Chapter 11:  Acetylcholine

Acetylcholine receptors have been classified into subtypes based on the pharmacology of the receptors

Initially, subtyping of the receptors in the cholinergic nervous system was based on the pharmacological activity of two alkaloids: nicotine and muscarine (Fig. 11-2). This classification occurred long before the structures of these naturally occurring agonists were determined (Fig. 11-3). The greatly different activities of the antagonists atropine on muscarinic receptors and d-tubocurarine on nicotinic receptors further supported the argument that multiple classes of receptors exist for ACh. Subsequently, it was found that all nicotinic receptors are not identical. Nicotinic receptors in the neuromuscular junction, sometimes denoted as N₁ receptors, show selectivity for phenyltrimethylam-monium as an agonist; elicit membrane depolarization in the presence of bisquaternary agents, with decamethonium being the most potent; are preferentially blocked by the competitive antagonist d-tubocurarine; and are blocked irreversibly by the snake α-toxins. Nicotinic receptors in ganglia, N₂ receptors, are stimulated preferentially by 1,1-dimethyl-4-phenylpiperazinium; blocked competitively by trimethaphan; blocked noncompetitively by bisquaternary agents, with hexamethonium being the most potent; and resistant to the snake α-toxins [9]. A large number of distinct neuronal nicotinic receptors are found in the central nervous system (CNS); they are closer relatives of the nicotinic receptors in ganglia than of those in muscle.

Muscarinic receptors also exhibit distinct subtypes. The antagonist pirenzepine (PZ) has the highest affinity for one subtype, M₁, which is found mainly in neuronal tissues. Another antagonist, methoctramine, has a higher affinity for M₂ receptors, which are the predominant muscarinic receptor subtype in mammalian heart. Hexahydrosiladifenidol is relatively selective for the M₃ receptors present in smooth muscle and glands, whereas himbacine exhibits high affinity for M₄ receptors. With this level of multiplicity of receptor subtypes, limitations on specificity preclude a single antagonist defining a distinct subtype.

The intrinsic complexity and the multiplicity of cholinergic receptors became evident upon elucidation of their primary structures

In the CNS, at least eight different sequences of α subunits and three different sequences of β subunits of the nicotinic receptor have been identified [10,11]. Expression of the cloned genes encoding certain subunit combinations yields functional receptors with different sensitivities toward various toxins and agonists.

At least five distinct muscarinic receptor genes have been cloned and sequenced. The genes are called m₁ to m₅. The m₁ to m₄ clones correlate with the M₁ to M₄ receptors identified pharmacologically. The subtypes differ in their ability to couple to different G proteins and, hence, to elicit cellular signaling events. The muscarinic and nicotinic receptor subtypes exhibit distinct regional locations of their mRNAs, based on in situ hybridization.

Thus, cholinergic receptor classification can be considered in terms of three stages of development. Initially, Dale [2] distinguished nicotinic and muscarinic receptor subtypes with crude alkaloids. Then, chemical synthesis and structure—activity relationships clearly revealed that nicotinic and muscarinic receptors were heterogeneous but could not come close to uncovering the true diversity of receptor subtypes. Lastly, analysis of subtypes comes from molecular cloning, which makes possible the classification of receptors on the basis of primary structure (Fig. 11-2).

Both muscarinic and nicotinic responses are found in brain and spinal cord

A few specific central cholinergic pathways have been characterized. For example, Renshaw cells in the spinal cord play a role in modulating motoneuron activity by a feedback mechanism. Stimulation of Renshaw cells occurs through branches of the motoneuron, and the transmitter is ACh acting on nicotinic receptors. Both nicotinic and muscarinic receptors are widespread in the CNS. Muscarinic receptors with a high affinity for PZ, M₁ receptors, predominate in the hippocampus and cerebral cortex, whereas M₂ receptors predominate in the cerebellum and brainstem and M₄ receptors are most abundant in the striatum. Nicotinic receptors may be largely prejunctional. The mapping of cholinergic pathways in the brain continues to be pursued actively and relies on several techniques [12]. Histochemical studies utilizing antibodies selective for ChAT and presynaptic transport proteins, along with receptor autoradiography with labeled ligands, have produced detailed maps of the CNS. In addition, the nerve cell bodies containing the mRNA encoding these proteins have been defined through in situ hybridization with a cDNA or antisense mRNA. Studies involving iontophoretic application of transmitter, local stimulation and intracellular or cell-surface measurements of responses establish appropriate functional correlates.

Neurotransmission in autonomic ganglia is more complex than depolarization mediated by a single transmitter

In autonomic ganglia, the primary electrophysiological event following preganglionic nerve stimulation is the rapid depolarization of postsynaptic sites by released ACh acting on nicotinic receptors. This activation gives rise to an initial excitatory postsynaptic potential (EPSP), which is due to an inward current through a cation channel (see Chaps. 6 and 10). This mechanism is virtually identical to that in the neuromuscular junction, with an immediate onset of the depolarization and decay within a few milliseconds. Nicotinic antagonists such as trimethaphan competitively block ganglionic transmission, whereas agents such as hexamethonium produce blockade by occluding the channel. An action potential is generated in the postganglionic nerve when the initial EPSP attains a critical amplitude.

Several secondary events amplify or suppress this signal. These include the slow EPSP; the late, slow EPSP; and an inhibitory postsynaptic potential (IPSP). The slow EPSP is generated by ACh acting on muscarinic receptors and is blocked by atropine. It has a latency of approximately 1 sec and a duration of 30 to 60 sec. The late, slow EPSP can last for several minutes and is mediated by peptides found in ganglia, including substance P, angiotensin, leutinizing hormone-releasing hormone (LHRH) and the enkephalins. The slow EPSP and late, slow EPSP result from decreased K⁺ conductance and are believed to regulate the sensitivity of the postsynaptic neuron to repetitive depolarization [13]. The IPSP seems to be mediated by the catecholamines, dopamine and/or norepinephrine and is blocked by α-adrenergic antagonists and atropine. ACh released from presynaptic terminals may act on a catecholamine-containing interneuron to stimulate the release of norepinephrine or dopamine. As in the case of the slow EPSP, the IPSP has a longer latency and duration of action than the fast EPSP. These secondary events vary with the individual ganglia and are believed to modulate the sensitivity to the primary event. Hence, drugs that selectively block the slow EPSP, such as atropine, will diminish the efficiency of ganglionic transmission rather than eliminate it. Similarly, drugs such as muscarine and the ganglion-selective muscarinic agonist McN-A-343 are not thought of as primary ganglionic stimulants. Rather, they enhance the initial EPSP under conditions of repetitive stimulation.

Since parasympathetic and sympathetic ganglia exhibit comparable sensitivities to nicotine and ACh in producing the initial EPSP, the pharmacological action of ganglionic stimulants depends on the profile of innervation to particular organs or tissues (Table 11-1). For example, blood vessels are innervated only by the sympathetic nervous system; thus, ganglionic stimulation should produce only vasoconstriction. Similarly, the pharmacological effects of ganglionic blockade will depend on which component of the autonomic nervous system is exerting the predominant tone at the effector organ.

