Medical Physiology A Cellular and Molecular Approach, Updated 2nd Ed.


Edward G. Moczydlowski

The ionic gradients that cells maintain across their membranes provide a form of stored electrochemical energy that cells can use for electrical signaling. The combination of a resting membrane potential of –60 to –90 mV and a diverse array of voltage-gated ion channels allows excitable cells to generate action potentials that propagate over long distances along the surface membrane of a single nerve axon or muscle fiber. However, another class of mechanisms is necessary to transmit such electrical information from cell to cell throughout the myriad neuronal networks that link the brain with sensory and effector organs. Electrical signals must pass across the specialized gap region between two apposing cell membranes that is called a synapse. The process underlying this cell-to-cell transfer of electrical signals is termed synaptic transmission. Communication between cells at a synapse can be either electrical or chemical. Electrical synapses provide direct electrical continuity between cells by means of gap junctions, whereas chemical synapses link two cells together by a chemical neurotransmitter that is released from one cell and diffuses to another.

In this chapter we discuss the general properties of synaptic transmission and then focus mainly on synaptic transmission between a motor neuron and a skeletal muscle fiber. This interface between the motor neuron and the muscle cell is called the neuromuscular junction. In Chapter 13, the focus is on synaptic transmission between neurons in the central nervous system (CNS).


Electrical continuity between cells is established by electrical or chemical synapses

Once the concept of bioelectricity had taken hold among physiologists of the 19th century, it became clear that the question of how electrical signals flow between cells posed a fundamental biological problem. Imagine that two cells lie side by side without any specialized device for communication between them. Furthermore, imagine that a flat, 20-μm2 membrane area of the first or presynaptic cell is separated—by 15 nm—from a similar area of the second or postsynaptic cell. In his classic book on electrophysiology, Katz calculated that a voltage signal at the presynaptic membrane would suffer 10,000-fold attenuation in the postsynaptic membrane. A similar calculation based on the geometry and cable properties of a typical nerve-muscle synapse suggests that an action potential arriving at a nerve terminal could depolarize the postsynaptic membrane by only 1 μV after crossing the synaptic gap—an attenuation of 105. Clearly, the evolution of complex multicellular organisms required the development of special synaptic mechanisms for electrical signaling to serve as a workable means of intercellular communication.

Two competing hypotheses emerged in the 19th century to explain how closely apposed cells could communicate electrically. One school of thought proposed that cells are directly linked by microscopic connecting bridges that enable electrical signals to flow directly. Other pioneering physiologists used pharmacological observations to infer that cell-to-cell transmission was chemical in nature. Ultimate resolution of this question awaited both the development of electron microscopic techniques, which permitted visualization of the intimate contact region between cells, and further studies in neurochemistry, which identified the small, organic molecules that are responsible for neurotransmission. By 1960, accumulated evidence led to the general recognition that cells use both direct electrical and indirect chemical modes of transmission to communicate with one another.

The essential structural element of intercellular communication, the synapse, is a specialized point of contact between the membranes of two different but connected, cells. Electrical and chemical synapses have unique morphological features, distinguishable by electron microscopy. One major distinction is the distance of separation between the two apposing cell membranes. At electrical synapses, the adjacent cell membranes are separated by ~3 nm and appear to be nearly sealed together by a plate-like structure that is a fraction of a micrometer in diameter. Freeze-fracture images of the intramembrane plane in this region reveal a cluster of closely packed intramembranous particles that represent a gap junction. As described in Chapter 6, a gap junction corresponds to planar arrays of connexons, each of which is made up of six connexin monomers (see Fig. 6-18). The multiple connexons from apposing cells physically connect the two cells together through multiple aqueous channels.

In contrast to the gap junction, the apposing cell membranes of the chemical synapse are separated by a much larger distance of ~30 nm at a neuronal chemical synapse and up to 50 nm at the vertebrate nerve-muscle synapse. An additional characteristic of a chemical synapse is the presence of numerous synaptic vesicles on the side of the synapse that initiates the signal transmission, termed the presynaptic side. These vesicles are sealed, spherical membrane-bound structures that range in diameter from 40 to 200 nm and contain a high concentration of chemical neurotransmitter.

The contrasting morphological characteristics of electrical and chemical synapses underline the contrasting mechanisms by which they function (Table 8-1). Electrical synapses pass voltage changes directly from one cell to another across the low-resistance continuity that is provided by the connexon channels. On the other hand, chemical synapses link two cells by the diffusion of a chemical transmitter across the large gap separating them. Key steps in chemical neurotransmission include release of transmitter from synaptic vesicles into the synaptic space, diffusion of transmitter across the cleft of the synapse, and activation of the postsynaptic cell by binding of transmitter to a specific receptor protein on the postsynaptic cell membrane.

Table 8-1 Summary of Properties of Electrical and Chemical Synapses


Direct evidence for the existence of chemical synapses actually predated the experimental confirmation of electrical synapses. The foundations of synaptic physiology can be traced to early studies of the autonomic nervous system. Early in the 1900s, researchers noted that adrenal gland extracts, which contain epinephrine, elicited physiological effects (e.g., an increase in heart rate) that were similar to those elicited by stimulation of sympathetic nerve fibers. In 1904, Elliot proposed that sympathetic nerves might release a substance analogous to epinephrine that would function in chemical transmission between a nerve and its target organ. Similar studies suggested that the vagus nerve, which is parasympathetic, produces a related substance that is responsible for depression of the heartbeat.

A classic experiment performed by Loewi in 1921 is widely cited as the first definitive evidence for chemical neurotransmission. Loewi used an ingenious bioassay to test for the release of a chemical substance by the vagus nerve. He repeatedly stimulated the vagus nerve of a cannulated frog heart and observed a slowing of the heartbeat. At the same time, he collected the artificial saline that emerged from the ventricle of this overstimulated heart. When he later applied the collected fluid from the vagus-stimulated heart to a different heart, he observed that this perfusate slowed the second heart in a manner that was identical to direct vagal stimulation. He also later identified the active compound in the perfusion fluid, originally called Vagusstoff, as acetylcholine (ACh).

Efforts by Dale and coworkers to understand the basis of neurotransmission between motor nerves and skeletal muscle culminated in the identification of ACh as the endogenous excitatory neurotransmitter. Thus, the inherent complexity of chemical synaptic transmission was evident from these earliest investigations, which indicated that the same neurotransmitter (ACh) could have an inhibitory action at one synapse (vagus nerve–heart) and an excitatory action at another synapse (motor nerve–skeletal muscle). For their work on nerve transmission across chemical synapses, Otto Loewi and Sir Henry Dale received the Nobel Prize in Medicine in 1936. (See Note: Sir Henry H. Dale and Otto Loewi)

Electrical synapses directly link the cytoplasm of adjacent cells

Whereas overwhelming support for chemical synaptic transmission accumulated in the first half of the 20th century, the first direct evidence for electrical transmission came much later from electrophysiological recordings of a crayfish nerve preparation. In 1959, Furshpan and Potter used two pairs of stimulating and recording electrodes to show that depolarization of a presynaptic nerve fiber (the crayfish abdominal nerve) resulted in excitation of a postsynaptic nerve cell (the motor nerve to the tail muscle) with virtually no time delay. In contrast, chemical synapses exhibit a characteristic delay of ~1 ms in the postsynaptic voltage signal after excitation of the presynaptic cell. The demonstration of an electrical synapse between two nerve membranes highlighted an important functional difference between electrical and chemical synapses—immediate signal propagation (electrical) versus briefly delayed communication (chemical) through the junction.

An electrical synapse is a true structural connection formed by connexon channels of gap junctions that link the cytoplasm of two cells (Fig. 8-1). These channels thus provide a low-resistance path for electrotonic current flow and allow voltage signals to flow with little attenuation and no delay between two or more coupled cells. Many types of gap junctions pass electrical current with equal efficiency in both directions (reciprocal synapses). In other words, the current passing through the gap junction is ohmic; it varies linearly with the transjunctional voltage (i.e., the Vm difference between the two cells). However, the crayfish synapse described by Furshpan and Potter allows depolarizing current to pass readily only in one direction, from the presynaptic cell to the postsynaptic cell. Such electrical synapses are called rectifying synapses to indicate that the underlying junctional conductance is voltage dependent. Studies of cloned and expressed connexins have shown that the voltage dependence of electrical synapses arises from unique gating properties of different connexin isoforms. Some isoforms are voltage dependent; others are voltage independent. Intrinsic rectification can also be altered by the formation of a gap junction that is composed of two hemichannels, each made up of a different connexin monomer. Such hybrid connexins are called heterotypic channels.


