Calcium-Dependent Desensitizing Function of the Postsynaptic Neuronal-Type Nicotinic Acetylcholine Receptors at the Neuromuscular Junction

Pharmacol. Ther. Vol. 77, No. 3, pp. 183–202, 1998 Copyright © 1998 Elsevier Science Inc.

ISSN 0163-7258/98 $19.00 PII S0163-7258(97)00113-7

Associate Editor: M. Kimura

Calcium-Dependent Desensitizing Function of the Postsynaptic Neuronal-Type Nicotinic Acetylcholine Receptors at the Neuromuscular Junction Ikuko Kimura DEPARTMENT OF CHEMICAL PHARMACOLOGY, FACULTY OF PHARMACEUTICAL SCIENCES, TOYAMA MEDICAL AND PHARMACEUTICAL UNIVERSITY, 2630 SUGITANI, TOYAMA 930-0194, JAPAN

ABSTRACT. Several subunits that commonly have been regarded as neuronal-type nicotinic acetylcholine receptor (nAChR) subtypes, have been found in the postjunctional endplate membrane of adult skeletal muscle fibres. The postsynaptic function of these neuronal-type nAChR subtypes at the neuromuscular junction has been investigated by using aequorin luminescence and fluorescence confocal imaging. A biphasic elevation of intracellular Ca21 is elicited by prolonged nicotinic action at the mouse muscle endplates. The fast and slow Ca21 components are operated by a postsynaptic muscle- and colocalized neuronal-type nAChR, respectively. Neuromuscular functions may be regulated by a dual nAChR system to maintain the normal postsynaptic excitability. Certain neuronal-type nAChR may be endowed with the same functional role in the central nervous system also. pharmacol. ther. 77(3):183–202, 1998. © 1998 Elsevier Science Inc. KEY WORDS. Muscle endplate, neuronal-type nicotinic acetylcholine receptor, receptor-activity modulating intracellular Ca21 (RAMIC), desensitization, calcitonin gene-related peptide, protein kinase C. CONTENTS 1. INTRODUCTION . . . . . . . . . . . . . . 184 2. COEXISTENCE OF NEURONAL- WITH MUSCLE-TYPE NICOTINIC ACETYLCHOLINE RECEPTORS AT THE POSTSYNAPTIC MEMBRANE . . . . . 185 2.1. NEURONAL NICOTINIC

3.

ACETYLCHOLINE RECEPTOR SUBUNITS . . . . . . . . . . . . . . . 185 2.2. INDICATION OF COEXISTENCE OF NEURONAL- AND MUSCLE-TYPE NICOTINIC ACETYLCHOLINE RECEPTORS AT THE NEUROMUSCULAR JUNCTION . . . . 185 2.3. NONCONTRACTILE SLOW Ca21 (RECEPTOR-ACTIVITY MODULATING INTRACELLULAR Ca21) MOBILIZATION: RELATIONSHIP WITH NEURONAL-TYPE NICOTINIC ACETYLCHOLINE RECEPTOR LOCATED AT THE NEUROMUSCULAR POSTSYNAPSE . . 186 Ca21-DEPENDENT FUNCTION OF NEURONAL-TYPE NICOTINIC ACETYLCHOLINE RECEPTOR . . . . . . . . 188 3.1. DESENSITIZATION OF MUSCLE- BY NEURONAL-TYPE NICOTINIC ACETYLCHOLINE RECEPTOR . . . . . 188 3.1.1. Ca21-DEPENDENT DESENSITIZATION OF MUSCLE-TYPE NICOTINIC ACETYLCHOLINE RECEPTOR . . . . . . . . . . . 189 3.1.2. DESENSITIZATION BY

