Update on the acetylcholine receptor and the neuromuscular junction

1 Update on the acetylcholine receptor and the neuromuscular junction I A N G. M A R S H A L L CHRIS PRIOR

THE PROCESS OF N E U R O M U S C U L A R TRANSMISSION

The purpose of neuromuscular transmission, involving the release of the chemical transmitter acetylcholine, is to amplify the electrical signal from the tiny nerve ending to a level at which an electrical signal can be produced in the relatively huge muscle fibre. In other words, it is a mechanism for translating the nerve action potential into a muscle action potential. Central or reflex activity initiates the nerve action potential, which results from the rapid opening of sodium ion channels resulting in the depolarization of the nerve membrane from its resting negative potential. The opening of the sodium channels is followed by the opening of potassium channels, causing membrane repolarization. These voltage-operated sodium and potassium channels are separate channels, differentiated by time of occurrence of activation and by different pharmacological sensitivities. The sodium channels are blocked by the puffer fish toxin (tetrodotoxin), and the potassium channels by aminopyridines. At the nerve terminal the depolarization results in an opening of voltage-operated calcium channels. The resultant influx of extracellular calcium is crucial to transmitter release and indirectly results in the simultaneous fusion of many acetylcholine-containing synaptic vesicles with the presynaptic membrane, releasing their contents into the synaptic gap. After release, the acetylcholine diffuses across the synaptic gap and interacts with nicotinic acetylcholine receptors embedded in the postjunctional membrane directly opposite the release sites. This interaction causes the receptor-ion channel complex to undergo a conformational change from a closed to an open state. In the open stage the channel can conduct both sodium and potassium ions, plus other less important cations, down their respective chemical and electrical gradients; i.e. it is a relatively non-selective cation channel. The opening of the channel therefore results in a localized fall in the membrane potential in the region of the chemically excitable nicotinic receptors. In electrophysiological experiments this is measured as the endplate potential. The localized fall in membrane potential results in local circuit currents which serve to lower the membrane potential in the electrically excitable membrane in the area of the muscle adjacent to the motor end-plate. Bailli~re's Clinical Anaesthesiology-Vol. 8, No. 2, June 1994 ISBN 0-7020-1847-3

299 Copyright 9 1994, by Bailli~re Tindall All rights of reproduction in any form reserved

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This rapid fall in membrane potential, if large enough in amplitude, causes sodium ion channels to open. This represents the initiation of the muscle action potential in the same way that a central generator potential initiates a nerve action potential. The muscle action potential, like the nerve action potential, is produced by selective and separate increases in permeability to sodium and potassium ions. Its purpose is to activate the contractile machinery of the skeletal muscle, resulting in a twitch response. This chapter deals firstly with two aspects of prejunctional physiology and pharmacology--mechanisms of acetylcholine release and the role of prej unctional autoreceptors--and secondly, at the postjunctional level, with the structure and function of the nicotinic receptor, including details of its pharmacology at the molecular level.

A C E T Y L C H O L I N E STORAGE AND RELEASE

The vesicular theory of neurotransmitter release was first proposed in the early 1950s to explain observations relating to the morphology of motor nerve terminals and acetylcholine release. Thus Katz and his co-workers observed that the release of acetylcholine from motor nerve endings occurred in discrete packages or quanta (Fatt and Katz, 1952). This, coupled to the existence of a large number of small membrane-bound vesicles within the nerve terminal (de Robertis and Bennett, 1955), led to the now widely accepted theory that transmitter release is an exocytotic process whereby each quantum of acetylcholine released from the nerve terminal represents the exocytotic liberation of the contents of a single synaptic vesicle. The subsequent demonstration that synaptic vesicles do indeed contain highly concentrated levels of acetylcholine further strengthened the notion that it is the synaptic vesicles that are the source of acetylcholine released following excitation of the motor nerve terminal. In spite of the widely accepted nature of the vesicular theory of transmitter release, it is only in the last 5-10 years that the molecular mechanisms of the processes of vesicular storage and release of acetylcholine have started to become a little clearer. Thus, if acetylcholine is stored in synaptic vesicles prior to its release, there must be a system within the nerve terminal to pump the acetylcholine, synthesized in the cytoplasm by the enzyme choline acetyl-O-transferase, into the interior of synaptic vesicles against its concentration gradient. Similarly, if acetylcholine is released from the synaptic vesicles by an exocytotic process then there must be (a) a mechanism that links the initiation of exocytosis to the stimulus-induced influx of calcium ions through the nerve terminal voltage-activated calcium channels, and (b) a mechanism that promotes the fusion of the synaptic vesicle membrane with the nerve terminal membrane and the formation of a 'pore' linking the inside of the synaptic vesicle to the extracellular space of the synaptic cleft. Much progress has been made in identifying these processes following the recent characterization of a number of nerve terminal and synaptic constituents.