Figure 8-1 An electrical synapse. An electrical synapse consists of one or more gap junction channels permeable to small ions and molecules.

Chemical synapses use neurotransmitters to provide electrical continuity between adjacent cells

By their very nature, chemical synapses are inherently rectifying or polarized. They propagate current in one direction: from the presynaptic cell that releases the transmitter to the postsynaptic cell that contains the receptors that recognize and bind the transmitter. However, the essentially vectorial nature of chemical synaptic transmission belies the possibility that the postsynaptic cell can influence synapse formation or transmitter release by the presynaptic cell. Studies of synapse development and regulation have shown that postsynaptic cells also play an active role in synapse formation. In the CNS, postsynaptic cells may also produce retrograde signaling molecules, such as nitric oxide, that diffuse back into the presynaptic terminal and modulate the strength of the synaptic connection (see Chapter 13). Furthermore, the presynaptic membrane at some synapses contains receptors that may either inhibit or facilitate the release of transmitter by biochemical mechanisms. Thus, chemical synapses should be considered a unidirectional pathway for signal propagation that can be modulated by bidirectional chemical communication between two interacting cells.

The process of chemical transmission can be summarized by the following series of steps (Fig. 8-2):

Step 1: Neurotransmitter molecules are packaged into synaptic vesicles. Specific transport proteins in the vesicle membrane use the energy of an H+ gradient to energize uptake of the neurotransmitter in the vesicle.

Step 2: An action potential, which involves voltage-gated Na+ and K+ channels (see Chapter 7), arrives at the presynaptic nerve terminal.

Step 3: Depolarization opens voltage-gated Ca2+ channels, which allows Ca2+ to enter the presynaptic terminal.

Step 4: The increase in intracellular Ca2+ concentration ([Ca2+]i) triggers the fusion of synaptic vesicles with the presynaptic membrane. As a result, packets (quanta) of transmitter molecules are released into the synaptic cleft.

Step 5: The transmitter molecules diffuse across the synaptic cleft and bind to specific receptors on the membrane of the postsynaptic cell.

Step 6: The binding of transmitter activates the receptor, which in turn activates the postsynaptic cell.

Step 7: The process is terminated by (1) enzymatic destruction of the transmitter (e.g., hydrolysis of ACh by acetylcholinesterase), (2) uptake of transmitter into the presynaptic nerve terminal or into other cells by Na+-dependent transport systems, or (3) diffusion of the transmitter molecules away from the synapse.


Figure 8-2 A chemical synapse. Synaptic transmission at a chemical synapse can be thought of as occurring in seven steps.

The molecular nature of chemical synapses permits enormous diversity in functional specialization and regulation. Functional diversity occurs at the level of the transmitter substance, receptor protein, postsynaptic response, and subsequent electrical and biochemical processes. Many different small molecules are known—or proposed—to serve as neurotransmitters (see Chapter 13). These molecules include both small organic molecules, such as norepinephrine, ACh, serotonin (5-hydroxytryptamine [5-HT]), glutamate, γ-aminobutyric acid (GABA), and glycine, and peptides such as endorphins and enkephalins.

Neurotransmitters can activate ionotropic or metabotropic receptors

Neurotransmitter receptors transduce information by two molecular mechanisms: some are ligand-gated ion channels and others are G protein–coupled receptors (see Chapter 3). Several neurotransmitter molecules, such as glutamate and ACh, serve as ligands (agonists) for both types of receptors. In the particular case of glutamate, glutamate receptors that are ion channels are known as ionotropic receptors, and glutamate receptors coupled to G proteins are called metabotropic receptors. This nomenclature is often used to describe the two major functional classes of receptors for transmitters other than glutamate.

Ionotropic and metabotropic receptors determine the ultimate functional response to transmitter release. Activation of an ionotropic receptor causes rapid opening of ion channels. This channel activation in turn results in depolarization or hyperpolarization of the postsynaptic membrane, the choice depending on the ionic selectivity of the conductance change. Activation of a metabotropic G protein–linked receptor results in the production of active α and βγ subunits, which initiate a wide variety of cellular responses by direct interaction with either ion channel proteins or other second-messenger effector proteins (see Chapter 3). By their very nature, ionotropic receptors mediate fast ionic synaptic responses that occur on a millisecond time scale, whereas metabotropic receptors mediate slow, biochemically mediated synaptic responses in the range of seconds to minutes.

Figure 8-3 compares the basic processes mediated by two prototypic ACh receptors (AChRs): (1) the ACh-activated ion channel at the neuromuscular junction of skeletal muscle, an ionotropic receptor also known as the nicotinicAChR (Fig. 8-3A), and (2) the G protein–linked AChR at the atrial parasympathetic synapse of the heart, a metabotropic receptor also known as the muscarinic AChR (Fig. 8-3B). The nicotinic versus muscarinic distinction was a classic pharmacological classification based on whether the AChR is activated by nicotine or muscarine, two natural products that behave like agonists. In the case of the ionotropic (nicotinic) receptor, opening of the AChR channel results in a transient increase in permeability to Na+ and K+, which directly produces a brief depolarization that activates the muscle fiber. In the case of the metabotropic (muscarinic) receptor, activation of the G protein–coupled receptor opens an inward rectifier K+ channel, or GIRK (see Chapter 7), through βγ subunits released from an activated heterotrimeric G protein. Enhanced opening of these GIRKs produces membrane hyperpolarization and leads to inhibition of cardiac excitation (see Chapter 21). These two functionally distinct mechanisms are the molecular basis for the seemingly conflicting observations of early physiologists that ACh (Vagusstoff) activates skeletal muscle but inhibits heart muscle.


Figure 8-3 Ionotropic and metabotropic ACh receptors. A, This example illustrates a nicotinic AChR, which is a ligand-gated channel on the postsynaptic membrane. In a skeletal muscle, the end result is muscle contraction. B, This example illustrates a muscarinic AChR, which is coupled to a heterotrimeric G protein. In a cardiac muscle, the end result is decreased heart rate. Note that the presynaptic release of ACh is similar here and in A.


Neuromuscular junctions are specialized synapses between motor neurons and skeletal muscle

The chemical synapse between peripheral motor nerve terminals and skeletal muscle fibers is the most intensely studied synaptic connection in the nervous system. Even though the detailed morphology and the specific molecular components (e.g., neurotransmitters and receptors) differ considerably among different types of synapses, the basic electrophysiological principles of the neuromuscular junction are applicable to many other types of chemical synapses, including neuronal synaptic connections in the brain, to which we will return in Chapter 13. In this chapter, we focus on the neuromuscular junction in discussing the basic principles of synaptic transmission.

Motor neurons with cell bodies in the spinal cord have long axons that branch extensively near the point of contact with the target muscle (Fig. 8-4). These axon processes each innervate a separate fiber of skeletal muscle. The whole assembly of muscle fibers innervated by the axon from one motor neuron is called a motor unit.


Figure 8-4 The vertebrate neuromuscular junction or motor end plate. A motor neuron, with its cell body in the ventral horn of the spinal cord, sends out an axon that progressively bifurcates to innervate several muscle fibers (a motor unit). The neuron contacts a muscle fiber at exactly one spot called a neuromuscular junction or motor end plate. The end plate consists of an arborization of the nerve into many presynaptic terminals, or boutons, as well as the specializations of the postsynaptic membrane. A high-magnification view of a bouton shows that the synaptic vesicles containing the neurotransmitter ACh cluster and line up at the active zone of the presynaptic membrane. The active zones on the presynaptic membrane are directly opposite the secondary postsynaptic clefts that are created by infoldings of the postsynaptic membrane (postjunctional folds). Depolarization of the bouton causes the vesicles to fuse with the presynaptic membrane and to release their contents into the synaptic cleft. The ACh molecules must diffuse at least 50 nm before reaching nicotinic AChRs. Note the high density of AChRs at the crests of the postjunctional folds. The activity of the released ACh is terminated mainly by an acetylcholinesterase. The bouton reloads its discharged synaptic vesicles by resynthesizing ACh and transporting this ACh into the vesicle through an ACh-H exchanger.

