Synaptic Transmission and the Neuromuscular Junction

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-µm ² 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, Bernard 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 10 ⁵ . 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, 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 via multiple aqueous channels.

In contrast to those of 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 pre synaptic 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.

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 (or adrenaline), elicit physiological effects (e.g., increases in heart rate and blood pressure) that are similar to those elicited by stimulation of sympathetic nerve fibers. In 1904, Thomas R. Elliot proposed that sympathetic nerves might release a substance analo­gous to epinephrine—later identified as norepinephrine—that would function in chemical transmission between a nerve and its target organ.

Subsequent studies by Otto Loewi suggested that the vagus nerve, which is parasympathetic, produces a substance responsible for depression of the heartbeat. His classic 1921 experiment 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 Henry 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 Henry Dale received the Nobel Prize in Physiology or Medicine in 1936. N8-1

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) via 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—these are termed reciprocal synapses. In other words, the current passing through the gap junction is ohmic; it varies linearly with the transjunctional voltage (i.e., the V _m difference between the two cells). However, the crayfish synapse described by Furshpan and Potter allows depolarizing current to pass readily in only 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.

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 (see pp. 315–317 ), that diffuse back into the presynaptic terminal and modulate the strength of the synaptic connection. Furthermore, the presynaptic membrane at many synapses has receptors that may either inhibit or facilitate the release of transmitter. 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. Vesicular transporters concentrate neuro­transmitters inside the vesicle using the energy of an H ⁺ electrochemical gradient.

• Step 2: An action potential, which involves voltage-gated Na ⁺ and K ⁺ channels (see pp. 176–177 ), arrives at the presynaptic nerve terminal.

• Step 3: Depolarization opens voltage-gated Ca ²⁺ channels, which allows Ca ²⁺ to enter the presynaptic terminal.

• Step 4: The increase in intracellular Ca ²⁺ concentration ([Ca ²⁺ ] _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 (a) enzymatic destruction of the transmitter (e.g., hydrolysis of ACh by ace­tylcholinesterase), (b) uptake of transmitter into the presynaptic nerve terminal or into other cells by Na ⁺ -dependent transport systems, or (c) diffusion of the transmitter molecules away from the synapse.

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 pp. 314–322 ). These molecules include both small organic molecules, such as norepinephrine, ACh, serotonin (5-hydroxytryptamine [5-HT]), glutamate, gamma-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 pp. 51–66 ). Several neurotransmitter molecules—such as ACh, glutamate, serotonin, GABA, and glycine—serve as ligands (agonists) for both types of receptors. Agonist-gated receptors that are also ion channels are known as ionotropic receptors. Receptors coupled to G proteins are called metabotropic receptors because their activation initiates a metabolic process involving GTP.

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, 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. 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 nicotinic AChR (see Fig. 8-3 A ), and (2) the G protein–linked AChR at the atrial parasympathetic synapse of the heart, a metabotropic receptor also known as the muscarinic AChR (see Fig. 8-3 B ). The nicotinic versus muscarinic nomenclature is pharmacological 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 pp. 197–198 ) via 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 p. 492 ). These two receptor types provide the molecular explanation for the puzzling observations of early physiologists that ACh (Vagusstoff) activates skeletal muscle but inhibits heart muscle.

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 located in the ventral horn of the spinal cord have long axons that branch extensively near the point of contact with the target muscle ( Fig. 8-4 ). Each axon process innervates 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.

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 (see 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 p. 25 ) translocates them to the nerve terminal. The quantal nature of transmitter release (described below 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 via 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 pp. 118–119 ). 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.

ACh activates nicotinic AChRs 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 ( V _m ) at the postsynaptic region (i.e., muscle cell) of the neuromuscular junction. Nerve stimulation normally drives the V _m of the muscle above threshold and elicits an action potential (see p. 173 ). 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 below. N8-2 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 V _m at one location of the muscle cell called the neuromuscular junction or NMJ.

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: V _m 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 V _m 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 (see pp. 201–203 ) 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 V _m above threshold and consequently trigger a regenerating action potential (see p. 173 ).

What ions pass through the AChR channels during generation of the EPP? This question can be answered by the same voltage-clamp technique N7-4 that was used to study the basis of the action potential (see Fig. 7-5 B ). Figure 8-6 A illustrates 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 V _m . 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 (see Fig. 8-6 B ). 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.

As shown in Figure 8-6 B , 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 V _m is made more positive, and the current reverses direction to become an outward current at positive values of V _m . A plot of the peak current versus the clamped V _m shows that the reversal potential for the EPC is close to 0 mV (see Fig. 8-6 C ). 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 Ca ²⁺ but not to anions such as Cl ⁻ . Because of its low extracellular concentration, the current attributable to Ca ²⁺ is small under physiological conditions and its contribution to reversal potential can be ignored. N8-3 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 P _Na / P _K ratios of ~20, and voltage-gated K ⁺ channels, which have P _K / P _Na ratios of >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 V _m 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 V _m shifts to a value between E _K (approximately −80 mV) and E _Na (approximately +50 mV).

