are present in the nerve terminal, including voltage-gated and calcium-activated potassium channels. Potassium channels limit the duration of nerve terminal depolarization and hence the entry of calcium and the release of transmitter. Alterations in entry of calcium into the nerve ending can also alter the release of transmitter. The Eaton-Lambert myasthenic syndrome, which should not be confused with myasthenia gravis, is an acquired autoimmune disease in which antibodies are directed against voltage-gated calcium channels at nerve endings. In this syndrome, decreased function of the calcium channel causes decreased release of transmitter, which results in inadequate depolarization and muscle weakness. Patients with Eaton-Lambert myasthenic syndrome exhibit increased sensitivity to depolarizing and nondepolarizing relaxants.
Higher-than-normal concentrations of bivalent inorganic cations (e.g., magnesium, cadmium, manganese) can also block the entry of calcium through P channels and profoundly impair neuromuscular transmission. This mechanism is behind the typical muscle weakness and potentiation of the effect of muscle relaxants in a mother and fetus when magnesium sulfate is administered to treat preeclampsia. P channels, however, are not affected by calcium entry-blocking drugs such as verapamil, diltiazem, and nifedipine. These drugs have profound effects on the slower L channels present in the cardiovascular system. As a result, the L-type calcium channel blockers have no significant effect at therapeutic doses on the normal release of acetylcholine or on the strength of normal neuromuscular transmission. However, calcium entry-blocking drugs may increase the block in neuromuscular transmission induced by NDMRs. The effect is small, and not all investigators have been able to observe it. The explanation may lie in the fact that nerve endings also contain L-type calcium channels. The effects of calcium channels on depolarizing relaxants, if any, are unknown.
Synaptic Vesicles and Recycling
Two pools of vesicles seem to release acetylcholine, a readily releasable pool and a reserve pool, sometimes called VP1 and VP2, respectively. Electron microscopic studies have demonstrated that the majority of synaptic vesicles (VP1) are sequestered in the reserve pool and tethered to the cytoskeleton in a filamentous network made up of primarily actin, synapsin (an actin-binding protein), synaptotagmin, and spectrin. Vesicles in VP2 are a bit smaller and limited to an area very close to the nerve membrane, where they are bound to the active zones. These vesicles are the ones that ordinarily release transmitter. Release occurs when calcium ions enter the nerve through the P channels lined up on the sides of the active zones by soluble N -ethylmaleimide-sensitive attachment protein receptor (SNARE) proteins. The SNARE proteins are involved in fusion, docking, and release of acetylcholine at the active zone. Calcium needs to move only a very short distance (i.e., a few atomic radii) to encounter a vesicle and activate the proteins in the vesicle wall involved in a process known as docking (see section on “Process of Exocytosis”). The activated proteins seem to react with the nerve membrane to form a pore through which the vesicle discharges its acetylcholine into the junctional cleft. Studies using fluorescent proteins have visualized how synaptic vesicles fuse with release sites and release their contents, which are then retrieved. Some vesicles stay open briefly before retrieval and do not completely collapse into the surface membrane (“kiss and run”). Others stay open longer and probably do not completely collapse (“compensatory”). Still others completely collapse and are not retrieved until another stimulus is delivered (“stranded”).
The larger reserve (VP1) vesicles, from their position deep from the nerve ending and firmly tethered to the cytoskeleton by many proteins, including actin, synapsin (an actin-binding protein), synaptotagmin, and spectrin, may be moved to the readily releasable store to replace worn-out vesicles or to participate in transmission when the nerve is called on to work especially hard (e.g., when it is stimulated at very high frequencies or for a very long time). Under such strenuous circumstances, calcium may penetrate more deeply than normal into the nerve or may enter through L channels to activate calcium-dependent enzymes that break the synapsin links holding the vesicles to the cytoskeleton, thereby allowing the vesicles to be moved to the release sites. Repeated stimulation requires the nerve ending to replenish its store of vesicles filled with transmitter, a process known as mobilization . The term is commonly applied to the aggregate of all steps involved in maintaining the nerve ending’s capacity to release transmitter—everything from the acquisition of choline and the synthesis of acetate to the movement of filled vesicles to release sites. Uptake of choline and the activity of choline acetyltransferase, the enzyme that synthesizes acetylcholine, are probably the rate-limiting steps.