Typically, an axon makes a single point of synaptic contact with a skeletal muscle fiber, midway along the length of the muscle fiber. This specialized synaptic region is called the neuromuscular junction or the end plate (Fig. 8-4). An individual end plate consists of a small tree-like patch of unmyelinated nerve processes that are referred to as terminal arborizations. The bulb-shaped endings that finally contact the muscle fiber are called boutons. Schwann cells are intimately associated with the nerve terminal and form a cap over the face of the nerve membrane that is located away from the muscle membrane. The postsynaptic membrane of the skeletal muscle fiber lying directly under the nerve terminal is characterized by extensive invaginations known as postjunctional folds. These membrane infoldings greatly increase the surface area of the muscle plasma membrane in the postsynaptic region. The intervening space of the synaptic cleft, which is ~50 nm wide, is filled with a meshwork of proteins and proteoglycans that are part of the extracellular matrix. A particular region of the muscle basement membrane called the synaptic basal lamina contains various proteins (e.g., collagen, laminin, agrin) that mediate adhesion of the neuromuscular junction and play important roles in synapse development and regeneration. The synaptic basal lamina also contains a high concentration of the enzyme acetylcholinesterase (AChE), which ultimately terminates synaptic transmission by rapidly hydrolyzing free ACh to choline and acetate.

Electron micrographs of the bouton region demonstrate the presence of numerous spherical synaptic vesicles, each with a diameter of 50 to 60 nm. The cell bodies of motor neurons in the spinal cord produce these vesicles, and the microtubule-mediated process of fast axonal transport (see Chapter 2) translocates them to the nerve terminal. The quantal nature of transmitter release (described later in more detail) reflects the fusion of individual synaptic vesicles with the plasma membrane of the presynaptic terminal. Each synaptic vesicle contains 6000 to 10,000 molecules of ACh. The ACh concentration in synaptic vesicles is ~150 mM. ACh is synthesized in the nerve terminal—outside the vesicle—from choline and acetyl coenzyme A by the enzyme choline acetyltransferase. The ACh moves into the synaptic vesicle through a specific ACh-H exchanger, which couples the inward transport of ACh to the efflux of H+. Energetically, this process is driven by the vesicular proton electrochemical gradient (positive voltage and low pH inside), which in turn is produced by a vacuolar-type H+ pump fueled by ATP (see Chapter 5). The nerve terminal also contains numerous mitochondria that produce the ATP required to fuel energy metabolism.

The process of fusion of synaptic vesicles and release of ACh occurs at differentiated regions of the presynaptic membrane called active zones. In electron micrographs, active zones appear as dense spots over which synaptic vesicles are closely clustered in apposition to the membrane. High-resolution images of active zones reveal a double, linear array of synaptic vesicles and intramembranous particles. These zones are oriented directly over secondary post-synaptic clefts that lie between adjacent postjunctional folds. Molecular localization studies have shown that the density of ionotropic (nicotinic) AChRs is very high at the crests of postjunctional folds. Examination of the detailed microarchitecture of the neuromuscular synapse thus reveals a highly specialized structure for delivery of neurotransmitter molecules to a precise location on the postsynaptic membrane.

Acetylcholine activates nicotinic acetylcholine receptors to produce an excitatory end-plate current

Electrophysiological experiments on muscle fibers have characterized the electrical nature of the postsynaptic response at the muscle end plate. Figure 8-5 illustrates results obtained from a classic experiment performed by Fatt and Katz in 1951. Their work is the first description of how stimulation of the motor nerve affects the membrane potential (Vm) at the postsynaptic region (i.e., muscle cell) of the neuromuscular junction. Nerve stimulation normally drives the Vm of the muscle above threshold and elicits an action potential (see Chapter 7). However, Fatt and Katz were interested not in seeing the action potential but in studying the small, graded electrical responses that are produced as ACh binds to receptors on the muscle cell membrane. Therefore, Fatt and Katz greatly reduced the response of the AChRs by blocking most of them with a carefully selected concentration of d-tubocurarine, which we discuss later. They inserted a KCl-filled microelectrode into the end-plate region of a frog sartorius muscle fiber. This arrangement allowed them to measure tiny changes in Vm at one particular spot of the muscle cell. (See Note: Tubocurarine)


Figure 8-5 End-plate potentials elicited at the frog neuromuscular junction by stimulation of the motor neuron. The magnitude of the excitatory postsynaptic potential is greatest near the end plate and decays farther away. (Data from Fatt P, Katz B: An analysis of the end-plate potential recorded with an intracellular electrode. J Physiol 1951; 115:320-370.)

When Fatt and Katz electrically excited the motor nerve axon, they observed a transient depolarization in the muscle membrane after a delay of a few milliseconds. The delay represents the time required for the release of ACh, its diffusion across the synapse, and activation of postsynaptic AChRs. The positive voltage change follows a biphasic time course: Vm rapidly rises to a peak and then more slowly relaxes back to the resting value, consistent with an exponential time course. This signal, known as the end-plate potential (EPP), is an example of an excitatory postsynaptic potential. It is produced by the transient opening of AChR channels, which are selectively permeable to monovalent cations such as Na+ and K+. The increase in Na+ conductance drives Vm to a more positive value in the vicinity of the end-plate region. In this experiment, curare blockade allows only a small number of AChR channels to open, so that the EPP does not reach the threshold to produce an action potential. If the experiment is repeated by inserting the microelectrode at various distances from the end plate, the amplitude of the potential change is successively diminished and its peak is increasingly delayed. This decrement with distance occurs because the EPP originates at the end-plate region and spreads away from this site according to the passive cable properties of the muscle fiber. Thus, the EPP in Figure 8-5 is an example of a propagated, graded response. However, without the curare blockade, more AChR channels would open and a larger EPP would ensue, which would drive Vm above threshold and consequently trigger a regenerating action potential (see Chapter 7).

What ions pass through the AChR channels during generation of the EPP? This question can be answered by the same voltage-clamp technique that was used to study the basis of the action potential (see Fig. 7-5B). Figure 8-6Aillustrates the experimental preparation for a two-electrode voltage-clamp experiment in which the motor nerve is stimulated while the muscle fiber in the region of its end plate is voltage clamped to a chosen Vm. The recorded current, which is proportional to the conductance change at the muscle end plate, is called the end-plate current (EPC). The EPC has a characteristic time course that rises to a peak within 2 ms after stimulation of the motor nerve and falls exponentially back to zero (Fig. 8-6B). The time course of the EPC corresponds to the opening and closing of a population of AChR channels, governed by the rapid binding and disappearance of ACh as it diffuses to the postsynaptic membrane and is hydrolyzed by AChE. (See Note: Two-Electrode Voltage Clamping)


Figure 8-6 End-plate currents obtained at different membrane potentials in a voltage-clamp experiment. A, Two-electrode voltage clamp is used to measure the EPC in a frog muscle fiber. The tips of the two microelectrodes are in the muscle fiber. B, The six records represent EPCs that were obtained while the motor nerve was stimulated and the postsynaptic membrane was clamped to Vm values of –120, –91, –68, –37, +24, and +38 mV. Notice that the peak current reverses from inward to outward as the holding potential shifts from –37 to +24 mV. C, The reversal potential is near 0 mV because the nicotinic AChR has a poor selectivity for Na+ versus K+(Data from Magleby KL, Stevens CF: The effect of voltage on the time course of end-plate current. J Physiol 1975; 223:151-171.)

As shown in Figure 8-6B, when the muscle fiber is clamped to a “holding potential” of –120 mV, we observe a large inward current (i.e., the EPC). This inward current decreases in magnitude as Vm is made more positive, and the current reverses direction to become an outward current at positive values of Vm. A plot of the peak current versus the clamped Vm shows that the reversal potential for the EPC is close to 0 mV (Fig. 8-6C). Because the EPC specifically corresponds to current through AChR channels, this reversal potential reflects the ionic selectivity of these channels when extracellular Na+ and K+concentrations ([Na+]o and [K+]o) are normal.

By varying the concentrations of the extracellular ions while monitoring the shift in the reversal potential of the EPC, researchers found that the AChR channel is permeable to Na+, K+, and Ca2+ but not to anions such as Cl. Because of its low extracellular concentration, the current attributable to Ca2+ is small under physiological conditions and its contribution can be ignored. By plugging the values for the various cations into the Goldman-Hodgkin-Katz voltage equation (see Equation 6-9), one can obtain the permeability of the AChR channel to various alkali monovalent ions, relative to Na+ permeability. The result is the following sequence of relative permeability: 0.87 (Li+), 1.00 (Na+), 1.11 (K+), and 1.42 (Cs+). This weak ionic selectivity stands in marked contrast to typical voltage-gated Na+ channels, which have PNa/PK ratios of ~20, and voltage-gated K+ channels, which have PK/PNa ratios greater than 100. On this basis, the ionotropic (nicotinic) AChR channel at the muscle end plate is often classified as a nonselective cation channel. Nevertheless, the weak ionic selectivity of the AChR is well suited to its basic function of raising Vm above the threshold of about –50 mV, which is necessary for firing of an action potential. When the nicotinic AChR channel at the muscle end plate opens, the normally high resting permeability of the muscle plasma membrane for K+ relative to Na+ falls so that Na+ and K+ become equally permeant and Vm shifts to a value between EK (approximately –80 mV) and ENa (approximately +50 mV). (See Note: Contribution of Ca2+ to the Resting Membrane Potential)

As we shall see in Chapter 13, which focuses on synaptic transmission in the CNS, similar principles hold for generation of postsynaptic currents by other types of agonist-gated channels. For example, the receptor-gated channels for serotonin and glutamate are cation selective and give rise to depolarizingexcitatory postsynaptic potentials. In contrast, the receptor-gated channels for glycine and GABA are anion selective and drive Vm in the hyperpolarizing direction, toward the equilibrium potential for Cl. These hyperpolarizing postsynaptic responses are called inhibitory postsynaptic potentials.