Muscarinic receptors are widely distributed at postsynaptic parasympathetic effector sites

The response of systemically administered ACh is characteristic of stimulation of postganglionic effector sites rather than of ganglia. This is a consequence of the greater abundance of muscarinic receptors at effector sites in innervated tissues and the relatively poor blood flow to ganglia. Muscarinic receptors are found in visceral smooth muscle, in cardiac muscle, in secretory glands and in the endothelial cells of the vasculature. Except for endothelial cells, each of these sites receives cholinergic innervation. Responses can be excitatory or inhibitory, depending on the tissue. Even within a single tissue the responses may vary. For example, muscarinic stimulation causes gastrointestinal smooth muscle to depolarize and contract, except at sphincters, where hyperpolarization and relaxation are seen (Table 11-2). Smooth muscle in many tissues innervated by the cholinergic nervous system exhibits intrinsic electrical and/or mechanical activity. This activity is modulated rather than initiated by cholinergic nerve stimulation. Cardiac muscle and smooth muscle exhibit spikes of electrical activity that are propagated between cells. These spikes are initiated by rhythmic fluctuations in resting membrane potential. In intestinal smooth muscle, cholinergic stimulation will cause a partial depolarization and increase the frequency by spike production. In contrast, cholinergic stimulation of atria will decrease the generation of spikes through hyperpolarization of the membrane.

Membrane depolarization typically results from an increase in Na⁺ conductance. In addition, mobilization of intracellular Ca²⁺ from the endoplasmic or sarcoplasmic reticulum and the influx of extracellular Ca²⁺ appear to be elicited by ACh acting on muscarinic receptors (see Chap. 23). The resulting increase in intracellular free Ca²⁺ is involved in activation of contractile, metabolic and secretory events. Stimulation of muscarinic receptors has been linked to changes in cyclic nucleotide concentrations. Reductions in cAMP concentrations and increases in cGMP concentrations are typical responses (see Chap. 22). These cyclic nucleotides may facilitate contraction or relaxation, depending on the particular tissue. Inhibitory responses also are associated with membrane hyperpolarization, and this is a consequence of an increased K⁺ conductance. Increases in K⁺ conductance may be mediated by a direct receptor linkage to a K⁺ channel or by increases in intracellular Ca²⁺, which in turn activate K⁺ channels. The mechanisms through which muscarinic receptors couple to multiple cellular responses are considered later.

Stimulation of the motoneuron for skeletal muscle results in the release of acetylcholine and contraction of the skeletal muscle fibers

Contraction and associated electrical events can be produced by intra-arterial injection of ACh close to the muscle. Since skeletal muscle does not possess inherent myogenic tone, the tone of apparently resting muscle is maintained by spontaneous and intermittent release of ACh. The consequences of spontaneous release at the motor endplate of skeletal muscle are small depolarizations from the quantized release of ACh, termed miniature endplate potentials (MEPPs) [14] (see Chap. 10). Decay times for the MEPPs range between 1 and 2 msec, a value of about the same duration as the mean channel open time seen with ACh stimulation of individual receptor molecules. Stimulation of the motoneuron results in the release of several hundred quanta of ACh. The summation of MEPPs gives rise to a postsynaptic excitatory potential (PSEP), also termed motor endplate potential. A sufficiently large and abrupt potential change at the endplate will elicit an action potential by activating voltage-sensitive Na⁺ channels. The action potential propagates in two-dimensional space across the surface of the muscle to release Ca²⁺ and elicit contraction. Therefore, the PSEP may be thought of as a generator potential. It is found only in junctional regions and arises from the opening of the receptor channel. Normal resting potentials in endplates are about −70 mV. The PSEP causes the endplate to depolarize partially to about −55 mV. It is the rapid and transient changes from −70 to −55 mV in localized areas of the endplate that triggers action potential generation [9,14].

Competitive blocking agents cause muscle paralysis by preventing access of acetylcholine to its binding site on the receptor

Competitive blockade with agents such as d-tubocurarine result in maintenance of the endplate potential at −70 mV. Without frequent PSEPs, action potentials are not triggered and there is flaccid paralysis of the muscle. The actions of competitive blocking agents can be surmounted by excess ACh. Depolarizing neuromuscular blocking agents, such as decamethonium and succinylcholine, produce depolarization of the endplate such that the endplate potential is −55 mV. The high concentrations of depolarizing agent that are maintained in this synapse do not allow regions of the endplate to repolarize, as would occur with a labile transmitter such as ACh. Since it is the transition between −70 and −55 mV that triggers the action potential, flaccid paralysis also will occur with a depolarizing block [9]. Excess ACh will not reverse the paralysis by depolarizing blocking agents. As might be expected if depolarization occurs in a nonuniform manner in microscopic areas within individual endplates and in individual motor units, the onset of depolarization blockade is characterized by muscle twitching and fasciculations that are not evident in competitive block. Once paralysis occurs, the overall pharmacological actions of competitive and depolarizing blocking agents are similar, yet intracellular measurements of endplate potential can distinguish these two classes of agent.

Acetylcholine is synthesized from its two immediate precursors, choline and acetyl coenzyme A

The synthesis reaction is a single step catalyzed by the enzyme ChAT (EC 2.3.1.6):

ChAT, first assayed in a cell-free preparation in 1943, subsequently has been purified and cloned from several sources [15]. The purification of ChAT has allowed production of specific antibodies. Whereas acetylcholinesterase (AChE), the enzyme responsible for degradation of ACh, is produced by cells containing cholinoreceptive sites as well as in cholinergic neurons, ChAT is found in the nervous system specifically at sites where ACh synthesis takes place. Within cholinergic neurons, ChAT is concentrated in nerve terminals, although it is also present in axons, where it is transported from its site of synthesis in the soma. When subcellular fractionation studies are carried out, ChAT is recovered in the synaptosomal fraction, and within synaptosomes it is primarily cytoplasmic. It has been suggested that ChAT also binds to the outside of the storage vesicle under physiological conditions and that ACh synthesized in that location may be situated favorably to enter the vesicle.

Brain ChAT has a K _D for choline of approximately 1 mM and for acetyl coenzyme A (CoA) of approximately 10 μM. The activity of the isolated enzyme, assayed in the presence of optimal concentrations of cofactors and substrates, appears far greater than the rate at which choline is converted to ACh in vivo. This suggests that the activity of ChAT is repressed in vivo. Inhibitors of ChAT do not decrease ACh synthesis when used in vivo; this may reflect a failure to achieve a sufficient local concentration of inhibitor but also suggests that this step is not rate-limiting in the synthesis of ACh.

The acetyl CoA used for ACh synthesis in mammalian brain comes from pyruvate formed from glucose. It is uncertain how the acetyl CoA, generally thought to be formed at the inner membrane of the mitochondria, accesses the cytoplasmic ChAT, and it is possible that this is a rate-limiting step.