Process of Exocytosis
The readily releasable pool of synaptic vesicles constitutes the vesicles directly available for release. During an action potential and calcium influx, neurotransmitter is released. Studies have shed some light on the inner workings by which the vesicle releases its contents. The whole process is called exocytosis . The SNARE proteins include ( Fig. 12.3 A ) the synaptic-vesicle protein, synaptobrevin; the plasmalemma-associated protein, syntaxin; and the synaptosome-associated protein of 25-kd (SNAP-25). The current model of protein-mediated membrane fusion in exocytosis is as follows. When there is an action potential and calcium ions enter, synapsin becomes phosphorylated, which frees the vesicle from its attachment to the cytoskeleton. Syntaxin and SNAP-25 are complexes attached to the plasma membrane. After the initial contact, the synaptobrevin on the vesicle forms a ternary complex with syntaxin and SNAP-25. Synaptotagmin is the protein on the vesicular membrane that acts as a calcium sensor, localizes the synaptic vesicles to synaptic zones rich in calcium channels, and stabilizes the vesicles in the docked state. Assembly of the ternary complex forces the vesicle to move close to the underlying nerve terminal membrane (i.e., the active zone), and the vesicle is then ready for release (see Fig. 12.3 B ). The close proximity of release sites, calcium channels, and synaptic vesicles and the use of the calcium sensor lead to a burst of release of new transmitter synchronous with the stimulus (see Fig. 12.3 C ). The vesicle can release part or all of its contents, some of which can be recycled to form new vesicles as previously described (“kiss and run,” “compensatory,” “stranded”).
Botulinum neurotoxin selectively digests one or all these SNARE proteins and blocks exocytosis of the vesicles, which ultimately results in muscle weakness or more profound muscle paralysis. This toxin may produce a partial or complete chemical denervation. Botulinum toxin is therapeutically used to treat spasticity or spasm in several neurologic and surgical diseases, to prevent hyperhidrosis in patients with excessive sweating, and cosmetically to correct wrinkles. Botulinum toxin consists of two protein segments known as heavy and light chains (see Fig. 12.3 D and E ). The heavy chain interacts with lipid molecules called polysialogangliosides in the cell membrane and synaptotagmin on the vesicle to enter the vesicle. Once in the vesicle, the light chain inactivates neuromuscular transmission by breakdown and thereby inhibits the function of SNARE proteins (see Fig. 12.3 F ). Some reports indicate an increased incidence of clostridial infections in both Canada and the United States, with Clostridium botulinum infection being particularly common after traumatic injuries, in drug abusers, and after musculoskeletal allografts. Thus, systemic paralysis can occur after clostridial infection. Local injection for therapeutic purposes will usually result in localized paresis, although systemic effects have been reported.
The acetylcholine released from the nerve diffuses across the junctional cleft and reacts with nicotinic AChRs in the end plate to initiate muscle contraction. Transmitter molecules that do not immediately react with a nicotinic AChR or those released after binding to the receptor are almost instantly destroyed by acetylcholinesterase in the junctional cleft. Acetylcholinesterase at the junction is the asymmetric or A12 form protein made in the muscle under the end plate. Acetylcholinesterase (enzyme classification 188.8.131.52) is type B carboxylesterase enzyme. A smaller concentration of the enzyme is found in the extrajunctional area. The enzyme is secreted from the muscle but remains attached to it by thin stalks of collagen fastened to the basement membrane. Most of the molecules of acetylcholine released from the nerve initially pass between the enzymes to reach the postjunctional receptors; however, as they are released from the receptors, they invariably encounter acetylcholinesterase and are destroyed. Under normal circumstances, a molecule of acetylcholine reacts with only one receptor before it is hydrolyzed. Acetylcholine is a potent messenger, but its actions are very short lived because it is destroyed in less than 1 ms after it is released.