The nicotinic acetylcholine receptor is a member of the pentameric Cys-loop receptor family of ligand-gated ion channels

The molecular nature of the nicotinic AChR channel was revealed by studies that included protein purification, amino acid sequencing of isolated subunits, and molecular cloning. Purification of the receptor was aided by the recognition that the electric organs of certain fish are a particularly rich source of the nicotinic AChR. In the electric eel and torpedo ray, the electric organs are embryologically derived from skeletal muscle. The torpedo ray can deliver large electrical discharges by summating the simultaneous depolarizations of a stack of many disk-like cells called electrocytes. These cells have the skeletal muscle isoform of the nicotinic AChR, which is activated by ACh released from presynaptic terminals.

The purified torpedo AChR consists of four subunits (α, β, γ, and δ) in a pentameric stoichiometry of 2α:1β:1γ:1δ (Fig. 8-7). Each subunit has a molecular mass of ~50 kDa and is homologous to the other subunits. The primary sequences of nicotinic AChR subunits are ~90% identical between the torpedo ray and human.


Figure 8-7 Structure of the nicotinic AChR. The nicotinic AChR is a heteropentamer with the subunit composition of α2βγδ. These subunits are homologous to one another, and each has four membrane-spanning segments (M1 to M4).

The α, β, γ, and δ subunits each have four distinct hydrophobic regions known as M1 to M4, which correspond to membrane-spanning segments. For each of the subunits, the M2 transmembrane segment lines the aqueous pore through which Na+ and K+ cross the membrane.

The pentameric complex has two agonist binding sites. One ACh binding site is formed at the interface of the extracellular domain of one α subunit and the extracellular domain of the γ subunit. The other site is located between the extracellular domain of the other α subunit and the extracellular domain of the δ subunit. (See Note: Ligand Binding Sites of the Nicotinic Acetylcholine Receptors)

AChRs of normal adult muscle fibers are present in high density in the junctional folds of the postsynaptic membrane. However, in developing muscle fibers of the mammalian embryo and in denervated fibers of adult skeletal muscle, AChRs are also widely distributed in the membrane outside the end-plate region. The two types of AChRs, called junctional and nonjunctional receptors, have different functional properties. The unitary conductance of nonjunctional receptors is ~50% larger and the single-channel lifetime is longer in duration than that of junctional receptors. The basis for this phenomenon is a difference in subunit composition. The nonjunctional (or fetal) receptors are a pentameric complex with a subunit composition of α2βγδ in mammals, just as in the electric organ of the torpedo ray. For the junctional AChR in adult skeletal muscle, substitution of an γ subunit for the fetal γ subunit results in a complex with the composition α2βimageδ.

The functional properties of the two types of receptors have been studied by coexpressing the cloned subunits in Xenopus oocytes. Figure 8-8A shows patch-clamp recordings of single ACh-activated channels in oocytes that had been injected with mRNA encoding either α, β, γ, δ or α, β, image, δ. Measurements of currents at different voltages yielded single-channel I-V curves (Fig. 8-8B) showing that the channel formed with the image subunit had a unitary conductance of 59 pS, whereas that formed with the γ subunit had a conductance of 40 pS. The mean lifetime of single-channel openings at 0 mV was 1.6 ms for image-type and 4.4 ms for γ-type receptors, closely corresponding to values found in native fetal and adult muscle, respectively. The different functional properties of junctional and nonjunctional nicotinic AChRs presumably reflect their specialized roles in synaptic transmission versus development and synapse formation.


Figure 8-8 Properties of fetal and adult AChRs from skeletal muscle. A, The results of patch-clamp experiments, with the patch pipettes in the outside-out configuration and the patch exposed to 0.5 μM ACh, are summarized. In the upper panel, the investigators expressed the fetal acetylcholine receptor channel (AChR), which has the subunit composition α2βγδ, in Xenopus oocytes. In the lower panel, the investigators expressed the adult AChR, which has the subunit composition α2βγδ. Notice that the mean open times are greater for the fetal form, whereas the unitary currents are greater for the adult form. B, The two lines summarize data that are similar to those obtained in A. The single-channel conductance of the adult form (59 pS) is higher than that of the fetal form (40 pS). (Data from Mishina M, Takai T, Imoto K, et al: Molecular distinction between fetal and adult forms of muscle acetylcholine receptor. Nature 1986; 321:406-411.)

Humans have nine genes that encode homologous α subunits of nicotinic ACh-activated receptors (see Fig. 6-21L). The α subunit of the skeletal muscle receptor (α1) is encoded by the CHRNA1 gene. The eight other α subunits (α2to α9), which are expressed in neuronal tissues, are encoded by genes CHRNA2 to CHRNA9. Only the protein products of genes α1, α7, and α8 bind a venom protein called α-bungarotoxin, a venom protein from a snake called the Taiwanese banded krait. In addition, at least four β subunits exist, encoded by human genes CHRNB1 to CHRNB4 (see Fig. 6-21). Besides the β subunit of the skeletal muscle AChR—which is called β1—there are three neuronal homologues (β2, β3, β4). Heteromeric association of different combinations of these subunits could potentially produce a large number of functional receptor isoforms. Although the exact physiological role of nicotinic AChR channels in various neuronal pathways remains to be established, AChRs in the brain play a role in addiction to the nicotine contained in tobacco.

Besides nicotinic AChRs, three other related classes of agonist-activated channels are recognized, including ionotropic receptor channels that are activated by serotonin (5-HT3 receptor), glycine (GlyR), and GABA (GABAAreceptor). As mentioned previously, AChR and 5-HT3 receptor channels are both permeable to cations and thus produce excitatory currents, whereas glycine-activated and GABAA channels are permeable to anions such as Cl and produce inhibitory currents. Figure 8-9 shows examples of macroscopic and unitary Cl currents mediated by glycine-activated and GABAA channels. Cloned genes encoding subunits of these receptor channels encode proteins that are homologous to AChR subunits. Their primary amino acid sequences share a common arrangement of M1, M2, M3, and M4 transmembrane segments, as described earlier for the nicotinic AChR (Fig. 8-7). These proteins all belong to the pentameric Cys-loop receptor family of ligand-gated ion channels (see Fig. 6-21), so named because they contain a highly conserved pair of disulfide-bonded cysteine residues. Sequence analysis of these genes indicates that they evolved from a common ancestor. The basis for cation versus anion selectivity appears to reside solely within the M2 segment. Mutation of only three residues within the M2 segment of a cation-selective α subunit of a neuronal nicotinic AChR is sufficient to convert it to an anion-selective channel activated by ACh.


Figure 8-9 Currents activated by glycine and GABA. A, These experiments were performed on cultured mouse spinal cord neurons by patch-clamp techniques. The left panel shows the macroscopic Cl current, which is measured in the whole-cell configuration and carried by glycine receptor (GlyR) channels when exposed to glycine. The right panel shows single-channel currents that are recorded in the outside-out patch configuration. In both scenarios, the holding potential was –70 mV. B, The left panel shows the macroscopic Cl current that is carried by GABAA receptor channels when exposed to GABA. The right panel shows single-channel currents. (Data from Bormann J, Hamill OP, Sakmann B: Mechanism of anion permeation through channels gated by glycine and γ-aminobutyric acid in mouse spinal neurones. J Physiol 1987; 385:243-286.)