Acetylcholine formation is limited by the intracellular concentration of choline, which is determined by uptake of choline into the nerve ending

Choline is present in the plasma at a concentration of about 10 μM. A “low-affinity” choline uptake system with a K _m of 10 to 100 μM is present in all tissues, but cholinergic neurons also have an Na⁺-dependent “high-affinity” choline uptake system, with a K _m for choline of 1 to 5 μM [16]. The high-affinity uptake mechanisms should be saturated at 10 μM choline, so the plasma choline concentration is probably adequate for sustained ACh synthesis even under conditions of high demand, as observed in ganglia. Since the plasma concentration of choline is above the K _m of the high-affinity choline-transport system, it is not expected that choline concentrations in the nerve ending would be increased by increasing the plasma concentration of choline or by changing the K _m of the uptake system. However, neuronal choline content might be changed by altering the capacity of the high-affinity choline-uptake mechanism, such as changing the maximum velocity (V _max) for transport, and this has been reported to occur in some brain regions in response to increased or decreased neuronal activity. There is some dispute about whether the capacity of the uptake system is increased or whether choline influx is regulated by changes in the intraterminal concentration of choline; it is agreed, however, that some event associated with neuronal activity enhances choline entry into neurons [16]. If the K _m of ChAT for choline in vivo is as high as that seen with the purified enzyme, one would expect ACh synthesis to increase in proportion to the greater availability of choline. Conversely, ACh synthesis should be diminished when high-affinity choline uptake is blocked. Hemicholinium-3 is a potent inhibitor of the high-affinity choline-uptake system, with a K _i in the submicromolar range (Fig. 11-4). Treatment with this drug decreases ACh synthesis and leads to a reduction in ACh release during prolonged stimulation; these findings lend support to the notion that choline uptake is the rate-limiting factor in the biosynthesis of ACh. To date, the high-affinity choline-uptake system has not been cloned successfully.

A second transport system concentrates acetylcholine in the synaptic vesicle

ACh is transported into storage vesicles following its synthesis by ChAT in the nerve ending [16]. The vesicular ACh transporter (VAChT) has been cloned and expressed. Its sequence places it in the 12-membrane-spanning family characteristic of other biogenic amine transporters found in adrenergic nerve endings [17,18]. Interestingly, the gene encoding the transporter is located within an intron of the ChAT gene, suggesting a mechanism for coregulation of gene expression for ChAT and VAChT. ACh uptake in the vesicle is driven by a proton-pumping ATPase. Coupled countertransport of H⁺ and ACh allows the vesicle to remain isoosmotic and electroneutral [16].

A selective inhibitor of ACh transport, vesamicol (Fig. 11-4), inhibits vesicular ACh uptake with an IC₅₀ of 40 nM [16–18]. Inhibition appears noncompetitive, suggesting that it acts on some site other than the ACh-binding site on the transporter. Vesamicol blocks the evoked release of newly synthesized ACh without significantly affecting high-affinity choline uptake, ACh synthesis or Ca²⁺ influx. The fact that ACh release is lost secondary to the blockade of uptake by the vesicle strongly suggests that the vesicle is the site of ACh release. The expressed transporter from the cloned cDNA also is inhibited by vesamicol [17,18].

Choline is supplied to the neuron either from plasma or by metabolism of choline-containing compounds

At least half of the choline used in ACh synthesis is thought to come directly from recycling of released ACh, hydrolyzed to choline by cholinesterase. Presumably, uptake of this metabolically derived choline occurs rapidly, before the choline diffuses away from the synaptic cleft. Another source of choline is the breakdown of phosphatidylcholine, which may be stimulated by locally released ACh. Choline derived from these two sources becomes available in the extracellular space and is then subject to high-affinity uptake into the nerve ending. In the CNS, these metabolic sources of choline appear to be particularly important because choline in the plasma cannot pass the blood—brain barrier. Thus, in the CNS, the high-affinity uptake of choline into cholinergic neurons might not be saturated and ACh synthesis could be limited by the supply of choline, at least during sustained activity. This would be consistent with the finding that ACh stores in the brain are subject to variation, whereas ACh stores in ganglia and muscles remain relatively constant.

A slow release of acetylcholine from neurons at rest probably occurs at all cholinergic synapses

This was described first by Fatt and Katz, who recorded small, spontaneous depolarizations at frog neuromuscular junctions that were subthreshold for triggering action potentials. These MEPPs were shown to be due to the release of ACh. When the nerve was stimulated and endplate potentials recorded and analyzed, the magnitude of these potentials always was found to be some multiple of the magnitude of the MEPPs. It was suggested that each MEPP resulted from a finite quantity or quantum of released ACh and that the endplate potentials resulted from release of greater numbers of quanta during nerve stimulation (see Chap. 10).

A possible structural basis for these discrete units of transmitter was discovered shortly thereafter when independent electron microscopic and subcellular fractionation studies by de Robertis and Whittaker revealed the presence of vesicles in cholinergic nerve endings. Subcellular fractionation of mammalian brain and Torpedo electric organs yields resealed nerve endings, or synaptosomes, that can be lysed to release a fraction enriched in vesicles. More than half of the ACh in the synaptosome is associated with particles that look like the vesicles seen under an electron microscope. Therefore, it is clear that ACh is associated with a vesicle fraction, and it is likely that it is contained within the vesicle. The origin of the free ACh within the synaptosome is less clear. It may be ACh that is normally in the cytosol of the nerve ending, or it may be an artifact of release from the vesicles during their preparation (see Chap. 10).

The relationship between the amount of acetylcholine in a vesicle and the quanta of acetylcholine released can only be estimated

Estimates of the amount of ACh contained within cholinergic vesicles vary, and there is obviously some subjectivity in correcting the values obtained, such as the percent of vesicles that are cholinergic or how much ACh may be lost during their preparation [19]. Whittaker estimated that there are about 2,000 molecules of ACh in a cholinergic vesicle from the CNS. A similar estimate of about 1,600 molecules of ACh per vesicle was made using sympathetic ganglia. The most abundant source of cholinergic synaptic vesicles is the electric organ of Torpedo. Vesicles from Torpedo are far larger than those from mammalian species and are estimated to contain up to 100 times more ACh, that is, 200,000 molecules per vesicle. The Torpedo vesicle also contains ATP and, in its core, a proteoglycan of the heparin sulfate type. Both of these constituents may serve as counter-ions for ACh, which otherwise would be at a hyperosmotic concentration.

The amount of ACh in a quantum has been estimated by comparing the potential changes associated with MEPPs to those obtained by iontophoresis of known quantities of ACh. Based on such analysis, the amount of ACh per quantum at the snake neuromuscular junction was estimated to be something less than 10,000 molecules [19]. Given the possible error in these calculations, this would be within the range of that estimated to be contained in a vesicle. Therefore, it is likely that quanta are defined by the amount of releasable ACh in the vesicle. An alternative favored by some investigators is that ACh is released directly from the cytoplasm. In this model, definable quanta are evident because channels in the membrane are open for finite periods of time when Ca²⁺ is elevated. A presynaptic membrane protein suggested to mediate Ca²⁺-dependent translocation of ACh has been isolated [18]. Although there are some compelling arguments in support of this model, most investigators favor the notion that the vesicle serves not only as a unit of storage but also as a unit of release. The vesicle hypothesis and release of neurotransmitters are discussed also in Chapter 10.