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Concentrative uptake of acetylcholine by synaptic vesicles As with the postjunctional nicotinic acetylcholine receptor (see subsequent sections), much of the information concerning the cholinergic synaptic vesicle storage process comes from the study of the specialized synapse found in the electric organ of marine rays such as Torpedo. High-speed centrifugation of homogenized electric organ tissue can be used to prepare extracts which contain only the nerve terminal synaptic vesicles (Nagy et al, 1976). These isolated vesicles can be used in radiotracer experiments to determine the biochemistry of the synaptic vesicle system. Such experimental approaches have revealed the existence of a two-stage concentrative uptake or transport process by which acetylcholine is loaded into synaptic vesicles (Figure 1). Firstly, protons are actively transported into the vesicle by a vesicular membrane V-type proton-pumping ATPase. This is an energy-dependent process requiring the hydrolysis of ATP to ADP for translocation of the proton. The second stage of the process involves the exchange of the intravesicular protons for cytoplasmic acetylcholine. This second process is mediated by a vesicular membrane protein, the acetytcholine transporter (AChT). ATP

"~

~

AChT-VR complex

~

ADP

Protonpumping V-type ATPase

Figure 1. The two-stage process by which cholinergic synaptic vesicles accumulate acetylcholine against its concentration gradient. The pH of the inside of the vesicle is reduced through the action of a vesicular membrane proton-pumping V-type ATPase and the internal protons are exchanged for acetylcholine by the synaptic vesicle acetylcholine transporter (AChT). Reprinted from GeneralPhrmacology23, Prior C et al, pp 1017-1022, copyright 9 (1992), with kind permission from Pergamon Press Ltd, Headington Hill Hall, Oxford OX3 0BW, UK.

Any disruption of nerve terminal energy metabolism will prevent the establishment of the transvesicular membrane proton gradient and will consequently inhibit the vesicular storage of acetylcholine. In addition, certain experimental pharmacological agents, such as quinacrine, tetraphenylboron and vesamicol, have been shown to be specific inhibitors of the acetylcholine transporter. While these agents have no clinical value in themselves, vesamicol in particular is proving valuable in providing insights

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into the physiological processes involved in transmitter mobilization (see later) and the effects of neuromuscular blocking drugs on this process. The physiology and pharmacology of the synaptic vesicle acetylcholine transport system is reviewed in detail by Parsons et al (1993). Molecular mechanism of vesicular exocytosis