Activation of acetylcholine receptor channels requires binding of two acetylcholine molecules

The EPC is the sum of many single-channel currents, each representing the opening of a single AChR channel at the neuromuscular junction. Earlier we described the random opening and closing of an idealized channel in a two-state model in which the channel could be either closed or open (see Chapter 7):


In the case of an agonist-activated channel, such as the AChR channel, binding of an agonist to the channel in its closed state favors channel opening. This gating process may be represented by the following kinetic model:


In this two-step scheme, the closed state (C) of the channel must bind one molecule of the agonist ACh to form a closed, agonist-bound channel (AC) before it can convert to an open, agonist-bound channel (AO). However, studies of the dependence of the probability of channel opening on agonist concentration indicate that binding of two molecules of ACh is required for channel opening. This feature of nicotinic receptor gating is described by the following modification of Equation 8-2:


Understanding the kinetics of channel opening can be very important for clarifying the mechanism by which certain channel inhibitors work. For example, a competitive inhibitor could prevent binding of the agonist ACh. However, many noncompetitive antagonists of the AChR channel, including some local anesthetics, act by entering the lumen of the channel and blocking the flow of ionic current. Figure 8-10Ashows the results of a patch-clamp experiment in which a single AChR channel opened and closed in response to its agonist, ACh. After the addition of QX-222, an analogue of the local anesthetic agent lidocaine (see Chapter 7), to the extracellular side, the channel exhibits a rapidly flickering behavior. This flickering represents a series of brief interruptions of the open state by numerous closures (Fig. 8-10B). This type of flickering block is caused by rapid binding and unbinding of the anesthetic drug to a site in the mouth of the open channel. When the drug binds, it blocks the channel to the flow of ions (A2B). Conversely, when the drug dissociates, the channel becomes unblocked (A2O):



Figure 8-10 The effect of a local anesthetic on the AChR. A, Single-channel recording of nicotinic AChR expressed in a Xenopus oocyte. The patch was in the outside-out configuration, and the holding potential was –150 mV. The continuous presence of 1 μM ACh caused brief channel openings. B, This experiment is similar to that in A except that in addition to the ACh, the lidocaine analogue QX-222 (20 μM) was present at the extracellular surface of the receptor channel. Note that the channel opening is accompanied by rapid flickering caused by many brief channel closures. The time scale of the lower panel is expanded 10-fold. (Data from Leonard RJ, Labarca CG, Charnet P, et al: Evidence that the M2 membrane-spanning region lines the ion channel pore of the nicotinic receptor. Science 1988; 242:1578-1581.)

Channel blockers are often used as molecular tools to study the mechanism of ion permeation. For example, in combination with site-directed mutagenesis, QX-222 was helpful in locating amino acid residues on the M2 transmembrane segment that form part of the blocker binding site, thus identifying residues that line the aqueous pore.

Miniature end-plate potentials reveal the quantal nature of transmitter release from the presynaptic terminals

Under physiological conditions, an action potential in a presynaptic motor nerve axon produces a depolarizing postsynaptic EPP that peaks at ~40 mV more positive than the resting Vm. This large signal results from the release of ACh from only about 200 synaptic vesicles, each containing 6000 to 10,000 molecules of ACh. The neuromuscular junction is clearly designed for excess capacity inasmuch as a single end plate is composed of numerous synaptic contacts (~1000 at the frog muscle end plate), each with an active zone that is lined with dozens of mature synaptic vesicles. Thus, a large inventory of ready vesicles (>104), together with the ability to synthesize ACh and to package it into new vesicles, allows the neuromuscular junction to maintain a high rate of successful transmission without significant loss of function as a result of presynaptic depletion of vesicles or ACh.

The original notion of a vesicular mode of transmitter delivery is based on classic observations of EPPs under conditions of reduced ACh release. In 1950, Fatt and Katz observed an interesting kind of electrophysiological “noise” in their continuous, high-resolution recordings of Vm with a microelectrode inserted at the end-plate region of a frog muscle fiber. Their recordings from resting muscle fibers that were not subjected to nerve stimulation revealed the occurrence of tiny depolarizations of ~0.4 mV that appeared at random intervals. These small depolarizations were blocked by curare, an antagonist of AChR channels, and they increased in size and duration with the application of neostigmine, an inhibitor of AChE. Because the spontaneous Vm fluctuations also exhibited a time course similar to that of the normal EPP, they were named miniature end-plate potentials (also known as MEPPs or minis). These observations suggested that even in the absence of nerve stimulation, there is a certain low probability of transmitter release at the presynaptic terminal, resulting in the opening of a small number of AChRs in the postsynaptic membrane. An examination of the size of individual MEPPs suggested that they occur in discrete multiples of a unitary amplitude. This finding led to the notion that ACh release is quantized, with the quantum event corresponding to ACh release from one synaptic vesicle.

Another way of studying the quantal release of ACh is to stimulate the presynaptic motor neuron and to monitor Vm at the end plate under conditions when the probability of ACh release is greatly decreased. How can we decrease the probability of ACh release? The amplitude of the EPP that is evoked in response to nerve stimulation is decreased by lowering [Ca2+]o and increasing [Mg2+]o. A low [Ca2+]o decreases Ca2+ entry into the presynaptic terminal (Fig. 8-2, step 3). A high [Mg2+]o partially blocks the presynaptic Ca2+ channels and thus also decreases Ca2+ entry. Therefore, the consequence of either decreased [Ca2+]o or increased [Mg2+]o is a fall in [Ca2+]i in the presynaptic terminal, which reduces transmitter release and thus the amplitude of the EPP (Fig. 8-11). Del Castillo and Katz exploited this suppression of transmitter release under conditions of low [Ca2+]o and high [Mg2+]o to observe the Vm changes caused by the quantal release of transmitter. Figure 8-12A shows seven superimposed records of MEPPs that were recorded from a frog muscle fiber during seven repetitive trials of nerve stimulation under conditions of reduced [Ca2+]o and elevated [Mg2+]o. The records are aligned at the position of the nerve stimulus artifact. The amplitudes of the peak responses occur in discrete multiples of ~0.4 mV. Among the seven records were one “nonresponse,” two responses of ~0.4 mV, three responses of ~0.8 mV, and one response of ~1.2 mV. One of the recordings also revealed a spontaneous MEPP with a quantal amplitude of ~0.4 mV that appeared later in the trace. Del Castillo and Katz proposed that the macroscopic EPP is the sum of many unitary events, each having a magnitude of ~0.4 mV. Microscopic observation of numerous vesicles in the synaptic terminal naturally led to the supposition that a single vesicle releases a relatively fixed amount of ACh and thereby produces a unitary MEPP. According to this view, the quantized MEPPs thus correspond to the fusion of discrete numbers of synaptic vesicles: 0, 1, 2, 3, and so on. (See Note: Quantal Nature of Transmitter Release)


Figure 8-11 The effect of extracellular Ca2+ and Mg2+ on EPPs. The data obtained by stimulating the motor neuron and monitoring the evoked subthreshold EPP show that the EPP is stimulated by increasing levels of Ca2+ but inhibited by increasing levels of Mg2+(Data from Dodge FA Jr, Rahaminoff R: Cooperative action of calcium ions in transmitter release at the neuromuscular junction. J Physiol 1967; 193:419-432.)


Figure 8-12 Evoked and spontaneous MEPPs. A, The investigators recorded Vm in frog skeletal muscle fibers that were exposed to extracellular solutions having a [Ca2+] of 0.5 mM and a [Mg2+] of 5 mM. These values minimize transmitter release, and therefore it was possible to resolve the smallest possible MEPP, which corresponds to the release of a single synaptic vesicle (i.e., 1 quantum). The investigators stimulated the motor neuron seven consecutive times and recorded the evoked MEPPs. In one trial, the stimulus evoked no response (0 quanta). In two trials, the peak MEPP was about 0.4 mV (1 quantum). In three others, the peak response was about 0.8 mV (2 quanta). Finally, in one, the peak was about 1.2 mV (3 quanta). In one case, a MEPP of the smallest magnitude appeared spontaneously. B, The histogram summarizes data from 198 trials on a cat neuromuscular junction in the presence of 12.5 mM extracellular Mg2+. The data are in bins with a width of 0.1 mV. The distribution has eight peaks. The first represents stimuli that evoked no responses. The other seven represent stimuli that evoked MEPPs that were roughly integral multiples of the smallest MEPP. The curve overlying each cluster of bins is a gaussian or “normal” function and facilitates calculation of the average MEPP for each cluster of bins. The peak values of these gaussians follow a Poisson distribution. (Data from Magleby KL: Neuromuscular transmission. In Engel AG, Franzini-Armstrong C [eds]: Myology, Basic and Clinical, 2nd ed, pp 442-463. New York, McGraw-Hill, 1994.)