Depolarization of the nerve terminal by an action potential increases the number of quanta released per unit time

Release of ACh requires the presence of extracellular Ca²⁺, which enters the neuron when it is depolarized. Most investigators are of the opinion that a voltage-dependent Ca²⁺ current is the initial event responsible for transmitter release, which occurs about 200 μsec later. The mechanism through which elevated Ca²⁺ increases the probability of ACh release is not yet known; phosphorylation or activation of proteins that causes the vesicle to fuse with the neuronal membrane, are among the possibilities. Dependence on Ca²⁺ is a common feature of all exocytotic release mechanisms, and it is likely that exocytosis is a conserved mechanism for transmitter release. There is good evidence that adrenergic vesicles empty their contents into the synaptic cleft because norepinephrine and epinephrine are released along with other contents of the storage vesicle. Although less rigorous data are available for cholinergic systems, cholinergic vesicles contain ATP, and release of ATP has been shown to accompany ACh secretion from these vesicles. Furthermore, Heuser and Reese demonstrated, in electron microscopic studies at frog nerve terminals, that vesicles fuse with the nerve membrane and that vesicular contents appear to be released by exocytosis; it has been difficult to ascertain, however, whether the fusions are sufficiently frequent to account for release on stimulation. The nerve ending also appears to endocytose the outer vesicle membrane to form vesicles that subsequently are refilled with ACh [19].

All of the acetylcholine contained within the cholinergic neuron does not behave as if in a single compartment

Results of a variety of neurophysiological and biochemical experiments suggest that there are at least two distinguishable pools of ACh, only one of which is readily available for release. These have been referred to as the “readily available,” or “depot,” pool and the “reserve,” or “stationary,” pool. The reserve pool refills the readily available pool as it is utilized. Unless the rate of mobilization of ACh into the readily available pool is adequate, the amount of ACh that can be released may be limited. It is also likely that newly synthesized ACh is used to fill the readily available pool of ACh because it is the newly synthesized ACh that is released preferentially during nerve stimulation. The precise relationship between these functionally defined pools and ACh storage vesicles is not known. It is possible that the readily available pool resides in vesicles poised for release near the nerve ending membrane, whereas the reserve pool is in more distant vesicles. Although cholinergic vesicles appear to be homogeneous, there may be subpopulations of vesicles that differ in size and density.

Cholinesterases are widely distributed throughout the body in both neuronal and non-neuronal tissues

Based largely on substrate specificity, the cholinesterases are subdivided into the acetylcholinesterases (AChEs) (EC 3.1.1.7) and the butyryl or pseudocholinesterases (BuChE) (EC 3.1.1.8) [20,21]. Acetylcholines with an acyl group the size of butyric acid or larger are hydrolyzed very slowly by the former enzyme; selective inhibitors for each enzyme have been identified. BuChE is made primarily in the liver and appears in plasma; however, it is highly unlikely that appreciable concentrations of ACh diffuse from the locality of the synapse and elicit a systemic response. The distribution of BuChE mutations showing resistance to naturally occurring inhibitors suggests that this enzyme hydrolyzes dietary esters of potential toxicity. Although BuChE is localized in the nervous system during development, the existence of nonexpressing mutations in the BuChE gene within the human population demonstrates that this enzyme is not essential for nervous system function. In general, AChE distribution correlates with innervation and development in the nervous system. The AChEs also exhibit synaptic localization upon synapse formation. Acetyl- and butyrylcholinesterases are encoded by single, but distinct, genes.

Acetylcholinesterases exist in several molecular forms

These forms differ in solubility and mode of membrane attachment rather than in catalytic activity. One class of molecular forms exists as a homomeric assembly of catalytic subunits that appear as monomers, dimers or tetramers (Fig. 11-5). These forms also differ in their degree of hydrophobicity, and their amphiphilic character arises from a post-translational addition of a glycophospholipid on the carboxyl-terminal amino acid. The glycophospholipid allows the enzyme to be tethered on the external surface of the cell membrane. Soluble globular forms of the enzyme have been identified in brain.

The second class of AChEs exists as heteromeric assemblies of catalytic and structural subunits. One form consists of up to 12 catalytic subunits linked by a disulfide bond to filamentous, collagen-containing structural subunits. These forms are often termed asymmetric, since the tail unit imparts substantial dimensional asymmetry to the molecule. The asymmetric species are localized to synaptic areas. The collagenous tail unit is responsible for this molecular form being associated with the basal lamina of the synapse rather than the plasma membrane. Asymmetric forms are particularly abundant in the neuromuscular junction. A second type of structural subunit, to which a tetramer of catalytic subunits is linked by disulfide bonds, has been characterized in brain. This subunit contains covalently attached lipid, enabling this form of the enzyme to associate with the plasma membrane. The different subunit assemblies and post-translational modifications lead to distinct localization of AChE on the cell surface but appear not to affect the intrinsic catalytic activities of the individual forms.

The primary and tertiary structures of the cholinesterases are known

The primary structures of the cholinesterases define a large and functionally eclectic family of extracellular proteins that function not only catalytically as hydrolases and dehalogenases but also in forming heterologous cell contacts, as seen in the structurally related proteins neurotactin, glutactin, gliotactin and neuroligin. A sequence homologous to the cholinesterases and a presumed common structural matrix are found in thyroglobulin, in which tyrosine residues become iodinated and conjugated to form thyroid hormone [20]. The heterologous contacts formed by the tactin and neuroligin members of the family suggest that the cholinesterases also may have nonhydrolase functions.

The initial solution of the crystal structure of the Torpedo enzyme [22], followed by the mammalian enzyme [23], revealed that the active center serine lies at the base of a rather narrow gorge that is lined heavily with aromatic residues (Fig. 11-6). The enzyme carries a net negative charge, and an electrostatic dipole is oriented on the enzyme to facilitate diffusional entry of cationic ligands. Crystal structures of several inhibitors in a complex with AChE also have been elucidated.

The open reading frame in mammalian AChE genes is encoded by three invariant exons (exons 2, 3 and 4) followed by three splicing alternatives. Continuation through exon 4 gives rise to a monomeric species. Splicing to exon 5 gives rise to the carboxyl-terminal sequence signal for addition of glycophospholipid, while splicing to exon 6 encodes a sequence containing a cysteine that links to other catalytic or structural subunits. These species of AChE differ only in the last 40 residues in their carboxy-termini.

The catalytic mechanism for acetylcholine hydrolysis involves formation of an acyl enzyme, followed by deacylation

The acylation step proceeds through the formation of a tetrahedral transition state. Alkylphosphate inhibitors, such as diisopropylfluorophosphate, are tetrahedral in configuration, and this geometric resemblance to the transition state in part accounts for their effectiveness as inhibitors of AChE. Acylation occurs on the active-site serine, which is rendered nucleophilic by proton withdrawal by Glu 334 through His 447. The acetyl enzyme that is formed is short-lived, lasting approximately 10 μsec; this accounts for the high catalytic efficiency of the enzyme (Fig. 11-6). The availability of a crystal structure of AChE has enabled investigators to assign residues and domains in the cholinesterase responsible for catalysis and inhibitor specificity [22,23].

Inhibition of acetylcholinesterase occurs by several distinct mechanisms

Some AChE inhibitors are useful therapeutically, whereas others have proven useful as insecticides. Still others have been manufactured for a more insidious use in chemical warfare. Inhibitors such as edrophonium bind reversibly to the active site of the enzyme and prevent access of the substrate. Other reversible inhibitors, such as gallamine, propidium and the three-fingered peptide from snake venom, fasciculin, bind to a peripheral site on the enzyme. The carbamoylating agents, such as neostigmine and physostigmine, form a carbamoyl enzyme by reacting with the active-site serine. The carbamoyl enzymes are more stable than the acetyl enzyme; their deacylation occurs over several minutes. Since the carbamoyl enzyme will not hydrolyze ACh, the carbamoylating agents are alternative substrates that are effective inhibitors of ACh hydrolysis. The alkylphosphates, such as diisopropylfluorophosphate or echothiophate, act in a similar manner; however, the alkylphosphorates and alkylphosphonates form extremely stable bonds with the active-site serine on the enzyme. The time required for their hydrolysis often exceeds that for biosynthesis and turnover of the enzyme. Accordingly, inhibition with the alkylphosphates is typically irreversible.