Over the last few years, several protein components specific to synaptic vesicles and the nerve terminal membrane have been identified that are important with respect to the function of synaptic vesicles. Broadly speaking, these molecules can be divided into two main groups: those involved in the movement of synaptic vesicles around the nerve terminal, such as the synapsins and synaptophysin, and those thought to be involved directly in vesicular release process, such as the terminal membrane voltage-activated calcium channels, the ~-latrotoxin receptor, synaptotagmin and synaptobrevin and the guanosine triphosphate (GTP) binding protein rab3A. The former of these two groups is considered in the subsequent section on the control of mobilization. The observation that vesicular release occurs at specific locations on the nerve terminal membrane, the so-called active zones, implies the existence of specialized structures for the initiation and control of vesicular exocytosis. Active zones themselves consist of a number of active zone particles. These particles are arranged in an ordered fashion which, although there is some slight species variation, generally consists of four rows of particles arranged in two pairs (Figure 2). The fact that by using appropriate electron microscopy techniques similar ordered arrangements of synaptic vesicles can be seen in close proximity to the nerve terminal membrane led to the suggestion that each active zone particle represents a site of attachment for a synaptic vesicle in anticipation of its release. Further, the observation that in the muscle weakness disorder, Lambert-Eaton myasthenic syndrome, there is a functional loss of nerve terminal voltage-activated calcium channels (Lung et al, 1987) and a morphological loss of active zone particles (Fukunaga et al, 1982) suggests that the active zone particles are closely associated with the nerve terminal calcium channels. Thus a picture emerges of a system whereby the influx of calcium ions upon nerve terminal depolarization can be linked to the initiation of vesicular exocytosis. Presumably, calcium ions entering the nerve terminal through voltage-activated calcium channels bind to an acceptor site on either a vesicular or a nerve terminal membrane component, and this binding initiates exocytosis. The fact that in most mammalian muscle preparations, at low levels of transmitter release, the quantal release of acetylcholine is proportional to the fourth power of the extracellular calcium ion concentration, suggests the cooperative binding of four calcium ions to the intraterminal acceptor site is required for the initiation of vesicular exocytosis (for review see Silinsky, 1985). With respect to the vesicular exocytotic process itself there is much controversy and uncertainty. The vesicular theory, as originally presented, proposed that, upon exocytosis, the synaptic vesicle membrane fully merges with the nerve terminal membrane. Indeed there are several lines of evi-

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d e n c e s u p p o r t i n g this n o t i o n . T h u s , t h e n e u r o t o x i n o~-latrotoxin, w h i c h is i s o l a t e d f r o m t h e v e n o m of t h e b l a c k w i d o w s p i d e r Latrodectus mactans, i n d u c e s t h e m a s s i v e d i s c h a r g e of q u a n t a of a c e t y l c h o l i n e f r o m m o t o r n e r v e t e r m i n a l s a n d this is a c c o m p a n i e d b y a large i n c r e a s e in t h e surface a r e a o f t h e n e r v e t e r m i n a l ( F r o n t a l i et al, 1976). This is c o n s i s t e n t with t h e incorp o r a t i o n of s y n a p t i c vesicle m e m b r a n e into t h e s y n a p t i c m e m b r a n e . S e c o n dly, t h e r e is e v i d e n c e t h a t c e r t a i n s y n a p t i c vesicle m e m b r a n e c o m p o n e n t s such as t h e a c e t y l c h o l i n e t r a n s p o r t e r can b e c o m e i n c o r p o r a t e d into the

Figure 2. Freeze fracture electron micrograph showing the P-face of the nerve terminal membrane from a human biceps muscle. Note the appearance of large intramembranous particles (active zone particles) arranged in groups (active zones) comprising four parallel rows (arrowed). In the lower micrograph, two active zones can be seen located opposite the acetylcholine-receptor-richcrests of the postjunctional folds. Bars = 0.1 p~m.From Fukunaga H et al, Muscle and Nerve, copyright 9 1982 John Wiley & Sons Inc., reprinted by permission of John Wiley & Sons Inc.