For elucidating the mechanism of synaptic transmission at the neuromuscular junction, Bernard Katz shared the 1970 Nobel Prize in Physiology or Medicine. (See Note: Sir Bernard Katz)

Direct sensing of extracellular transmitter also shows quantal release of transmitter

Instead of using the postsynaptic AChR as a biological detector of quantum release, one can use a microscopic electrochemical sensor to measure neurotransmitter levels directly. Figure 8-13 shows results from an experiment in which a fine carbon fiber electrode was placed very close to the presynaptic terminal membrane of a leech neuron that uses serotonin as its only neurotransmitter. The carbon fiber is an electrochemical detector of serotonin (Fig. 8-13A); the current measured by this electrode corresponds to four electrons per serotonin molecule oxidized at the tip. Stimulation of the leech neuron to produce an action potential also elicits an oxidation current, as measured by the carbon fiber, that corresponds to the release of serotonin. At a [Ca2+]o of 5 mM, the current is large and composed of many small spikes (Fig. 8-13B, top). On the other hand, reducing [Ca2+]o to 1 mM—presumably reducing Ca2+influx into the nerve terminal and thus reducing the number of quanta released—reveals individual spikes of serotonin release. The release spikes come in two sizes, small and large (Fig. 8-13B, bottom), corresponding to two separate classes of synaptic vesicles that are evident on electron micrographs. Injection of the cell with tetanus toxin, which blocks the release of synaptic vesicles, abolishes the serotonin release spikes. Thus, the spikes represent genuine events of synaptic exocytosis.


Figure 8-13 Detection of serotonin that is released from synaptic vesicles. A, The serotonin that is released from a synaptic terminal of a leech neuron can be detected electrochemically by use of a carbon fiber microelectrode. The current carried by the carbon fiber increases with the amount of serotonin that is released, reflecting the oxidation of serotonin molecules on the surface of the carbon fiber. B, The top panel shows the action potential recorded from the stimulated motor neuron. The middle panel shows the evoked serotonin release (measured as a current) at both a [Ca2+]o of 5 mM (high level of serotonin release) and a [Ca2+]o of 1 mM (lower level or release). The bottom panel shows five consecutive trials at a [Ca2+]o of 1 mM and illustrates that the release of serotonin can occur in either small quanta or very large quanta. These two sizes of quanta correspond to small clear vesicles and large dense-core vesicles, both of which can be observed by electron microscopy. (Data from Bruns D, Jahn R: Real-time measurement of transmitter release from single synaptic vesicles. Nature 1995; 377:62-65.)

The nearly immediate appearance of the small release spikes after electrical stimulation of the cell shows that this type of vesicular release is extremely rapid. From the height and duration of the small and large spikes in Figure 8-13B, one can estimate the amount of electrical charge and thus the number of serotonin molecules oxidized at the carbon fiber per spike. A unitary small event corresponds to the release of ~4700 serotonin molecules, whereas a unitary large event corresponds to the release of 15,000 to 300,000 serotonin molecules. Thus, the amount of serotonin released by the small synaptic vesicles of the leech neuron is about half the number of ACh molecules contained in a synaptic vesicle at the frog neuromuscular junction. These and other observations of the synaptic function of nerve-muscle and nerve-nerve synapses have led to the conclusion that chemical neurotransmission operates by a fundamentally similar mechanism at many types of synapses in different animal species (see Chapter 13).

Short-term or long-term changes in the relative efficiency of neurotransmitter release can increase or decrease the strength of a particular synapse and thereby give rise to an alteration in behavior. Three types of synaptic modulation occur at the neuromuscular junction, and they differ in how they affect the quantal release of neurotransmitter. (See Note: Modulation of Quantal Release)

Facilitation is a short-lived enhancement of the EPP in response to a brief increase in the frequency of nerve stimulation. One way that facilitation may occur is by a transient increase in the mean number of quanta per nerve stimulus.

Potentiation (or post-tetanic potentiation) is a long-lived and pronounced increase in transmitter release that occurs after a long period of high-frequency nerve stimulation. This effect can last for minutes after the conditioning stimulus. Potentiation may be caused by a period of intense nerve firing, which increases [Ca2+]i in the presynaptic terminal and thus increases the probability of exocytosis.

Synaptic depression is a transient decrease in the efficiency of transmitter release and, consequently, a reduction in the EPP in response to a period of frequent nerve stimulation. Depression may result from temporary depletion of transmitter-loaded vesicles from the presynaptic terminal, that is, a reduction in the number of available quanta. Thus, these three temporal changes in synaptic strength and efficiency appear to reflect changes at different steps of synaptic transmission. Similar modulation of synaptic strength in the CNS provides a mechanistic paradigm to understand how individual nerve terminals may “learn” (see Chapter 13).

Synaptic vesicles package, store, and deliver neurotransmitters

The physiology of synaptic vesicles in the nervous system is a variation on the universal theme used by endocrine-like cells in animals from the most primitive invertebrates up to mammals (see Chapter 3). Many of the proteins involved in synaptic vesicle movement and turnover are related to those involved in the intracellular membrane trafficking processes that take place in almost all eukaryotic cells. This trafficking involves vesicular translocation from the endoplasmic reticulum to the Golgi network and fusion with the plasma membrane. Genetic analysis of the yeast secretory pathway has identified various gene products that are homologous to those associated with synaptic vesicles of higher vertebrates. Thus, the processes underlying synaptic function are inherently quite similar to cellular exocytosis and endocytosis.

As shown in Figure 8-14nascent synaptic vesicles are produced in the neuronal cell body by a process similar to the secretory pathway. Thus, the membrane proteins of synaptic vesicles are synthesized in the rough endoplasmic reticulum and are then directed to the Golgi network, where processing, maturation, and sorting occur. Nascent synaptic vesicles—which are, in fact, secretory vesicles—are then transported to the nerve terminal by fast axonal transport mediated by the microtubule system, which also carries mitochondria to the terminal (see Chapter 2).


Figure 8-14 Synthesis and recycling of synaptic vesicles and their content.

Vesicles destined to contain peptide neurotransmitters travel down the axon with the presynthesized peptides or peptide precursors already inside. On arrival at the nerve terminal (Fig. 8-14), the vesicles—now called dense-core secretory granules (100 to 200 nm in diameter)—become randomly distributed in the cytoplasm of the terminal as discussed in more detail in Chapter 13.

Vesicles destined to contain non-peptide neurotransmitters (e.g., ACh) travel down the axon with no transmitter inside. On arrival at the nerve terminal (Fig. 8-14), the vesicles take up the non-peptide neurotransmitter that is synthesized locally in the nerve terminal. These non-peptide synaptic vesicles, which are clear and 40 to 50 nm in diameter, then attach to the actin-based cytoskeletal network. At this point, the mature clear synaptic vesicles are functionally ready for Ca2+-dependent transmitter release and become docked at specific release sites in the active zones of the presynaptic membrane. After exocytotic fusion of the clear synaptic vesicles, endocytosis through clathrin-coated vesicles (see Chapter 2) recovers membrane components and recycles them to an endosome compartment in the terminal. Synaptic vesicles may then be re-formed within the terminal for reuse in neurotransmission, or they may be transported back to the cell body for turnover and degradation.

The purification of synaptic vesicles has made it possible to analyze their composition, which has facilitated the molecular characterization of many proteins that are intrinsic to synaptic vesicle function. Figure 8-15 summarizes a number of the major classes of synaptic vesicle proteins.


Figure 8-15 Membrane-associated proteins of synaptic vesicles.

The uptake of non-peptide neurotransmitters is accomplished by the combination of a vacuolar-type H+-ATPase and a neurotransmitter transport protein. The vacuolar-type H+ pump is a large, multisubunit complex that catalyzes the inward movement of H+ into the vesicle, coupled to the hydrolysis of cytosolic ATP to ADP and inorganic phosphate (see Chapter 5). The resulting pH and voltage gradients across the vesicle membrane energize the uptake of neurotransmitters into the vesicle by a unique family of neurotransmitter transport proteins that exchange neurotransmitters in the cytosol for H+ in the vesicle. This family of transporters includes members specific for ACh, monoamines (e.g., serotonin), catecholamines (e.g., norepinephrine), glutamate, and GABA/glycine.

Another cloned synaptic vesicle protein named SV2 (for synaptic vesicle protein 2) structurally resembles a transport protein. However, a transport substrate for SV2 has not been identified, and its function is unknown.

Synaptobrevin is a 19-kDa synaptic vesicle protein containing one transmembrane segment. Synaptobrevin, which is a v-SNARE (see Chapter 2), is essential for transmitter release. As discussed in the next section, synaptobrevin on the vesicle membrane forms a complex with two proteins on the presynaptic membrane and helps drive vesicle fusion. Tetanus toxin or botulinum toxins B, D, F, and G are endoproteinases that digest synaptobrevin and are potent inhibitors of synaptic vesicle exocytosis.

Rab3 is a member of a large family of low-molecular-weight GTP-binding proteins that appears to be universally involved in cellular membrane trafficking (see Chapter 2) through the binding and hydrolysis of GTP. Synaptotagmin is the synaptic vesicle Ca2+ receptor, a protein with two external repetitive domains that are homologous to the C2 domain of protein kinase C. The C2 domains appear to mediate binding of Ca2+, a process that also depends on the presence of acidic phospholipids. Synaptotagmin senses a local rise in [Ca2+]i and triggers the exocytosis of docked vesicles.