Consequences of acetylcholinesterase inhibition differ between synapses

At postganglionic parasympathetic effector sites, AChE inhibition enhances or potentiates the action of administered ACh or ACh released by nerve stimulation. In part, this is a consequence of stimulation of receptors extending over a larger area from the point of transmitter release. Similarly, ganglionic transmission is enhanced by cholinesterase inhibitors. Since atropine and other muscarinic antagonists are effective antidotes of the toxicity of inhibitors of AChE, at least some CNS manifestations result largely from excessive muscarinic stimulation.

By prolonging the residence time of ACh in the synapse, AChE inhibition in the neuromuscular junction promotes a persistent depolarization of the motor endplate. The decay of endplate currents or potentials resulting from spontaneous release of ACh is prolonged from 1 to 2 msec to 5 to 30 msec. This indicates that the transmitter activates multiple receptors before diffusing from the synapse. Excessive depolarization of the endplate, resulting from slowly decaying endplate potentials, leads to a diminished capacity to initiate coordinated action potentials. In a fashion similar to depolarizing blocking agents, fasciculations and muscle twitching are observed initially with AChE inhibition, followed by flaccid paralysis.

The nicotinic acetylcholine receptor is the best characterized neurotransmitter receptor

The nicotinic receptor was purified about a decade before purification of other neurotransmitter receptors. The electric organ of Torpedo, consisting of stacks of electrocytes that have differentiated from tissue of embryonic origin common to that of skeletal muscle, is a rich source of nicotinic receptors. Upon differentiation, the electrogenic bud in the electrocyte proliferates, but the contractile elements atrophy. The excitable membrane encompasses the entire ventral surface of the electrocyte rather than being localized to small, focal junctional areas, as found in skeletal muscle. The electrical discharge in Torpedo relies solely on a PSEP resulting from depolarization of the postsynaptic membrane, rather than propagation from an action potential. The density of receptors in the Torpedo electric organ approaches 100 pmol/mg protein, which may be compared with 0.1 pmol/mg protein in skeletal muscle.

In the early 1960s, it was established that snake α-toxins, such as α-bungarotoxin, irreversibly inactivate receptor function in intact skeletal muscle, and this finding led directly to the identification and subsequent isolation of the nicotinic ACh receptor from Torpedo [3]. By virtue of their high affinity and very slow rates of dissociation, labeled α-toxins serve as markers of the receptor during solubilization and purification.

Purification of the nicotinic acetylcholine receptor facilitated examination of its overall structure

Antibodies were raised to the purified protein, and sufficient amino acid sequence of the receptor itself became available to permit the cloning and sequencing of the genes encoding the individual subunits of the receptor [4]. As a consequence of the high density of nicotinic ACh receptors in the postsynaptic membranes of Torpedo, sufficient order of the receptor molecules is achieved in isolated membrane fragments such that image reconstructions from electron microscopy have allowed a more detailed analysis of structure [24]. Finally, labeling of functional sites, determination of subunit composition and structure modification through mutagenesis contributed to our understanding of the structure of nicotinic receptors [25].

The nicotinic acetylcholine receptor consists of five subunits arranged around a pseudoaxis of symmetry

The subunits display homologous amino acid sequences with 30 to 40% identity of amino acid residues [4]. In muscle, one subunit, designated α, is expressed in two copies; the other three, β, γ and δ, are present as single copies (Fig. 11-7). Thus, the receptor is a pentamer of molecular mass of approximately 280 kDa. Structural studies show the subunits to be arranged around a central cavity, with the largest portion of the protein exposed toward the extracellular surface. The central cavity is believed to lead to the ion channel, which in the resting state is impermeable to ions; upon activation, however, it opens to a diameter of 6.5 Å. The open channel is selective for cations. The two α subunits and the opposing face of the γ and δ subunits form the two sites for binding of agonists and competitive antagonists and provide the primary surface with which the larger snake α-toxins associate. The sites for ligand binding are localized toward the external perimeter of each of the α subunits; occupation of both sites is necessary for receptor activation. Electrophysiological and ligand-binding measurements together with analysis of the functional states of the receptor indicate positive cooperativity in the association of agonists; Hill coefficients greater than unity have been described for agonist-elicited channel opening, agonist binding and agonist-induced desensitization of the receptor [3,25]. Noncompetitive inhibitor sites within various depths of the internal channel also have been defined and are the sites of local anesthetic inhibition of receptor function.

Sequence identity among the subunits appears to be greatest in the hydrophobic regions. Various models for the disposition of the peptide chains have been proposed on the basis of hydropathy and reactivity of certain residues to modifying agents and antibodies (Fig. 11-8). Four candidate membrane-spanning regions are predicted, although only one clear α-helical segment is evident in the electron microscopic reconstruction of the channel [24]. All of these potential membrane-spanning domains appear after residue 210, with the amino-terminal portion of the molecule on the extracellular surface. The homology among the four subunits strongly suggests that the same folding pattern is found in all subunits.

Site-directed labeling, chemical cross-linking, homology modeling, antibody association, fluorescence energy transfer and site-specific mutagenesis represent techniques that have made incremental contributions to the understanding of nicotinic receptor structure [25]. Analysis with techniques achieving atomic level resolution has not been possible for an integral membrane protein of this size.

A disulfide loop between Cys 128 and 142 in the α subunit is conserved in the entire receptor channel family (Fig. 11-8A). A second disulfide is found in the α subunits between vicinal Cys 192 and 193, and this structural feature has been used to identify α subunits. Early studies showed that reduction of the Cys 192–193 bond allowed for labeling by the site-directed sulfhydryl-reactive agonist and antagonist, respectively, bromoacetylcholine and m-maleimidobenzyl trimethylammonium (Fig. 11-8B) [25]. Subsequent studies involving photolytic labeling, labeling by the natural coral toxin lophotoxin and site-specific mutagenesis identified the region between residues 185 and 200 in the α subunit as being important for forming part of the agonist- and antagonist-binding surface. Two other segments of sequence in the α subunit and four discrete segments on the opposing face of the γ and δ subunits also have been identified as forming loops that contribute to the binding surfaces at the αγ and αδ interfaces [26].

Four candidate membrane-spanning regions are found after residue 210 with a large cytoplasmic loop between membrane spans 3 and 4 (Fig. 11-8A). Based on labeling experiments and site-specific mutagenesis, membrane span 2 was found to be proximal to the ion channel. This span, when constructed as an α helix, is amphipathic, with an abundance of serine and threonine residues pointed toward the channel lumen. Positions corresponding to α-Thr 244, α-Leu 251, α-Val 255 and α-Glu 262 in this transmembrane span have been labeled with the noncompetitive, channel-blocking inhibitors chlorpromazine and tetraphenyl phosphonium [3,25]. Mutation of several of the hydroxyl groups on residues at these positions affects channel kinetics. The channel gate, or constriction, is thought to lie deep within the channel either at the boxed leucine in Figure 11-8C or even farther to the cytoplasmic side. The ion selectivity of the channels appears to be controlled in part by rings of charges formed by all five subunits at the extracellular surface of the channel corresponding to α-Glu 262 and at the cytoplasmic exit corresponding to α-Glu241. Exposed amide backbone hydrogens and carbonyl groups and a ring of hydroxylated amino acids corresponding to α-Thr 244 also contribute to ion selectivity and permeation [25].