PAIRED PULSE . . . . . . . . . 190 3.1.3. DESENSITIZATION BY ACETYLCHOLINE RECEPTOR CHANNEL BLOCKER . . . . . . 191 3.2. DESENSITIZATION OF NEURONAL-TYPE NICOTINIC ACETYLCHOLINE RECEPTORS . . . . . 191 3.3. OTHER Ca21-DEPENDENT FUNCTIONS OF MUSCLE NICOTINIC ACETYLCHOLINE RECEPTOR . . . . . 192 4. INTERACTION OF CALCITONIN GENERELATED PEPTIDE RECEPTOR WITH NEURONAL-TYPE NICOTINIC ACETYLCHOLINE RECEPTOR LOCATED AT THE NEUROMUSCULAR POSTSYNAPSE . . . . . 192 4.1. CALCITONIN GENE-RELATED PEPTIDE, PROTEIN KINASE A, AND NONCONTRACTILE SLOW Ca21 (RECEPTOR-ACTIVITY MODULATING INTRACELLULAR Ca21) MOBILIZATION . . . . . . . . . . . 192 4.2. RECEPTOR-ACTIVITY MODULATING INTRACELLULAR Ca21, PROTEIN KINASE C, AND DESENSITIZATION BY PAIRED PULSE . . . . . . . . . . . 193 5. ABNORMALITY INDUCED BY NEURONALTYPE NICOTINIC ACETYLCHOLINE RECEPTOR MUTATION . . . . . . . . . . . 193 6. OTHER ABNORMALITIES IN DISEASES . . . 194 7. CONCLUSION AND PERSPECTIVE . . . . . 196 ACKNOWLEDGEMENTS . . . . . . . . . . . . . 196 REFERENCES . . . . . . . . . . . . . . . . . . 196

ABBREVIATIONS. ACh, acetylcholine; AChR, acetylcholine receptor; BgTX, bungarotoxin; CGRP, calcitonin gene-related peptide; FDB, flexor digitorum brevis; GABA, g-aminobutyric acid; H-89, N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinoline sulfonamide; mAb, monoclonal antibody; nAChR, nicotinic acetylcholine receptor; RAMIC, receptor-activity modulating intracellular Ca21; TPA, 12-O-tetradecanoyl phorbol 13-acetate.

184

1. INTRODUCTION This review deals with Ca21 entry mediated by neuronaltype nicotinic acetylcholine receptors (nAChRs) located postjunctionally at the neuromuscular junction. The other major pathway for Ca21 entry in muscle, i.e., the voltagedependent Ca21 channels (L types) located at transverse tubules and responsible for the slow inward calcium current (Kerr and Sperelakis, 1982; Walsh et al., 1986), is not considered in this review. We recently have demonstrated that neuronal-type nAChRs are expressed both pre- and postjunctionally at the rodent neuromuscular junction, and we have shown that they play a modulatory, extracellular Ca21-dependent role in the desensitization process of the muscle-type nAChR responsible for transmission (Dezaki et al., 1996; Kimura et al., 1994, 1995a; Tsuneki et al., 1995). Muscle-type nAChRs are pentameric supramolecular complexes of four different subunits arranged in the precise stoichiometric ratio of (a1)2, b1, d and g/e to form a cationselective channel. Binding of acetylcholine (ACh) causes the ion channel to open within microseconds (Colquhoun and Sakmann, 1985). The three-dimensional structure of this receptor, the molecular aspects of the agonist and antagonist binding sites, and the properties of the ion channel and its selectivity filter have been described in detail (Unwin, 1993, 1995; Hucho et al., 1996). When viewed from the synaptic cleft, the subunits are arranged in the following clockwise order around the channel pore: aH-g-aL-d-b (Machold et al., 1995), where aH and aL indicate the highand low-affinity binding sites for (1)-tubocurarine. The ion channel is formed by the alignment of the M2 helices of all the five subunits (Hucho et al., 1996). Charged residues bordering the extra- and intracellular portion of the M2 segment are major determinants of channel conductance (Imoto et al., 1988). The g- and e-subunits are mutually exclusive subunits of nAChR (Mishina et al., 1986). The e-subunit is expressed postnatally and is restricted to the neuromuscular junction; the g-subunit is expressed throughout the entire length of the muscle fiber during fetal life or in the adult following denervation (Goldman and Staple, 1989; Brenner et al., 1990). The g- and e-subunits endow the receptor with distinct electrophysiological properties: single- and multiple-point mutations in M1-M4 segments of the g- and e-subunits indicate that the major determinants of the differences in conductance between fetal and adult endplate channels are located in the membrane-spanning M2 segment (Herlitze et al., 1996). Four of the neuronaltype genes (a4, a5, a7 and b4) are expressed in developing chick skeletal muscle (Corriveau et al., 1995). The a7-subunit binds the snake toxin a-bungarotoxin (BgTX), an irreversible blocker of the muscle-type receptor; however, in muscle, a7 and a1 segregate during assembly and form distinct nAChRs (Corriveau et al., 1995). In contrast to skeletal muscle-type nAChRs, where the primary sequences of the a- and b-subunits are highly conserved, the primary structures of the neuronal-type nAChR a- and b-subunits are diverse (Le Novere and Changeux,