Some congenital and acquired diseases are caused by altered activity of acetylcholinesterase. The congenital absence of the secreted enzyme (in knock-out mice) leads to impaired maintenance of the motor neuronal system and organization of nerve terminal branches. Many syndromes caused by congenital abnormalities in cholinesterase function have been described and result in neuromuscular disorders whose symptoms and signs usually resemble those of myasthenia gravis or myasthenic syndromes. Denervation decreases acetylcholinesterase at the junctional and extrajunctional areas. Other acquired diseases involving cholinesterases are related to chronic inhibition of acetylcholinesterase by organophosphate pesticides or nerve gas (e.g., sarin) or to chronic pyridostigmine therapy given as prophylaxis against nerve gas poisoning. Symptoms ranging from chronic fatigue to muscle weakness have been attributed to chronic cholinesterase inhibition, thus underscoring the importance of acetylcholinesterase in normal and abnormal neuromuscular function. A rodent study confirms that the muscle weakness associated with chronic pyridostigmine therapy is related to both AChRs downregulation and to receptor-independent factors.
Postjunctional Acetylcholine Receptors
The similarity of AChRs among many species and the abundance of AChRs from Torpedo electric fish have greatly facilitated research in this area. The availability of messenger RNA from humans and other species and DNA has allowed the study of the receptor in artificial systems such as oocytes from frogs and in mammalian cells that do not express the receptor, such as COS or fibroblast cells. Receptors can also be mutated by molecular techniques to simulate pathologic states; the receptor function in these artificial systems can then be studied. By using these and related techniques, much has been learned about the synthesis, composition, and biologic function and mechanisms that underlie the physiologic and pharmacologic responses in AChRs. Three isoforms of postjunctional nicotinic AChRs exist: a junctional or mature receptor, an extrajunctional or immature (fetal) receptor, and the more recently described neuronal α7 nicotinic receptor (see section on “Biology of Prejunctional and Postjunctional Nicotinic Acetylcholine Receptors”). The differences between receptor subtypes, however, can be neglected in a general discussion of the role of receptors in neuromuscular transmission.
AChRs are synthesized in muscle cells and are anchored to the end-plate membrane by a special 43-kd protein known as rapsyn . This cytoplasmic protein is associated with the AChR in a 1:1 ratio. The receptors, formed of five subunit proteins, are arranged like the staves of a barrel into a cylindrical receptor with a central pore for ion channeling (the key features are illustrated in Fig. 12.4 ). The receptor protein has a molecular mass of approximately 250,000 daltons. The mature receptor consists of α1-, β1-, δ-, and ε-subunits, and the fetal (immature, extrajunctional) receptor consists of α1-, β1-, δ-, and γ-subunits; there are two subunits of α and one each of the others. The neuronal α7 AChR consists of five α7-subunits. Each of all receptor subunits consists of approximately 400 to 500 amino acids. The receptor-protein complex passes entirely through the membrane and protrudes beyond the extracellular surface of the membrane and into the cytoplasm. The binding site for acetylcholine is on each of the α1- or α7- subunits, is located on the extracellular component of the α-subunit protein, and these are the sites of competition between receptor agonists and antagonists. Agonists and antagonists are attracted to the binding site, and either may occupy the site, which is located near cysteine residues (unique to the α-chain) at amino acid positions 192 to 193 of the α-subunit. Radiolabeled α-bungarotoxin from the cobra, used to quantitate or fluorescent stain the receptor, binds to heptapeptide region 185 to 199 of the α-subunit. Motor neuron-derived neuregulin-1β (NRβ-1), originally described as AChR-inducing activity (ARIA), induces AChR gene transcription in subsynaptic myonuclei by activating ErbB receptors.
Synthesis and Stabilization of Postjunctional Receptors
Muscle tissue is formed from the mesoderm and initially appears as myoblasts. Myoblasts fuse to produce myotubes, which therefore have multiple nuclei. As the myotubes mature, the sarcomere, which is the contractile element of the muscle consisting of actin and myosin, develops. The protein β-integrin seems to be essential for myoblast fusion and sarcomere assembly. Shortly afterward, motor nerve axons grow into the developing muscle, and these axons bring in nerve-derived signals (i.e., growth factors), including agrin and neuregulins (NRβ-1 and NRβ-2), which are key to the maturation of myotubes to muscle. Agrin is a protein from the nerve that stimulates postsynaptic differentiation by activating muscle-specific tyrosine kinase (MuSK), a tyrosine kinase expressed selectively in muscle. With signaling from agrin, the AChRs, which have been scattered throughout the muscle membrane, cluster at the area immediately beneath the nerve. Agrin, together with neuregulins and other growth factors, induce the clustering of other critical muscle-derived proteins, including MuSK, rapsyn, and ErbB proteins, all of which are necessary for maturation and stabilization of AChRs at the junction. In addition to the effects on postsynaptic differentiation, agrin and MuSK display effects on presynaptic differentiation as well. Agrin and MuSK induce retrograde signals that instruct axons to undergo neuron outgrowth and terminal differentiation. Current understanding of presynaptic development of the neuromuscular junction, however, is significantly less advanced than the understanding of postsynaptic development. Just before and shortly after birth, the immature, γ-subunit-containing AChRs are replaced by the mature, ε-subunit-containing receptors. Although the mechanism of this change is unclear, a neuregulin, NRβ-1, also called ARIA , that binds to one of the ErbB receptors seems to play a role.