Analysis of the opening and closing events of individual channels has provided information about ligand binding and activation of the receptor

Electrophysiological studies utilize high-resistance patch electrodes of 1 to 2 μM diameter, which form tight seals on the membrane surface [27]. They have the capacity to record conductance changes of individual channels within the lumen of the electrode (see Chap. 10). The patch of membrane affixed to the electrode may be excised, inverted or studied on the intact cell. The individual opening events for ACh achieve a conductance of 25 pS across the membrane and have an opening duration that is distributed exponentially around a value of about 1 msec. The duration of channel opening is dependent on the particular agonist, whereas the conductance of the open-channel state is usually agonist-independent. Analyses of the frequencies of opening events have permitted an estimation of the kinetic constants for channel opening and ligand binding, and these numbers are in reasonable agreement with estimates of ligand binding and activation from rapid kinetic, or stopped-flow, studies. Overall, activation events can be described by Scheme 1 [3,27].

Two ligands (L) associate with the receptor (R) prior to the isomerization step to form the open-channel state L₂R*. For ACh, the forward rate constant for binding, k₊₁, is 1 to 2 × 10⁸ M ⁻¹ sec⁻¹; k₊₂ and k₋₂, forward and reverse rate constants for isomerization, yield rates of isomerization consistent with opening events in the millisecond time frame. Since k₊₂ and k₋₂ are greater than k₋₁, the rate constant for ligand dissociation, several opening and closing events with the fully liganded receptor occur prior to dissociation of the first ligand. Binding of the first and second ligands appears not to be identical, even allowing for the statistical differences arising from the two sites. Such a conclusion is consistent with receptor structure since different subunits, such as the γ and δ subunits in muscle, are adjacent to the same face of the α subunits in the pentamer.

Continued exposure of nicotinic receptors to agonist leads to desensitization of the receptors

This diminution of the response occurs even though the concentration of agonist available to the receptor has not changed. Katz and Thesleff examined the kinetics of desensitization with microelectrodes and found that a cyclic scheme in which the receptor existed in two states, R and R′, prior to exposure to the ligand best described the process.

To achieve receptor desensitization and activation by a single ligand, multiple conformational states of the receptor are required. The binding steps represented in horizontal equilibria are rapid; vertical steps reflect the slow, unimolecular isomerizations involved in desensitization (Scheme 2). Rapid isomerization to the open channel state (Scheme 1) should be added. To accommodate the additional complexities of the observed fast and slow steps of desensitization, additional states have to be included.

A simplified scheme, in which only one desensitized and one open-channel state of the receptor exist, is represented in Scheme 2, where R is the resting (activatable) state, R* the active (open channel) state and R′ the desensitized state of the receptor; M is an allosteric constant defined by R′/R, and K and K′ are equilibrium dissociation constants for the ligand.

In this scheme, M < 1 and K′ < K. Addition of ligand eventually will result in an increased fraction of R′ species due to the values dictated by the equilibrium constants. Direct binding experiments have confirmed the generality of this scheme for nicotinic receptors. Thus, distinct conformational states govern the different temporal responses that ensue on addition of a ligand to the nicotinic receptor. No direct energy input or covalent modification of the receptor channel is required.

Nicotinic receptor subunits are part of a large superfamily of ligand-gated channels

Nicotinic receptors on neurons, such as those originating in the CNS or neural crest, show ligand specificities distinct from the nicotinic receptor in the neuromuscular junction. One of the most remarkable differences is the resistance of most nicotinic neuronal receptors containing α2 through α6 subunits to α-bungarotoxin and related snake α-toxins. This fact and the lack of an abundant source of neuronal CNS receptors limited initial progress in their isolation and characterization. However, low-stringency hybridization with cDNAs encoding the subunits of electric organ and muscle receptors provided a means to clone neuronal nicotinic receptor genes. Isolation of the candidate cDNA clones, their expression in cell systems to yield functional receptors and the discrete regional localizations of the endogenous mRNAs encoding these receptor subunits revealed that the nicotinic receptor subunits are part of a large and widely distributed gene family. They are related in structure and sequence to receptors for inhibitory amino acids (GABA and glycine), to 5-hydroxytryptamine type 3 (5HT₃) receptors and, somewhat more distantly, to glutamate receptors.

At least 11 distinct genes encoding neuronal nicotinic receptor subunits α2 through α9 and β2 through β4 have been identified in the central and peripheral nervous systems (Fig. 11-2). The α subunits are similar in sequence to the muscle α1 subunit and contribute to the ligand-binding interface. The β subunits fulfill the role of β1, γ and δ subunits in the muscle receptor. When certain pairs or triplets of cDNAs encoding neuronal α and β subunits are cotransfected into cells or their corresponding mRNAs are injected into oocytes, characteristic ACh-gated channel function can be achieved. The α5 subunit appears unique in that it will not contribute to function in the absence of other α subunits; its global sequence features are more similar to those of the β subunits. The α7 and α8 subunits display function as homologous pentamers. Receptors containing α7 subunits have a high Ca²⁺ permeability, and Ca²⁺ entry may be integral to their function in vivo. While not all combinations of α and β mRNAs lead to the expression of functional receptors on the cell surface, the number of permutations is large [10,11,25]. A future challenge is the assignment of pharmacological and biophysical signatures to all of the subunit combinations found in vivo.

The α3 subunit is prevalent in peripheral ganglia, usually with β2, β4, and α5 subunits, while the α4β2 subunit combination predominates in the CNS. The α6 subunit appears to localize with biogenic amine-containing neurons, while α9 is found in vestibular sensory and cochlear hair cells. Receptors containing the α9 subunit may have some muscarinic receptor characteristics.

Substantial evidence points to nicotinic receptors in the CNS functioning at presynaptic locations to regulate release of several CNS transmitters [10]. Electrophysiological and microdialysis studies provide evidence that glutamatergic, dopaminergic, serotonergic, peptidergic and cholinergic pathways are under the control of presynaptic nicotinic receptors. Hence, nicotinic receptors appear to play an important amplification and modulatory role in the CNS.

Both nicotinic receptors and acetylcholinesterase are regulated tightly during differentiation and synapse formation

At present, we understand more about tissue-specific gene expression in muscle than in nerve [28,29]. Both of the above proteins show enhanced expression during myogenesis upon differentiating from a mononucleated myoblast to a multinucleated myotube. Curiously, enhanced receptor expression occurs largely by transcriptional activation, while the increase in cholinesterase expression arises from stabilization of the mRNA [30]. The receptor appears to cluster spontaneously, which involves a protein on the cytoplasmic side of the membrane, termed 43K or rapsyn [28,29]. This protein links the receptor to cytoskeletal elements and restricts its diffusional mobility. Following innervation and synaptic activity, expression of the receptor and AChE persists in endplate, or junctional, regions and disappears in extrajunctional regions. The collagen-tail-containing species of AChE is localized to the basal lamina in the neuromuscular synapse.