Another major constituent, synaptophysin, is an integral membrane protein with four transmembrane segments that exhibits channel-forming activity in planar bilayers. It may be involved in the formation of a fusion pore during exocytosis. The synapsins are a group of synaptic vesicle proteins that are phosphorylated by both cAMP-dependent and calmodulin-dependent protein kinases. Interactions of synapsins with cytoskeletal proteins and their inhibition by phosphorylation have led to the notion that synapsins normally mediate the attachment of synaptic vesicles to the actin cytoskeleton. With an increase in [Ca2+]iand subsequent phosphorylation, the synapsin detaches and permits vesicles to move to active sites at the synaptic membrane.

Neurotransmitter release occurs by exocytosis of synaptic vesicles

Although the mechanism by which synaptic vesicles fuse with the plasma membrane and release their contents is far from fully understood, we have working models (Fig. 8-16) for the function of various key components and steps involved in synaptic vesicle release. These models are based on a variety of in vitro experiments. The use of specific toxins that act at nerve synapses and elegant functional studies of genetic mutants in Drosophila, C. elegans, and gene knockout mice have provided important information on the role of various components.


Figure 8-16 Model of synaptic vesicle fusion and exocytosis. NSF, N-ethylmaleimide-sensitive factor; SNAP-25, synaptosome-associated protein 25 kDa; α-SNAP, soluble NSF attachment protein; SNARE, SNAP receptor.

We have already introduced the key proteins located in the synaptic vesicle. Of these, we now focus on the v-SNARE synaptobrevin and the Ca2+ sensor synaptotagmin. In addition, several other proteins—located in the target area of the presynaptic membrane of the nerve terminal—play an important role in the fusion process. Syntaxin is anchored in the presynaptic membrane by a single membrane-spanning segment. SNAP-25 is tethered to the presynaptic membrane by palmitoyl side chains. Both syntaxin and SNAP-25 are t-SNARES (see Chapter 2). Botulinum toxins A and E, which are endoproteinases, specifically cleave SNAP-25; another endoproteinase, botulinum toxin C1,specifically cleaves syntaxin. These toxins block the fusion of synaptic vesicles. (See Note: “SNAP” Nomenclature)

According to the model shown in Figure 8-16, docking of the vesicle to the presynaptic membrane occurs as n-Sec-1 dissociates from syntaxin. The free ends of synaptobrevin, syntaxin, and SNAP-25 begin to coil around each other. The result is a ternary complex, an extraordinarily stable rod-shaped structure of α helices. As the energetically favorable coiling of the three SNAREs continues, the vesicle membrane is pulled ever closer to the presynaptic membrane. Ca2+ enters through voltage-gated Ca2+ channels located in register with the active zone of the presynaptic membrane. A local increase in [Ca2+]itriggers the final event, fusion and exocytosis. The synaptic vesicle protein synaptotagmin is believed to be the actual sensor of increased [Ca2+]i because knockout mice and Drosophila mutants lacking the appropriate isoform of this protein have impaired Ca2+-dependent transmitter release. The soluble α-SNAP binds to the ternary complex formed by the intertwined SNAREs and promotes the binding of NSF (an ATPase), which uses the energy of ATP hydrolysis to disassemble the three tightly wound SNAREs. The now-free synaptobrevin presumably undergoes endocytosis, whereas the syntaxin and SNAP-25 on the presynaptic membrane are available for the next round of vesicle fusion. (See Note: “SNAP” Nomenclature)

The model just presented leaves unanswered some important questions. For example, what is the structure of the fusion pore detected by electrophysiological measurements as a primary event in membrane fusion? Also, the model does not fully explain the basis for the rapid catalysis of fusion by Ca2+. Neuroscientists are very interested in the details of synaptic vesicle fusion because this exocytotic process might be a target for controlling synaptic strength and may thus play a role in the synaptic plasticity that is responsible for changes in animal behavior.

Re-uptake or cleavage of the neurotransmitter terminates its action

Effective transmission across chemical synapses requires not only release of the neurotransmitter and activation of the receptor on the postsynaptic membrane but also rapid and efficient mechanisms for removal of the transmitter. At synapses where ACh is released, this removal is accomplished by enzymatic destruction of the neurotransmitter. However, the more general mechanism in the nervous system involves reuptake of the neurotransmitter mediated by specific, high-affinity transport systems located in the presynaptic plasma membrane and surrounding glial cells. These secondary active transport systems use the normal ionic gradients of Na+, K+, H+, or Cl to achieve concentrative uptake of transmitter. Vertebrates have two distinct families of neurotransmitter transport proteins. The first family is characterized by a common motif of 12 membrane-spanning segments and includes transporters with specificity for catecholamines, serotonin, GABA, glycine, and choline. Energy coupling of transport in this class of systems is generally based on cotransport of the substrate with Na+ and Cl. The second family is represented by transporters for the excitatory amino acids glutamate and aspartate; in these systems, substrate transport generally couples to cotransport of Na+ and H+ and to exchange of K+.

At the neuromuscular junction and other cholinergic synapses, immediate termination of the action of ACh is accomplished enzymatically by the action of AChE. Although AChE is primarily found at the neuromuscular junction, AChE activity can be detected throughout the nervous system. The enzyme occurs in a variety of physical forms. The globular or G forms exist as monomers, dimers, or tetramers of a common, ~72-kDa glycoprotein catalytic subunit. These molecules can be found either in soluble form or bound to cell membranes through a GPI linkage (see Chapter 2) in which a post-translational modification attaches the C terminus of the protein to a glycolipid moiety. The asymmetric or A forms consist of one to three tetramers of the globular enzyme coupled through disulfide bond linkage to a collagen-like structural protein. The largest asymmetric form, which has 12 catalytic subunits attached to the collagen-like tail, is the major species located at the neuromuscular junction. The triple-helical, collagen-like tail attaches the asymmetric AChE complex to extracellular matrix components of the synaptic basal lamina. The various physical forms of AChE are a result of the alternative splicing that occurs in the transcription of a single AChE gene. (See Note: Acetylcholinesterase)

The enzyme AChE rapidly hydrolyzes ACh to choline and acetate in a two-step process:


In the first step of the reaction, the enzyme cleaves choline from ACh, which results in the formation of an intermediate in which the acetate group is covalently coupled to a serine group on the enzyme. The second step is the hydrolysis and release of this acetate as well as the free enzyme. The nerve terminal recovers the extracellular choline through a high-affinity, Na+-coupled uptake system and uses it for the synthesis of ACh.


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 pharmacological 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.


Figure 8-17 Pharmacology of the vertebrate neuromuscular junction. Many of the proteins that are involved in synaptic transmission at the mammalian neuromuscular junction are the targets of naturally occurring or synthetic drugs. The antagonists are shown as minus signs highlighted in red. The agonists are shown as plus signs highlighted in green.

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 and saxitoxin (see Fig. 7-5C).

The mamba snake toxin dendrotoxin (see Chapter 7) has an effect that is precisely opposite that of tetrodotoxin: it facilitates the release of ACh that is evoked by nerve stimulation. Dendrotoxins are a family of ~59-residue proteins with three disulfide bonds that block certain isoforms 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.

Diseases of the Human Acetylcholine Receptor: Myasthenia Gravis and a Congenital Myasthenic Syndrome

The term myasthenia means muscle weakness (from the Greek mys and asthenia) and is usually used clinically to denote weakness in the absence of primary muscle disease, neuropathy, or CNS disorder. Myasthenia gravis, one specific type of myasthenia and the most common adult form, afflicts 25 to 125 of every 1 million people. It can occur at any age but has a bimodal distribution, with peak incidences occurring among people in their 20s and 60s. Those afflicted at an early age tend to be women with hyperplasia of the thymus; those who are older are more likely to be men with coexisting cancer of the thymus gland. The cells of the thymus possess nicotinic AChRs, and the disease arises as a result of antibodies directed against these receptors. The antibodies then lead to skeletal muscle weakness caused in part by competitive antagonism of AChRs. Symptoms include fatigue and weakness of skeletal muscle. Two major forms of the disease are recognized: one that involves weakness of only the extraocular muscles and another that results in generalized weakness of all skeletal muscles. In either case, myasthenia gravis is typified by fluctuating symptoms, with weakness greatest toward the end of the day or after exertion. In severe cases, paralysis of the respiratory muscles can lead to death. Treatment directed at enhancing cholinergic transmission, alone or combined with thymectomy or immunosuppression, is highly effective in most patients.