Basic Electrophysiology of Neurotransmission
Fig. 12.5 illustrates the results of the classic depolarizing action of acetylcholine on end-plate receptors. Normally, the pore of the channel is closed by approximation of the cylinders (i.e., subunits). When an agonist occupies both α-subunit sites, the protein molecule undergoes a conformational change with a twisting movement along the central axis of the receptor that results in the opening of the central channel through which ions can flow along a concentration gradient. When the central channel is open, sodium and calcium flow from the outside of the cell to the inside and potassium flows from the inside to the outside. The channel in the tube is large enough to accommodate many cations and electrically neutral molecules, but it excludes anions (e.g., chloride). The current transported by the ions depolarizes the adjacent membrane. The net current is depolarizing and creates the end-plate potential that stimulates the muscle to contract. In this instance, downward-going (i.e., depolarizing) current can be recorded by the patch-clamp electrophysiologic technique previously described (see Fig. 12.4 ).
The pulse stops when the channel closes by a reversed mechanical conformation (see earlier discussion), which is typically initiated when one or both agonist molecules detach from the receptor. In the activated, open state, the current that passes through each open channel is minuscule, only a few picoamperes (approximately 10 4 ions/ms). However, each burst of acetylcholine from the nerve normally opens approximately 500,000 channels simultaneously, and the total current is more than adequate to produce depolarization of the end plate and contraction of muscle. Opening of a channel causes conversion of chemical signals from a nerve to the flow of current on the muscle disease to cause end-plate potentials, thereby leading to muscle contraction. The end-plate potential has been viewed as a graded event that may be reduced in magnitude or extended in time by drugs, but, in reality, the end-plate potential is the summation of many all-or-nothing events simultaneously occurring at myriad ion channels. It is these tiny events that are affected by drugs.
Receptors that do not have two molecules of agonist (e.g., acetylcholine) bound remain closed. Both α-subunits must be simultaneously occupied by agonist; if only one of them is occupied, then the channel remains closed (see Fig. 12.5 ). This is the basis for preventing depolarization by antagonists. NDMRs act by binding to either or both α-subunits and thus preventing acetylcholine from binding and opening the channel. This interaction between agonists and antagonists is competitive, and the outcome—transmission or block—depends on the relative concentrations and binding characteristics of the drugs involved (see section on “Drug Effects on Postjunctional Receptors”).
Individual channels are also capable of a wide variety of conformational states. They may stay open or remain closed and thereby affect total current flow across the membrane, but they can do more. They may open for a longer or shorter time than normal, open or close more gradually than usual, open briefly and repeatedly (i.e., flickering), or pass fewer or more ions per opening than they usually do. Their function is also influenced by drugs, changes in fluidity of the membrane, temperature, electrolyte balance in the milieu, and other physical and chemical factors. Receptor channels are dynamic structures that are capable of a wide variety of interactions with drugs and of entering a wide variety of current-passing states. All these influences on channel activity are ultimately reflected in the strength or weakness of neuromuscular transmission and contraction of a muscle.