With innervation and the development of electrically excitable synapses, the γ subunit of the receptor is replaced by an ϵ subunit; small changes in the biophysical properties of the receptor occur concomitantly. Upon denervation, many of the developmental changes associated with innervation are reversed and there is again an increase in expression of extrajunctional receptors containing the γ subunit. In multinucleated muscle cells, particular subsynaptic nuclei drive the expression of these synapse-specific proteins. The factors controlling these regulatory events are incompletely understood, but calcitonin gene-related peptide (CGRP) and the protein ACh receptor-inducing activity (ARIA) may be extracellular mediators of expression. In addition, intracellular Ca²⁺, membrane depolarization and protein kinase C play distinct roles in maintaining junctional expression of synapse-localized proteins.

A neurally derived signaling protein, agrin, acts through a receptor tyrosine kinase, MuSK, in the formation of the specialized postsynaptic endplate by interaction with rapsyn. Thus, MuSK—rapsyn interactions are critical in forming the local scaffold for postsynaptic components in the motor endplate [29,31].

Muscarinic receptor stimulation causes inhibition of adenylyl cyclase, stimulation of phospholipase C and regulation of ion channels

Many types of neurons and effector cells respond to muscarinic receptor stimulation. Despite the diversity of responses that ensue, the initial event that follows ligand binding to the muscarinic receptor may be, in all cases, the interaction of the receptor with a G protein. Depending on the nature of the G protein, the receptor—G protein interaction can initiate any of several early biochemical events seen with muscarinic receptor occupation, including inhibition of adenylyl cyclase, stimulation of phosphoinositide hydrolysis or regulation of potassium channels (Fig. 11-9) [33].

Decreased cAMP formation is caused by muscarinic receptor stimulation. This effect is most apparent when adenylyl cyclase is stimulated, for example, by activation of adrenergic receptors with catecholamines or forskolin. Simultaneous addition of cholinergic agonists decreases the amount of cAMP formed in response to the catecholamine, in some tissues almost completely. The result is diminished activation of cAMP-dependent protein kinase (PKA) and decreased substrate phosphorylation catalyzed by this kinase. The mechanism by which the muscarinic receptor inhibits adenylyl cyclase is through activation of an inhibitory GTP-binding protein, G_i. The α subunit of G_i competes with the α subunit of the G protein activated by stimulatory agonists (G_S) for regulation of adenylyl cyclase (see Chaps. 20 and 22). Although muscarinic receptors do not interact with G_s, increases in cAMP formation are seen under some circumstances. These may result from stimulatory effects of βγ subunits released from G_i or effects of elevated intracellular Ca²⁺ on specific isoforms of adenylyl cyclase.

Activation of phosphoinositide-specific phospholipase C by muscarinic agonists stimulates phosphoinositide hydrolysis. Activation of the β₁ isoform of phosphoinositide-specific phospholipase C (PI-PLC) is mediated through the α subunit of a GTP-binding protein, G_q/11 [34]. This is the primary mechanism by which muscarinic receptors regulate this enzyme. However, some PLC isoforms, most clearly β₂, also are activated by βγ subunits. This probably accounts for the pertussis toxin-sensitive, G_i/G_o-mediated activation of PI-PLC seen when high levels of cloned M₂ receptors are expressed stably in some cell lines. The hydrolysis of phosphatidylinositol 4,5-bisphosphate yields two potential second messengers, inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG) (see Chap. 21). DAG increases the activity of the Ca²⁺ and phospholipid-dependent protein kinase (PKC). IP₃ mobilizes Ca²⁺ from intracellular stores in the endoplasmic reticulum and thereby elevates cytosolic free Ca²⁺. Subsequent responses are triggered by direct effects of Ca²⁺ on Ca²⁺-regulated proteins and by phosphorylation mediated through Ca²⁺/calmodulin-dependent kinases and PKC. Stimulation of a phospholipase D, which hydrolyzes phosphatidylcholine, also occurs in response to muscarinic receptor activation. This appears to be secondary to activation of PKC and contributes to a secondary rise in DAG.

Regulation of K ⁺ channels. Muscarinic agonists cause rapid activation of G protein-coupled, inwardly rectifying potassium channels (GIRKs). This muscarinic effect can be mimicked by GTP analogs in whole-cell clamp experiments, and the response is sensitive to pertussis toxin, which ribosylates and inactivates G_i and a related protein, G_o (see Chap. 20). It is now generally agreed that GIRK1 and GIRK2 are activated directly by binding βγ subunits released from G_i or G_o. This is a primary mechanism by which muscarinic agonists cause hyperpolarization of cardiac atrial cells, as well as of neurons [35]. This pathway contrasts with muscarinic inhibition of the M-current in sympathetic ganglia; suppression of this K⁺ channel is mediated indirectly through muscarinic formation of a diffusible second messenger.

Intracellular mediators of muscarinic receptor action. The three events described above, inhibition of adenylyl cyclase, stimulation of PLC and regulation of K⁺ channels, occur within the plasma membrane. They can be triggered directly by muscarinic receptor occupation independent of changes in cytosolic mediators. However, these primary events in turn affect the generation of diffusible second messengers such as cAMP, DAG, IP₃ and Ca²⁺, which generate other metabolic sequelae. For example, an increase in cytosolic free Ca²⁺ probably contributes to activation of phospholipase A₂, generating arachidonic acid, prostaglandins and related eicosanoids (see Chap. 35). These products in turn can stimulate cGMP formation and can regulate ion channel activity. Increased Ca²⁺ also can activate Ca²⁺-dependent ion channels (K⁺, Cl⁻), regulate cAMP phosphodiesterase and activate Ca²⁺/calmodulin kinase-dependent protein phosphorylation. PKC is activated by DAG, generally in concert with Ca²⁺, and has effects on ion-channel activity, as well as on cholinergic secretory and contractile responses. Given the obviously complex set of possible interactions between the intracellular mediators, it is easy to explain how diverse cellular responses can be mediated through a single receptor activating relatively few primary responses (see Chap. 10).

Radioligand-binding studies have been used to characterize muscarinic receptors

In membranes or homogenates from heart, brain and other tissues, muscarinic agonists compete for antagonist-binding sites with Hill slopes of less than unity, suggesting that these agonists interact with more than a single population of muscarinic receptors [32]. Direct binding experiments with radiolabeled agonists also show multiple binding sites for agonists. Competition curves are best fit by a model in which there are sites with low, high and in some cases, superhigh affinity for agonists. Addition of GTP to the binding assay can have a dramatic effect on the agonist competition curve or on direct agonist binding. The effect of GTP is to decrease the apparent affinity of the receptor for agonists. This results from a change in the interaction of the receptor with the GTP-binding protein that transduces its effects.

Agonists vary in their binding properties. Some, like ACh, carbamylcholine and methacholine, bind with high affinity to a large percentage of the total sites. Others, like oxotremorine and pilocarpine, appear to bind to a single class of sites and may show relatively little high-affinity binding. The capacity of an agonist to induce high-affinity binding correlates with the efficacy of that agonist for eliciting responses such as contraction or phosphoinositide breakdown. It therefore appears that interaction of the receptor and G protein is critical to production of the cellular response.