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nerve terminal membrane following a sustained period of acetylcoline release (Edwards et al, 1985; Zemkova et al, 1990). Finally, following sustained quantal acetylcholine release, extracellular markers such as horseradish peroxidase can be seen to be incorporated into synaptic vesicles, suggesting that there is a retrieval process which reforms synaptic vesicles from parts of the nerve terminal membrane (Heuser and Reese, 1972). However, in spite of all the evidence suggesting that full membrane fusion can and does occur upon vesicular exocytosis, there is now considerable evidence that certain, if not all, secretory processes occur via an exocytotic mechanism which, rather than involving full membrane fusion, involves the formation of a temporary 'fusion pore' between the inside of the secretory vesicle and the extracellular space, similar in many respects to a gap junction between two cells. Thus, by measuring the changes in transmembrane capacitance during secretion in mast cells, it has been shown that prior to membrane fusion there is a short period during which the interior of the secretory granule is reversibly connected to the extracellular space (Spruce et al, 1990). It is reasonable to propose that such a fusion pore mechanism may also be involved in neurotransmitter secretion. Indeed, evidence supporting the formation of a fusion pore at the motor nerve terminal membrane includes the observation that cholinergic synaptic vesicle recycling can apparently occur over a much smaller time scale than that required for vesicular membrane capture and retrieval. Following the formation of the fusion pore and the movement of acetylcholine into the extracellular space of the synaptic cleft, one of two events can take place. If the fusion pore closes then the vesicle remains intact, apart from the nerve terminal membrane, and release has been achieved by a so-called 'kiss-and-run' mechanism. Conversely, the fusion pore may simply represent a stage leading to subsequent full membrane merging along classical lines. The 'kiss-and-run' exocytotic process may in fact be the main mechanism for the release of acetylcholine, and full membrane fusion may only occur under unusual circumstances such as following prolonged stimulation or following treatment with oL-latrotoxin. Several molecules have been identified as being potentially involved in the formation of a release-mediating fusion pore. The two most promising candidates are the synaptic vesicle protein synaptotagmin and the nerve terminal membrane protein referred to as the c~-latrotoxin receptor. It has been shown that these two proteins bind to each other (Petrenko et al, 1991) and that synaptotagmin binds to the nerve terminal membrane calcium channel (Leveque et al, 1992). Also, synaptotagmin contains a cytoplasmic sequence homologous to the regulatory C2 region of protein kinase C capable of binding calcium ions (Perin et al, 1990), and phosphorylation sites susceptible to the activity of both protein kinase C and a synaptic vesicular membrane calcium/calmodulin-dependent protein kinase II (Takahashi et al, 1991; Popoli, 1993). The proposition that the nerve terminal membrane part of the fusion pore assembly is the binding site for e~-latrotoxin provides a convenient explanation for the effects of the toxin. This model for vesicular exocytosis, in which calcium ions enter through the voltage-activated calcium channels and bind to synaptotagmin on the synap-

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tic vesicle, promoting its binding to the cx-latrotoxin receptor and the formation of a fusion pore, is unlikely to be the complete picture. Thus, quantal release can still occur in the absence of synaptotagmin (Shoji-Kasai et al, 1992) and, in addition, synaptobrevin, another component of the vesicular membrane, must play an important (but necessarily obligatory) role in exocytosis, since it has recently been shown to be the target for the enzymatic activity of botulinum toxin (Schiavo et al, 1992), an extremely potent inhibitor of acetylcholine release from motor nerve terminals. Finally, there is an increasing body of evidence suggesting a role for the hydrolysis of GTP during some stage of the synaptic vesicle exocytotic to the process, mediated via the synaptic vesicle GTP binding protein rab3A (Fisher von Mollard et al, 1991; Oberhauser et al, 1992). It is clear from the above discussions that the long-established model for the quantal release of acetylcholine still serves as a good 'skeleton' outline of the physiological processes involved in transmitter exocytosis. However, it is likely that over the next few years modern experimental techniques will fill in the 'flesh' to give a detailed molecular description of the physiological processes involved in acetylcholine storage and release. Such experimental techniques will inevitably include both the study of the functional effects of drugs and toxins such as vesamicol, botulinum toxin and oL-latrotoxin and the use of molecular biology to determine the nature and interaction of the various components of synaptic vesicles and the nerve terminal membrane.

Transmitter mobilization and prejunctional autoreceptors Based on the ability of a wide range of drug and treatments to affect the release of acetylcholine from motor nerve terminals, it is clear that there exists, within the motor nerve terminal, a complex control system which can alter the amount of acetylcholine released in response to a nerve impulse. One key element of this control system is the phenomenon known as mobilization, orginally described by Birks and Macintosh (1961). As described above, the fact that the release of acetylcholine from synaptic vesicles can only occur at active zones means that there is a limited compartment of acetylcholine which can be defined as 'available for release'. Broadly speaking, mobilization is defined as the process or processes by which reserve supplies of either acetylcholine or acetylcholinecontaining synaptic vesicles are moved into the available compartment as it is depleted by the process of vesicular exocytosis. The importance of effective mobilization becomes critical at high nerve stimulation frequencies when the acetylcholine output of the nerve terminal is greatest. Therefore, it comes as no surprise that there is evidence that acetylcholine mobilization can be altered to accommodate the high levels of acetylcholine output associated with high-frequency nerve stimulation. A model has been proposed that nicotinic acetylcholine receptors exist on nerve terminals forming part of a positive feedback control system such that acetylcholine can enhance its own release (for review see Bowman et al, 1990). According to this model, at high frequencies of nerve stimulation the normal mobilization rate of the nerve terminal is insufficient to maintain a supply of acetylcholine