Drug Effects on Postjunctional Receptors
Classic Actions of Nondepolarizing Muscle Relaxants
Neurotransmission occurs when acetylcholine released by the nerve action potential binds to nicotinic AChRs. All NDMRs impair or block neurotransmission by competitively preventing the binding of acetylcholine to the muscle AChR. The final outcome—block or transmission—depends on the relative concentrations of the chemicals and their comparative affinities for the receptor. Fig. 12.5 shows a system exposed to acetylcholine and the nondepolarizing neuromuscular blocking compound. One receptor has attracted two acetylcholine molecules and has opened its channel, where current will flow to depolarize that segment of membrane. Another has attracted one molecule of NDMR; its channel will not open, and no current will flow, even if one acetylcholine molecule binds to the other site. The third receptor has acetylcholine on one α-subunit and nothing on the other. What will happen depends on which of the molecules binds. If acetylcholine binds, then the channel will open and the membrane will be depolarized; if a NDMR binds, then the channel will remain closed and the membrane will not be depolarized. At other times, one or two molecules of NDMR may attach to the receptor, in which case the receptor is not available to agonists; no current flow is recorded. In the presence of moderate concentrations of NDMR, the amount of current flowing through the entire end plate at any instant is reduced from normal, which results in a smaller end-plate potential and, if carried far enough, a block in neurotransmission or the production of neuromuscular paralysis.
Normally, acetylcholinesterase destroys acetylcholine and removes it from competition for a receptor; therefore, an NDMR has a better chance of inhibiting transmission. If, however, an inhibitor of acetylcholinesterase such as neostigmine is added, then the cholinesterase cannot destroy acetylcholine. The concentration of agonist in the cleft remains high, and this high concentration shifts the competition between acetylcholine and a NDMR in favor of the former, thereby improving the chance of two acetylcholine molecules binding to a receptor even though NDMR is still in the environment. This mechanism causes the cholinesterase inhibitors to overcome the neuromuscular paralysis produced by NDMRs. The channel opens only when acetylcholine attaches to both recognition sites. A single molecule of antagonist, however, is adequate to prevent depolarization of that receptor. This modifies the competition by strongly biasing it in favor of the antagonist (relaxant). Mathematically, if the concentration of a NDMR is doubled, then the concentration of acetylcholine must be increased fourfold if acetylcholine is to remain competitive. Paralysis produced by high concentrations of muscle relaxants (antagonist) is more difficult to reverse with cholinesterase inhibitors than that produced by low concentrations. After large doses of NDMRs, cholinesterase inhibitors may be ineffective until the concentration of relaxant in the perijunctional area decreases to a lower level by the redistribution or elimination of the drug. This is the molecular basis for the recommendation to not administer anticholinesterases too early (i.e., at a deep block). In contrast to reversal with a cholinesterase inhibitor, cyclodextrin encapsulation takes place at any concentration of a steroid-based compound, such as vecuronium or rocuronium, and reversal by this novel mechanism can therefore be achieved at any level of neuromuscular block provided the amount of cyclodextrin (sugammadex) is large enough.
Classic Actions of Depolarizing Muscle Relaxants
Depolarizing relaxants (e.g., succinylcholine, decamethonium) initially simulate the effect of acetylcholine and can be considered agonists, despite the fact that they block neurotransmission after the initial stimulation. Structurally, succinylcholine is very similar to the natural ligand acetylcholine and consists of two molecules of acetylcholine bound together through their backbones. It is thus not surprising that succinylcholine can mimic the effects of acetylcholine.
Succinylcholine or decamethonium can bind to the receptor, open the channel, pass current, and depolarize the end plate. These agonists, similar to acetylcholine, attach only briefly; each opening of a channel is very short in duration―1 ms or less. The response to acetylcholine, however, is over in milliseconds because of its rapid degradation by acetylcholinesterase, and the end plate resets to its resting state long before another nerve impulse arrives. In contrast, the depolarizing relaxants characteristically have a biphasic action on muscle—an initial contraction, followed by relaxation lasting from minutes to hours. Because they are not susceptible to hydrolysis by acetylcholinesterase, the depolarizing relaxants are not eliminated from the junctional cleft until after they are eliminated from plasma. The time required to clear the drug from the body is the principal determinant of how long the drug effect lasts. Whole-body clearance of the relaxant is very slow in comparison to acetylcholine, particularly when plasma (pseudo) cholinesterase is abnormal. Because the relaxant molecules are not quickly cleared from the cleft, compared with acetylcholine, they repeatedly react with receptors even with normal levels of plasma cholinesterase, almost immediately attaching to a receptor after separating from another, thereby repeatedly depolarizing the end plate and opening channels. For details on the effect of succinylcholine in patients with cholinesterase deficiency, also see Chapter 27 .