I. Kimura

1995; Ortells and Lunt, 1995). Moreover, the subunit stoichiometry differs substantially in the various parts of the CNS. Though the most widely spread combination in the CNS appears to be a3b2 (Vizi et al., 1987), the a3 is the only a-subunit that has been identified in the nerve terminals of motor neurons (Tsuneki et al., 1995) or in noradrenergic (Vizi et al., 1994) and dopaminergic nerves (Rapier et al., 1990; Grady et al., 1992). The a4b2 combination has been identified in neurons of the myenteric plexus, where it has a somatodendritic subcellular distribution. The nicotinebinding sites found in the CNS probably represent channels formed predominantly by the a4- and b2-subunits. In the CNS, the capacity to bind a-BgTX appears to be restricted largely to the a7-subunit containing nAChR, which does not appear to be involved in fast synaptic transmission (Flores et al., 1992). a8- and a9-subunits also confer the capacity to bind a-BgTX and are not involved in fast synaptic transmission (Clarke, 1992). The physiological properties of neuronal-type nAChRs are quite different from the muscle-type counterpart; for example, Ca21 permeability is usually larger for neuronal-type receptors. Though results obtained with in situ hybridization suggest that nAChRs are abundant in many parts of the brain (Boulter et al., 1986a; Sargent, 1993; Galzi and Changeux, 1995; McGehee and Role, 1995), their functional significance still remains obscure (Sivilotti and Colquhoun, 1995). A major question is the function of the a-BgTX-binding sites in the CNS. A clear difference with the well-studied muscle-type and Torpedo nAChR is that high-affinity nicotine-binding sites and a-BgTX-binding sites are quite separate in the CNS (Flores et al., 1992). In general, the presynaptic effects of nicotine in the CNS are not sensitive to a-BgTX (Wonnacott et al., 1990; Clarke, 1992; Lena et al., 1993; Vidal and Changeux, 1993; McMahon et al., 1994). On the other hand, pharmacological and subunit deletion experiments reveal that these presynaptic nAChRs may also include a7-containing receptors (McGehee et al., 1995). More recent data suggest that neuronal nAChR may play a modulatory role in the function of other neurotransmitter receptors. For example, iontophoretic applications of nicotinic agonists (nicotine, 1,1-dimethyl-4-phenylpiperazinium and cytisine) increase the amplitude of the monosynaptic excitatory postsynaptic potential mediated by glutamate receptors in some cells of the prelimbic area of the rat prefrontal cortex. This effect is abolished by the selective nicotinic blocker k-BgTX and by dihydro-b-erythroidine, whereas hexamethonium, mecamylamine, (1)-tubocurarine and a-BgTX are ineffective (Vidal and Changeux, 1993). Nanomolar concentrations of nicotine enhance both glutamatergic and cholinergic synaptic transmission, probably by activating presynaptic nAChRs. Nicotinic enhancement of the spontaneous release has been shown for g-aminobutyric acid (GABA) and dopamine, and enhancement of evoked excitatory synaptic transmission in the brain also has been reported (Lena et al., 1993). Though the modulatory role of presynaptic nAChRs is now clearly emerging, the major problem in elu-