N8-3

Contribution of Ca ²⁺ to the Resting Membrane Potential

Equation 6-9 in the text (shown here as Equation NE 8-1 ) is the Goldman-Hodgkin-Katz (GHK) voltage equation:

Of course, we could insert additional terms for other cations besides K ⁺ and Na ⁺ ; for example, if we included Ca ²⁺ , the equation would look something like the following *

* The GHK equation has dropped the z (valence) term, as if all ions were monovalent. In order to insert Ca ²⁺ into this simple equation, we treat the ion as if it were monovalent, which is clearly not the case. Thus, this equation merely serves to make the point that Ca ²⁺ contributes very little to V _m because of the small magnitude of the product of permeability and concentration.

:

A typical value for [Ca ²⁺ ] _i would be 10 ⁻⁷ M or 0.0001 mM, and a typical value for [Ca ²⁺ ] _o would be 1.2 mM. Thus, even though the concentration ratio for Ca ²⁺ across the plasma membrane is large, this ratio per se has no bearing on the GHK equation. What counts here are the magnitudes of the product P _Ca [Ca ²⁺ ], which are generally small compared to the other terms in both the numerator and denominator. Thus, Ca ²⁺ makes very little contribution to V _m in the resting state. However, if we were to reduce the size of the other terms in either the numerator or denominator, the Ca ²⁺ would begin to matter.

As we shall see in Chapter 13 , which focuses on synaptic transmission in the CNS, similar principles hold for the 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 depolarizing excitatory postsynaptic potentials. In contrast, the receptor-gated channels for glycine and GABA are anion selective and drive V _m in the hyperpolarizing direction, toward the equilibrium potential for Cl ⁻ . These hyperpolarizing postsynaptic responses are called inhibitory postsynaptic potentials.

The nicotinic AChR 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, molecular cloning, and cryoelectron microscopy. 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.

N8-14

Structure of the Nicotinic Acetylcholine Receptor

The nicotinic acetylcholine receptors (AChRs), which are all ligand-gated ion channels, come in two major subtypes, N ₁ and N ₂ . The N ₁ nicotinic AChRs are at the neuromuscular junction, whereas the N ₂ AChRs are found in the autonomic nervous system (on the postsynaptic membrane of the postganglionic sympathetic and parasympathetic neurons) and in the CNS. Both N ₁ and N ₂ are ligand-gated ion channels activated by ACh or nicotine. However, whereas the N ₁ receptors at the neuromuscular junction are stimulated by decamethonium and preferentially blocked by d -tubocurarine and α-bungarotoxin, the autonomic N ₂ receptors are stimulated by tetramethylammonium, blocked by hexamethonium, but resistant to α-bungarotoxin. When activated, N ₁ and N ₂ receptors are both permeable to Na ⁺ and K ⁺ , with the entry of Na ⁺ dominating. Thus, the nicotinic stimulation leads to rapid depolarization.

The nicotinic AChRs in skeletal muscle and autonomic ganglia are heteropentamers. That is, five nonidentical protein subunits surround a central pore, in a rosette fashion. N6-20 Because the five subunits are not identical, the structure exhibits pseudo symmetry, rather than true symmetry. There are at least ten α subunits (α ₁ to α ₁₀ ) and four β subunits (β ₁ to β ₄ ). As we will see below, the basis for these differences is a difference in subunit composition.

The N ₁ receptors have different subunit compositions depending upon location and developmental stage. The subunit composition of α ₂ βγδ is found in fetal skeletal muscle, as well as the nonjunctional regions of denervated adult skeletal muscle. The electric organ of the electric eel (Torpedo), from which the channel was first purified, has the same subunit composition. The subunit composition of α ₂ βεδ is found at the neuromuscular junction of adult skeletal muscle. Here, the ε subunit replaces the γ subunit. In both the α ₂ βγδ and α ₂ βεδ pentamers, the α subunits are of the α ₁ subtype and the β subunits are of the β ₁ subtype.

In the Torpedo N ₁ AChRs, the α, β, γ, and δ subunits have polypeptide lengths of 437 to 501 amino acids. eFigure 8-1 shows side and top views of this AChR.

The N ₂ receptors in the nervous system, like those in muscle, are heteromers, probably heteropentamers. N ₂ receptors use α ₂ to α ₁₀ and β ₂ to β ₄ .

Nicotinic Receptors
Receptor Type	Agonists	Antagonists
N 1 Nicotinic ACh	ACh (nicotine, decamethonium)	d -tubocurarine α-bungarotoxin
N 2 Nicotinic ACh	ACh (nicotine, tetramethylammonium)	Hexamethonium

References

• Unwin N: Refined structure of the nicotinic acetylcholine receptor at 4Å resolution. J Mol Biol 2005; 346: pp. 968-989.
• Unwin N, Fujiyoshi Y: Gating movement of acetylcholine receptor caught by plunge-freezing. J Mol Biol 2012; 422: pp. 617-634.

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. The two ACh binding sites are located at the extracellular α/γ interface of one α subunit and the α/δ interface of the other α subunit. N8-4