The quick shift from excitation of muscle contraction to block of transmission by depolarizing relaxants occurs because the end plate is continuously depolarized. This comes about as a result of the juxtaposition of the edge of the end plate with a different kind of ion channel, the sodium channel that does not respond to chemicals but opens when exposed to a transmembrane voltage change. Just as the AChR, the sodium channel is also a cylindrical transmembrane protein through which sodium ions can flow. Two parts of its structure act as gates that allow or stop the flow of sodium ions. Both gates must be open if sodium is to flow through the channel; closing of either cuts off the flow. Because these two gates act sequentially, a sodium channel has three functional conformational states and can progressively move from one state to another ( Fig. 12.6 ). This whole process is short lived when depolarization occurs with acetylcholine. The initial response of a depolarizing muscle relaxant resembles that of acetylcholine, but because the muscle relaxant is not rapidly hydrolyzed, depolarization of the end plate is not brief.
Depolarization of the end plate by the depolarizing relaxant initially causes the voltage gate in adjacent sodium channels to open, thereby producing a wave of depolarization that sweeps along the muscle and generates a muscle contraction. Shortly after the voltage-dependent gate opens, the time-dependent inactivation gate closes. Because the relaxant is not removed from the cleft, the end plate continues to be depolarized. Because the sodium channels immediately adjacent to the end plate are influenced by depolarization of the end plate, their voltage-dependent gates stay open and their inactivation gates stay closed. Since sodium cannot flow through a channel that has a closed inactivation gate, the perijunctional muscle membrane does not depolarize. When the flow of ions through sodium channels in the perijunctional zone stops because of a closure of the inactivation gates, the channels downstream (beyond the perijunctional zone) are freed of depolarizing influence. In effect, the perijunctional zone becomes a buffer that shields the rest of the muscle from events at the end plate. Consequently, the muscle membrane is separated into three zones: (1) the end plate, which is depolarized by succinylcholine; (2) the perijunctional muscle membrane, in which the sodium channels are frozen in an inactivated state; and (3) the rest of the muscle membrane, in which the sodium channels are in the resting state. Because a burst of acetylcholine from the nerve cannot overcome the inactivated sodium channels in the perijunctional zone, neuromuscular transmission is blocked. This phenomenon is also called accommodation . During accommodation, when the synapse is inexcitable through the nerve (transmitter), direct electrical stimulation of muscle causes muscle contraction because the sodium channels beyond the junctional area are in the resting excitable state.
The extraocular muscles are tonic muscles, which are multiply innervated and chemically excitable along most of their surfaces. Despite their innervated state, the ocular muscles express both mature and immature receptors. Accommodation does not occur, and these muscles can undergo a sustained contracture in the presence of succinylcholine. The tension that develops forces the eye against the orbit and accounts for part of the increase in intraocular pressure produced by depolarizing relaxants. The extraocular muscles contain a special type of receptor that does not become desensitized (see later discussion) during the continued presence of acetylcholine or other agonists. A single dose of succinylcholine can cause contracture lasting several minutes. Whether it is the immature γ-subunit AChR or the α7 AChR subunit that plays a role in this resistance to desensitization in the ocular muscles is unknown.
Nonclassic and Noncompetitive Actions of Neuromuscular Drugs
Several drugs can interfere with the receptor, directly or through its lipid environment, and can change transmission ( Box 12.1 ). These drugs react with the neuromuscular receptor to change its function and impair transmission, but they do not act through the acetylcholine binding site. These reactions cause drug-induced changes in the dynamics of the receptor; instead of sharply opening and closing, the modified channels are sluggish. They open more slowly and stay open longer, or they close slowly and in several steps, or both. These effects on channels cause corresponding changes in the flow of ions and distortions of the end-plate potential. The clinical effect depends on the molecular events. For example, procaine, ketamine, inhaled anesthetics, or other drugs that dissolve in the membrane lipid may change the opening or closing characteristics of the channel. If the channel is prevented from opening, then transmission is weakened. If, however, the channel is prevented from or slowed in closing, then transmission may be enhanced. These drugs do not fit the classic model, and the impaired neuromuscular function is not antagonized by increasing perijunctional acetylcholine concentrations with cholinesterase inhibitors. Such drugs can be involved in two clinically important reactions: receptor desensitization and channel block. The former occurs in the receptor molecule, whereas the latter occurs in the ion channel.