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for release. However, the released acetylcholine can act on the prejunctional nicotinic acetylcholine receptors to enhance the intraterminal mobilization of neurotransmitter so that release levels are maintained. The main piece of evidence to support a role for prejunctional nicotinic acetylcholine receptors in the control of acetylcholine mobilization is the observation that nicotinic antagonists such as tubocurarine depress acetylcholine release during high-frequency nerve stimulation. Thus, in nerve/muscle preparations both in vivo and in vitro, low concentrations of all clinically evaluated muscle relaxants (including tubocurarine) produce a phenomenon known as tetanic fade (Hutter, 1952; Paton and Zaimis, 1952; Liley and North, 1953), i.e. the inability of the muscle to sustain a constant tension in response to high-frequency motor nerve stimulation (Figure 3). The train-of-four fade used by anaesthetists to monitor neuromuscular block represents the same underlying phenomenon as does tetanic fade. That the tetanic fade phenomenon is a consequence of an inability of the motor nerve terminal to maintain a constant level of acetylcholine release during the high-frequency nerve stimulation can be demonstrated by measuring the amount of acetylcholine released using either electrophysiological techniques (Glavinovic, 1979; Magleby et al, 1981; Gibb and Marshall, 1984) or biochemical techniques (Wessler et al, 1986; Vizi et al, 1987). Under appropriate conditions it is

F

T

t

F

T

T u b o c u r a r i n e (2.5 x 10 -7 M)

Control

10 nA / 50 ms

50 ms

Figure 3. U p p e r half of figure shows the effects of the experimental steroidal nicotinic antagonist Org 8764 (5 x 10 -5 M) on the tension response to single and tetanic motor nerve stimulation. Trace shows single twitches at 0.1 Hz, train-of-four twitches at 2 Hz for 2 s (F) and tetanic contractions elicited at 50 Hz for 2 s (T). Arrows indicate exposure to the nicotinic antagonist. Lower half of figure shows the effect of the nicotinic antagonist tubocurarine (2.5 x 10-7M) on the amplitudes of end-plate current elicited at a high frequency of m o t o r nerve stimulation. End-plate currents were elicited in a single cell at 50 Hz, - 90 m V and 22~ in the absence (left) and presence (right) of tubocurarine. F r o m Gibb and Marshall (1984, 1987), with permission.

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possible to use intracellular microelectrodes to record the stimulus-evoked electrical signals, the end-plate potentials or end-plate currents, from motor end-plates. The amplitudes of these signals reflect the amount of quantally released acetylcholine activating postjunctional nicotinic acetylcholine receptors. Consequently, they can be used as an exquisitely sensitive bioassay of the nerve-evoked release of acetylcholine. In the absence of nicotinic antagonists, high-frequency nerve stimulation leads to a small decrease in the amplitude of successively recorded end-plate potentials or end-plate currents (Figure 3). The fact that this rundown is small, perhaps only 20-30%, indicates that acetylcholine mobilization is reasonably able to maintain a supply of synaptic vesicles for release during the high-frequency nerve stimulation. However, even very small concentrations of tubocurafine produce a profound enhancement of the rundown of end-plate signals (Figure 3). Analysis of the end-plate signals has shown that this tubocurarine-enhanced rundown is a consequence of decreased acetylcholine release and not of any changes in postjunctional receptor-ion channel activity. In conclusion, in the presence of tubocurarine, acetylcholine mobilization is not sufficient to sustain release during high-frequency motor nerve stimulation. According to the proposed model, tubocurarine itself does not impair acetylcholine mobilization. Rather, it acts to prevent acetylcholine increasing its own mobilization. So far, it has not been possible to show that this effect of tubocurarine is antagonized by acetylcholine agonists or that acetylcholine agonists can potentiate release. This is possibly a consequence of the acetylcholine agonist producing complicating effects such as postjunctional nicotinic acetylcholine receptor activation and prejunctional and postjunctional nicotinic acetylcholine receptor desensitization. However, it has been demonstrated, using radiochemical techniques to measure acetylcholine release from motor nerve terminals by overflow, that during high-frequency motor nerve stimulation (a) nicotinic antagonists depress acetylcholine release, (b) nicotinic agonists enhance release, and (c) nicotinic agonists can reverse the effects of nicotinic antagonists on release (for review see Wessler, 1989). Hence the biochemical observations are entirely consistent with the model of prejunctional control of acetylcholine mobilization described above. A complete molecular description of the processes controlling acetylcholine mobilization is still to be determined. Recent evidence has implicated a number of newly identified nerve terminal components as potentially being involved in these processes. The synapsins are a group of four homologous proteins found on the surface of synaptic vesicles (for review see Bahler et al, 1990; de Camilli et al, 1990). They have a number of properties that make them strong candidates for an involvement in mobilization. Thus, like synaptotagmin, the synapsins contain a binding site for a synaptic vesicleassociated calcium/calmodulin-dependent protein kinase II (Benfenati et al, 1992). Phosphorylation of synapsin by the vesicular membrane protein kinase to which it is attached uncouples this binding, i.e. it frees the synaptic vesicle from the synapsin. Further, the synapsins can bind to actin, one of the major components of the nerve terminal cytoskeleton. The implication is that dephosphorylated synapsin can act as an anchor fixing the synaptic