Progress toward achieving an understanding of the cause of myasthenia gravis was first made when electrophysiological analysis of involved muscle revealed that the amplitude of the miniature EPP was decreased, although the frequency of quantal events was normal. This finding suggested either a defect in the postsynaptic response to ACh or a reduced concentration of ACh in the synaptic vesicles. A major breakthrough occurred in 1973, when Patrick and Lindstrom found that symptoms similar to those of humans with myasthenia developed in rabbits immunized with AChR protein purified from the electric eel. This finding was shortly followed by the demonstration of anti-AChR antibodies in human patients with myasthenia gravis and a severe reduction in the surface density of AChR in the junctional folds. These anti-AChR antibodies are directed against one or more subunits of the receptor, where they bind and activate complement and accelerate destruction of the receptors. The most common target of these antibodies is a region of the AChR α subunit called MIR (main immunogenic region).

Myasthenia gravis is now recognized to be an acquired autoimmune disorder in which the spontaneous production of anti-AChR antibodies results in progressive loss of muscle AChRs and degeneration of postjunctional folds. Treatment is aimed at either reducing the potency of the immunological attack or enhancing cholinergic activity within the synapse. Reduction of the potency of the immunological attack is achieved by the use of immunosuppressants (most commonly corticosteroids) or plasmapheresis (removal of antibodies from the patient’s serum). Some patients with myasthenia gravis have a thymoma (a tumor of the thymus gland) that is often readily seen on routine chest radiographs. In these patients, removal of the thymoma leads to clinical improvement in nearly 75% of the cases. Enhancement of cholinergic activity is achieved through the use of AChE inhibitors; pyridostigmine is the most widely used agent. The dosage of these drugs must be carefully monitored to prevent overexposure of the remaining AChRs to ACh. Overexposure can lead to overstimulation of the postsynaptic receptors, prolonged depolarization of the postsynaptic membrane, inactivation of neighboring Na+channels, and thus synaptic blockade.

Another condition characterized by progressive muscle weakness and fatigue is the Lambert-Eaton syndrome (see the box titled Ca2+ Channel and Autoimmune Genetic Defects in Chapter 7). Lambert-Eaton syndrome is caused by antibodies that attack the presynaptic Ca2+channel and can be distinguished from myasthenia gravis in several ways. First, it primarily attacks the limb muscles, not the ocular and bulbar muscles. Second, repetitive stimulation of a particular muscle leads to enhanced amplitude of the postsynaptic action potential, whereas in patients with myasthenia, repetitive stimulation leads to progressive lessening of the action potential. Thus, repeated muscle stimulation leads to increasing contractile strength in patients with Lambert-Eaton syndrome and to decreasing strength in patients with myasthenia.

The term congenital myasthenic syndrome refers to a variety of inherited disorders, present at birth, that affect neuromuscular transmission in a variety of ways. Because specific cases can involve AChE deficiency, abnormal presynaptic release of ACh, or defective AChR function (without the presence of antireceptor antibodies), the signs and symptoms can also vary widely. In 1995, an unusual example of a congenital myasthenic syndrome disorder was traced to a mutation in the image subunit of the human AChR. Single-channel recordings from biopsy samples of muscle fibers of a young myasthenic patient revealed a profound alteration in AChR kinetics. The burst duration of AChR openings was greatly prolonged in comparison with that of normal human AChR channels. The molecular defect is a point mutation of Thr to Pro at position 264 in the adult image subunit of the AChR. This amino acid residue corresponds to an evolutionarily conserved position in the M2 membrane-spanning segment, which is involved in formation of the channel pore. Thus, a human mutation in the pore region of the AChR protein results in failure of the channel to close normally, thereby causing excessive depolarization and pathological consequences at the muscle end plate.

This mutation is only one of at least 53 mutations in 55 different kinships that have been identified in the AChR. Some of the other mutations result in electrophysiological changes similar to those described earlier. Thus, failure of neuromuscular transmission may be induced by multiple mechanisms, and even those related to the AChR can have many causes.

ω-Conotoxin blocks Ca2+ channels that mediate Ca2+ influx into nerve terminals, inhibiting synaptic transmission

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 (see Chapter 7) block N-type Ca2+ currents in a virtually 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 through 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.

Bacterial toxins such as tetanus and botulinum toxins cleave proteins involved in exocytosis, preventing fusion of 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 Clostridium 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 by 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 also have useful medical and cosmetic 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 to restore normal vision. A commercial preparation of botulinum toxin known as Botox has also gained popularity for the temporary treatment of facial wrinkles that occur in human aging.

Table 8-2 Neurotoxins That Block Fusion of Synaptic Vesicles





Botulinum B, D, F, G


Botulinum A/E


Botulinum C1


Both agonists and antagonists of the nicotinic acetylcholine receptor can prevent synaptic transmission

The ionotropic (nicotinic) AChR channel located in the postsynaptic muscle membrane (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; succinylcholine (or succinyldicholine) is a dimer of ACh linked together through 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 prolonged activation of AChRs.


Figure 8-18 Agonists and antagonists of the nicotinic AChR.

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 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 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 14), such as dry mouth, double vision, and difficulty in 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 27) 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, muscle 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 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. (See Note: Blockade of Muscle Na+Channels)

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 (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 (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). (See Note: Tubocurarine)

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 ~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 125I-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

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). Neostigmine (also called prostigmine), a synthetic anti-AChE drug that is partially analogous to physostigmine, is used to treat myasthenia gravis. As discussed earlier in the box about diseases of the human acetylcholine receptor, this disease is caused by the autoimmune destruction and loss of nicotinic AChRs at the muscle end plate. As shown in Equation 8-5, the acetyl-AChE must undergo hydrolysis to recycle AChE for its next round of catalysis. Physostigmine and neostigmine produce a carbamoylated form of AChE that is inactive. The slow hydrolysis of the carbamoylated enzyme relieves esterase inhibition.


Figure 8-19 Structures of AChE inhibitors.

Another important class of synthetic AChE inhibitors consists of organophosphorus compounds, which are irreversible inhibitors. These inhibitors are typified by diisopropylfluorophosphate (DFPFig. 8-19). Such compounds react with the serine residue of AChE and form an essentially irreversible covalent modification of the enzyme. Such agents rank high among the most potent and lethal of toxic chemicals. Their devastating effect is due to 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 lethality of these compounds dramatically underlines the essential role of AChE in terminating cholinergic neurotransmission. Chemical warfare agents (i.e., “nerve gas” such as sarin) are volatile forms of these compounds. Related compounds, such as Malathion (Fig. 8-19), which are relatively selective for insects, are widely used as agricultural insecticides. (See Note: Depolarization Blockade)

A natural organophosphorus neurotoxin is produced by Anabena flos-aquae, a toxic cyanobacterium (blue-green alga). Known as anatoxin-a(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.


Books and Reviews

Engel AG, Ohno K, Sine SM: Congenital myasthenic syndromes: A diverse array of molecular targets. J Neurocytol 2003; 32:1017-1037.

Jahn R: Principles of exocytosis and membrane fusion. Ann N Y Acad Sci 2004; 1014:170-178.

Katz B: Nerve, Muscle, and Synapse. New York: McGraw-Hill, 1966.

Lichman JW, Sanes JR: Watching the neuromuscular junction. J Neurocytol 2003; 32:767-775.

Südhof TC: The synaptic vesicle cycle. Annu Rev Neurosci 2004; 27:509-547.

Van der Kloot W, Molgó J: Quantal acetylcholine release at the vertebrate neuromuscular junction. Physiol Rev 1994; 74:899-989.

Journal Articles

Brejc K, van Dijk WJ, Klaassen RV, et al: Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors. Nature 2001; 411:269-276.

Del Castillo J, Katz B: Interaction at end-plate receptors between different choline derivatives. Proc R Soc Lond B Biol Sci 1957; 146:369-381.

Fatt P, Katz B: Spontaneous subthreshold activity at motor nerve endings. J Physiol 1952; 117:109-128.

Furshpan EJ, Potter DD: Transmission at the giant motor synapse of the crayfish. J Physiol 1959; 145:289-325.

Magleby KL, Stevens CF: A quantitative description of end-plate currents. J Physiol 1972; 233:173-197.

Noda M, Takahashi H, Tanabe T, et al: Structural homology of Torpedo californica acetylcholine receptor subunits. Nature 1983; 302:528.

Ohno K, Hutchinson DO, Milone M, et al: Congenital myasthenic syndrome caused by prolonged acetylcholine receptor channel openings due to a mutation in the M2 domain of the epsilon subunit. Proc Natl Acad Sci USA 1995; 92:758-762.


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