Unlike agonists, most muscarinic antagonists, such as quinuclidinylbenzilate, N-methylscopolamine and atropine, bind to the receptor with Hill slopes of unity, as expected for a mass-action interaction with a single receptor type. There is little difference in affinity for these ligands in various tissues. Similar findings with other antagonists initially suggested that all muscarinic receptors were the same. However, a number of functional studies have suggested that muscarinic receptors are heterogeneous, and several putative subtype-selective antagonists have been described throughout the years.

The binding properties of the antagonist pirenzepine led to the initial classification of muscarinic receptors

Pirenzepine (PZ) binds to muscarinic receptors in cortex, hippocampus and ganglia with relatively high affinity; these sites have been termed M₁, as mentioned earlier. Heart, gland and smooth muscle muscarinic receptors, as well as those in brainstem, cerebellum and thalamus, show 30- to 50-fold lower affinity for PZ [32,33]. The affinity for classic antagonists like N-methylscopolamine is the same in all of these regions, emphasizing the unique selectivity of PZ. Direct binding studies using [³H]PZ confirm that only certain tissues and brain regions have receptors with high affinity for this antagonist. Results of pharmacological studies also indicate that PZ blocks muscarinic responses in ganglia better than responses in heart. Brain and heart subsequently were used to purify M₁ and M₂ muscarinic receptors, and cDNA clones corresponding to these receptors were isolated from rat brain and heart libraries.

The cDNAs for the muscarinic receptors encode apparent glycoproteins of 55 to 70 kDa, which contain seven predicted transmembrane-spanning regions, similar to what is seen for the β-adrenergic receptor and other receptors that couple to G proteins (Fig. 11-10). There is only 38% amino acid identity between the proteins cloned from porcine brain and heart. The cDNA encoding the receptor initially cloned from the brain has been termed m₁, whereas that cloned from the heart has been termed m₂.

The human and rat homologs of these receptor genes, as well as three additional subtypes termed m₃, m₄ and m₅, subsequently have been cloned and expressed. Comparison of the amino acid sequences of the five muscarinic receptor subtypes suggests that they are members of a highly conserved gene family. The greatest sequence identity is in the transmembrane-spanning regions, whereas the long cytoplasmic loop (i₃) between transmembrane domains V and VI varies among the receptor subtypes [32,36]. The cloned receptors, expressed in mammalian cells, show differences in antagonist affinity similar to those of the pharmacologically defined receptors. Thus, the expressed m₁ receptor is blocked selectively by PZ, the m₂ receptor is blocked by AFDX-116 and methoctramine and the m₃ receptor is blocked by hexahydrosiladifenidol [33]. The regions in the receptor responsible for differences in antagonist affinity have not yet been identified clearly. Ligands are believed to bind to the receptor at sites facing the extracellular space but located in a central cavity deep within the bundle formed by transmembrane domains III through VII. The binding site for the covalent antagonist propylbenzilylcholine mustard has been mapped to a particular aspartic acid residue in the third transmembrane region. This amino acid is conserved in all biogenic amine G-protein-coupled receptors. Mutagenesis of this amino acid profoundly affects both agonist and antagonist binding to muscarinic receptors. It is hypothesized that this residue participates in ionic bonding with the ammonium headgroup of the cholinergic ligand [32].

Expression of the cloned receptors in Chinese hamster ovary cells, other mammalian cells and Xenopus oocytes has demonstrated differential coupling of these receptors to cellular responses. In general the m₁, m₃ and m₅ receptors regulate phosphoinositide hydrolysis by stimulating PLC. This occurs through selective coupling of the receptor to a pertussis toxin-insensitive G protein, probably G_q/11, which can activate the β isoform of PLC [34]. Calcium-dependent K⁺ and Cl⁻ channels are activated secondarily to the PLC-mediated increase in intracellular Ca²⁺. In contrast, the m₂ and m₄ receptors couple through a pertussis toxin-sensitive G protein (G_i) to inhibition of adenylyl cyclase. Regulation of K⁺ channels also is mediated through m₂ or m₄ receptor interaction with specific pertussis toxin-sensitive G proteins.

Chimeric receptors have been used to determine the regions critical for specifying coupling to particular responses. These studies demonstrate that it is the third intracellular (i₃) loop that defines functional specificity [33,36]. A series of amino acids proximal to the transmembrane domain, that is, at the amino- and the carboxy-terminal ends of the i₃ loop, carry most of this information. These particular regions are similar in the m₁, m₃ and m₅ receptors and in the m₂ and m₄ receptors but distinguish these two groups from one another. Both site-directed and random mutagenesis studies have identified specific amino acids at the amino-terminus of the i₃ loop which are required for G protein recognition and activation [36,37]. These critical noncharged amino acids are predicted to reside on the hydrophobic face of an α-helical extension of transmembrane domain V. Conserved amino acids in the carboxy-terminus of the i₃ loop and adjacent transmembrane domain VI also have been demonstrated to specify coupling to G_i- versus G_q-mediated responses. The hydrophobic regions at the two ends of the i₃ loop thus are suggested to form a surface that binds to and discriminates between different classes of G protein [36]. Other regions, including a portion of the second intracellular loop, also contribute to specifying correct G protein coupling.

The selectivity in muscarinic receptor coupling is not absolute. Overexpression of receptors or of particular G proteins supports interactions that may differ from those described above. For example, m₂ receptors expressed in Chinese hamster ovary cells not only inhibit adenylyl cyclase but also can stimulate phosphoinositide hydrolysis through a pertussis toxin-sensitive G protein [38]; this is not seen, however, when m₂ receptors are expressed in Y1 cells. These findings indicate that caution must be exercised in interpreting data obtained when receptors are expressed, often at high levels, in cells in which they normally do not function.

Muscarinic receptors of the m₁, m₃ and m₅ subclasses induce transformation or cell proliferation, a feature not shared by m₂ and m₄ receptors [39]. This property has been exploited to develop a high-throughput assay for screening effects of receptor mutations [37]. Mitogenactivated protein kinases (MAP kinases) also are activated by muscarinic receptors, but unlike transformation, this response occurs with receptors of the m₂/m₄ subtype as well as of the m₁/m₃ subtype. Notably, induction of cell growth by muscarinic receptor stimulation is cell type-specific and is seen only at high levels of receptor expression [39]. Thus, it is questionable whether there is a physiological role for ACh in growth regulation.

Transgenic mice are being generated to assess the functions of receptor subtypes in vivo

Knowledge of the anatomical distribution and coupling properties of receptor subtypes can indicate which physiological responses they mediate in vivo. However, the lack of good subtype-selective antagonists limits the use of pharmacological approaches to address this question. Generation of transgenic mice in which muscarinic receptors are overexpressed or receptor genes are disrupted by homologous recombination provides a new approach for evaluation of muscarinic receptor function. The m ₁ gene has been targeted selectively, and m₁ receptor expression in the forebrain was eliminated [40]. Homozygous m₁ receptor-deficient mice are completely resistant to seizures produced by pilocarpine, implicating the m₁ receptor in this model of epilepsy. Furthermore, inhibition of the M-current in sympathetic ganglia, suggested by previous pharmacological experiments to be m₁ receptor-coupled, is ablated in knockout mice. Future development of this approach should provide considerable insight into the distinct roles of the m₁, m₂ and other muscarinic receptor subtypes in peripheral and central nervous system function.