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Synapsin

II Synaptic Vesicle

'1

Oa,modu,in I §

Ca

Active Zone

Figure 4. Model illustrating th e potential role of the actin-rich cytoskeletal matrix, the synapsins and calcium/calmodulin-dependent protein kinase II (PKII) in the control of vesicular mobilization within cholinergic motor nerve terminals.

vesicle--via a vesicular membrane calcium/calmodulin-dependent protein kinase II--to the nerve terminal cytoskeletal matrix, hence reducing its 'availability' for release. Phosphorylation of the synapsin frees the vesicle from the actin chain and facilitates its movement towards, and attachment to, the active zone sites, increasing their availability for release without affecting the release mechanism itself (Figure 4). Thus, in this model the mobilization of synaptic vesicles is simply a function of the level of activity of the vesicular membrane calcium/calmodulin-dependent protein kinase II. In support of this model, it has been shown in the squid giant synapse that the intraterminal microinjection of activated calcium-calmodulindependent protein kinase enhances transmitter release, while the microinjection of dephosphorylated synapsin depresses release (Llinas et al, 1985; Lin et al, 1990). Calmodulin is found in abundance within nerve terminals, and so it is apparent that the ultimate control of mobilization may involve the binding of intraterminal calcium ions to this regulatory protein. The link between the putative prejunctional nicotinic acetylcholine receptor and the binding of calcium to calmodulin remains to be elucidated. POST JUNCTIONAL NICOTINIC ACETYLCHOLINE RECEPTORS Molecular construction of nicotinic acetylcholine receptors The present-day knowledge of the nicotinic receptor has been largely achieved with the help of two gifts from nature. The first is the abundance of nicotinic receptor material in the electric organs of electric fish such as the marine ray, Torpedo, and the electric eel, Electrophorus. The second is the

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very strongly binding nicotinic antagonist a-bungarotoxin (Chang and Lee, 1963), which has been used to label and isolate the nicotinic receptor material. The above, coupled with modern electrophysiological recording techniques and molecular biology techniques, have provided great insight into the structure and function of the receptor. All nicotinic receptors so far isolated and characterized function as cation channels, activation of which causes a change in membrane potential; for example, the end-plate potential at the neuromuscular junction. As such, the nicotinic acetylcholine receptor behaves in a similar way to receptors for several other known chemical neurotransmitters. There is, in fact, a family of closely related receptors of which the nicotinic receptor is one member, the others being the 5-HT3 receptor, the GABAA receptor, the glycine receptor and the kainate-type glutamate receptor. Nicotinic receptors themselves exist not only on muscle, but in neuronal tissue (e.g. ganglia), and although the exact structure of neuronal receptors is not as well documented as that for the muscle-type receptor, they also belong to the above receptor family (Lindstrom et al, 1990; Deneris et al, 1991). The common element in the receptor family is that the receptors are made up of five glycosylated protein subunits of varying molecular type. In mature skeletal muscle five different subunits have been identified, designated a, [3, % 8, and e. in Torpedo embryonic and denervated muscle, the subunit composition of the whole receptor protein is two o~subunits, each approximately 40kDa, one [~ subunit at 49kDa, one ~/subunit at 60kDa and one subunit at 67kDa. In mammalian muscle there is a change in subunit composition postnatally (Mishina et al, 1986), at a time when the size of the end-plates and the number of receptors are increasing. At this point the synthesis of the mRNA for the -/subunit is superseded by the synthesis of the mRNA for an e subunit (Takai et al, 1985). This change from ~ to e is accompanied by a decrease in the single channel conductance of the resultant ion channel (Schuetze, 1986). As a result, in adult mammalian muscle, where virtually all the receptors are concentrated at the end-plate, the subunit composition is two ~ units and one [3, ~ and e. The spread of denervation results in a reappearance of the ~/subunit at areas away from the original end-plate region. Each of the subunits traverses the muscle membrane at the end-plate region and the subunits are arranged to form the walls of an aqueous pore (Figure 5). This represents the ion channel through which mainly sodium and potassium ions flow to produce the single channel current measurable by the patch clamp technique. The open times of the square-wave single channel currents are exponentially distributed (Neher and Sakmann, 1976). As a result, when thousands of channels are activated simultaneously by normal quantal release of acetylcholine, the resultant end-plate current increases exponentially. The relationships between the single channel current, the basic unitary event of transmission, and the subsequent electrical events that lead to muscle contraction are summarized in Table 1. The receptor ion channel structure has a large extracellular component and a smaller intracellular component, both forming large openings to the outside and the inside of the fibre respectively, and a narrow transmembrane

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I. G. MARSHALL AND C. PRIOR mature/innervated

fetal/denervated

OUTSIDE

AChR membrane

I m

10nM INSIDE Figure 5. Schematic representation of the mature (left) and fetal (right) nicotinic acetylcholine receptors (AChR) showing the arrangement of the five protein subunits within the lipid membrane to create a central pore which acts as the cation channel. Adapted from Ward et al (1993), with permission.

Table 1. Electrical and mechanical events associated with neuromuscular transmission, their properties, and the effects of competitive antagonism. Event

Properties

Effect of competitive antagonist

Single channel opening

Uniform amplitude Open time exponentially distributed

Reduced frequency of occurrence Open time distribution unaffected

End-plate current

Many thousands of single channel events summated Exponential decay time Graded according to amount of ACh released

Reduced in amplitude Exponential decay time unaffected

End-plate potential (EPP)

As for EPC, i.e. graded Decay reflects capacitive properties of membrane

Reduced in amplitude EPP in some fibres will not reach threshold

Junctional action potential

Triggered by EPP reaching threshold All-or-nothing response

All-or-nothing In some fibres EPP will not reach action potential firing threshold

Contraction

All-or-nothing response in each fibre Maximal contraction results from all fibres in muscle contracting

All-or-nothing in each fibre Some fibres will not contract, therefore graded reduction in total population of contracting fibres Maximal twitch reduces in amplitude

(EPC)

Ach, acetylcholine.

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311

Figure 6. Arrangement of a single subunit of the nicotinic acetylcholine (ACh) receptor within the lipid bilayer. Each subunit comprises four membrane-spanning c~helices (M1-M4), a large N-terminal cytoplasmic domain containing the agonist and antagonist binding sites, and a large intracellular domain between M3 and M4 containing a number of potential modulatory phosphorylation sites.

region. Within each subunit of the receptor complex there is a considerable degree of similarity, each subunit having regions of high amino acid sequence homology, four transmembrane domains, a long extracellular N-terminal chain and a short intracellular chain. Within the subunit composition there are alternating hydrophilic and hydrophobic sequences, with the hydrophobic domains constituting the transmembrane components which are in close proximity to the lipid bilayer of the membrane. These transmembrane domains are designated M1 to M4 (Figure 6). Between the transmembrane domains M3 and M4 there is a long cytoplasmic portion of the subunit which constitutes the bulk of the intracellular part of each subunit, and hence of the receptor ion channel itself. Within this part of the subunit there are regions thought to be the sites of phosphorylation associated with the process of receptor desensitization. The M2 transmembrane domain is believed to be the part of each subunit that forms the inner wall of the ion channel (Imoto et al, 1986). Thus, the channel is lined by five c~helices of the M2 transmembrane domain, one from each subunit of the receptor.

Drug interaction with nicotinic acetylcholine receptors In Torpedo receptors the two c~ subunits are separated by the 8 subunit on one side and the y and ~ subunits on the other side. Thus one o~ subunit is

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adjoined by the [3 and the -/subunits, and the other by the [3 and g subunits. Each receptor possesses two binding or recognition sites for acetylcholine itself, other nicotinic agonists, and competitive antagonists such as tubocurarine and other clinically used muscle relaxant drugs. The two binding sites are situated on the two a subunits, on the large, extracellular Nterminal parts of the subunit molecules. The binding site has been localized to a 30 amino acid region of the N-terminal (positions 172-201) which forms a loop linked through a disulphide link between two cysteine components. Despite the fact that the two a subunits appear to be identical, their pharmacological binding properties are different (Popot and Changeaux, 1984; Colquhoun, 1986; Blount and Merlie, 1988). This leads us to the notion that pharmacological functioning of the receptors is related not just to the subunit containing the binding site itself, but to the relationship between this subunit and its neighbouring subunit. The common [3 subunit does not seem to play a part in defining the binding characteristics (Karlin, 1987), but when the a subunit is expressed with either the ~ or the g subunit, the dimers formed have different binding capabilities. Thus, it appears that the binding sites for agonists and antagonists lie on the a subunits close to the junctions between the a subunits and the ~ and ~ subunits respectively (Pederson and Cohen, 1990). Neurotransmitter acetylcholine must bind to both binding or recognition sites on the two c~ subunits for the ion channel to open and to produce the single channel current. When competitive antagonists such as tubocurarine are introduced, the resultant occupation of the binding sites results in acetylcholine having a reduced chance of binding and opening the channel. As a result, the frequency of opening of the channel is reduced. The resultant effects of competitive antagonists on the different components of transmission are shown in Table 1. It is known that tubocurarine has different binding affinities for the two o~ subunits and it is possible that differential binding of other chemical classes of muscle relaxants to the two subunits might help explain some of the potentiation interactions that can be observed clinically with such agents (Pedersen and Cohen, 1990). Another potential cause of drug interactions at the neuromuscular junction is end-plate ion channel block produced by other agents, or even muscle relaxants themselves, used during anaesthesia or as premedication. Such agents include general anaesthetics, local anaesthetics and antibiotics. The most widely documented type of channel block is the 'open channel block' (Lambert et al, 1983). This type of block can occur with drugs that have little or no affinity for the binding or recognition sites on the o~subunits of the receptor, but which, when the channel is opened by acetylcholine binding to the recognition sites, enter the channel and bind to the amino acid residues of the M2 transmembrane domains of the receptor subunits. The result is that the activated or open form of the receptor becomes blocked. This type of block is non-competitive in nature, as increasing the agonist concentration will lead to more open channels and hence provide more opportunity for the blocking compound to act. As a result, neuromuscular block of the open channel type is not reversible by anticholinesterase agents.

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SUMMARY The skeletal muscle neuromuscular junction is one of the most accessible synapses in the body, and hence is one of the most widely studied. Many of the concepts of neurochemical transmission have been built on observations made at the neuromuscular junction. As a result, there is widespread knowledge of the basic process of neuromuscular transmission and the effects of clinically used drugs that act on neuromuscular transmission are relatively well understood. Nevertheless, the process of neuromuscular transmission continues to provide a rich source of new information, much of which can be exploited at less accessible synapses. This chapter presents a brief overview of the established components of neuromuscular transmission, and is followed by more detailed descriptions of recent advances in the understanding of the vesicular storage of acetylcholine, the molecular mechanisms of synaptic vesicle mobilization and exocytosis, and the structure and function of the postjunctional nicotinic acetylcholine receptor.

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