Functions of Neuronal-Type Nicotinic ACh Receptors

cidating the function and the properties of these receptors is that they do not form fast synapses in the CNS. This is at variance with the neuromuscular junction (Colquhoun and Sakmann, 1985). On the other hand, the presence of neuronal-type nAChRs in this well-studied experimental model of the synapse may provide a unique experimental tool to address the question of the function and mode of action of neuronal nAChRs at the fast synapses. 2. COEXISTENCE OF NEURONAL- WITH MUSCLE-TYPE NICOTINIC ACETYLCHOLINE RECEPTORS AT THE POSTSYNAPTIC MEMBRANE 2.1. Neuronal Nicotinic Acetylcholine Receptor Subunits The genes encoding the various nAChR subunits have been named a1 (Boulter et al., 1985), a2 (Wada et al., 1988), a3 (Boulter et al., 1986a), a4 (Goldman et al., 1987), a5 (Boulter et al., 1990), a6 (Lamar et al., 1990), a7 (Couturier et al., 1990), a8 (Schoepfer et al., 1990), a9 (Elgoyhen et al., 1994), b1 (Noda et al., 1983), b2 (Deneris et al., 1988), b3 (Deneris et al., 1989), b4 (Duvoisin et al., 1989), g (Boulter et al., 1986b), d (Noda et al., 1983), and e (Mishina et al., 1986; Witzemann et al., 1987; Gu and Hall, 1988; Martinou and Merlie, 1991). The a2-, a3-, a4-, a5-, b2-, b3-, and b4-subunit gene products are expressed in the central and peripheral nervous system. In brain, the a5 gene product is presented both in b3- and in b4-based receptor subtypes, while in the ganglion, it is found in association with the b3-subunit and is concentrated in the postsynaptic membrane (Conroy et al., 1992). By in situ hybridization experiments, a4 transcripts have been found in several regions of the rat brain, while the a5 transcript has a more limited distribution. Channels containing the a5-subunit (a4a5b2) in oocytes are potently activated and desensitized by nanomolar concentrations of nicotine (Ramirez-Latorre et al., 1996). A class of nAChRs with a predominantly synaptic location is made up by combinations of at least three types of subunits encoded by the a3, b4, and a5 nAChR genes (Vernallis et al., 1993). The same authors have shown that nAChR containing the a7-subunit and lacking the others are located extrasynaptically in neurons. Some neuronal nAChRs may have an even more complex subunit composition, such as a3, a5, b2, and b4 (Conroy and Berg, 1995), as occurs in muscle and the electric organ of fishes. The b2-subunit is the most widely expressed neuronaltype nAChR subunit in the nervous system (Bessis et al., 1995). Most of the b2 gene product in brain is assembled with a4-subunits to make up the major receptor species with the ability to bind nicotine with high affinity (Whiting et al., 1987a,b; Schoepfer et al., 1988). In Xenopus oocytes, the receptors formed by the a3-, a4-, and b2-subunits are pharmacologically similar to the ganglionic-type neuronal nAChR (Boulter et al., 1987). Three of the nAChR subunit gene products, a5, a6 and b3, have failed to yield

185

agonist-gated currents when co-expressed in oocytes with any one of the other a or b genes. The EC50s for nicotine and ACh of the a4b2 receptor were increased by the coassociation with the a5-subunit (Ramirez-Latorre et al., 1996). Monoclonal antibodies (mAbs) to chicken brain nAChR subtypes have been used to identify two nAChR subtypes in chicken brains. These subtypes have very similar affinities for nicotine and other cholinergic agonists and antagonists. They are structurally distinct, having very similar or identical a-subunits, but different b-subunits (Whiting et al., 1987a). The nAChRs, which have high affinity for nicotine, but do not bind to a-BgTX chromatography columns, consist of two types of subunits: a3b2. Four of the neuronal-type genes (a4, a5, a7 and b4) are expressed in developing chick skeletal muscle (Corriveau et al., 1995). The a7-subunit binds the snake toxin a-BgTX, an irreversible blocker of the muscle-type receptor; however, in muscle, a7 and a1 segregate during assembly and form distinct AChRs (Corriveau et al., 1995). 2.2. Indication of Coexistence of Neuronal- and Muscle-Type Nicotinic Acetylcholine Receptors at the Neuromuscular Junction The serendipitous demonstration of the coexistence of neuronal- and muscle-type nAChRs at the postsynaptic membrane has been brought about by reconsidering the “eserinization effect.” Pretreatment with an anticholinesterase agent induces a state of high sensitivity to ACh in skeletal muscles, a phenomenon mainly explained by the inhibition of ACh hydrolysis. On the other hand, this simple explanation does not provide clues as to why anticholinesterase agents also sensitize to the blocking effect of pancuronium, (1)-tubocurarine, a-BgTX, decamethonium and suxamethonium on intracellular noncontractile Ca21 mobilization (Kimura et al., 1990b; Tsuneki et al., 1994). Suxamethonium is classified as a depolarizing neuromuscular blocker. The neuromuscular blockade is mainly due to desensitization of muscle-type nAChRs (Thesleff, 1955a,b; Nojima et al., 1992) rather than to the sustained depolarization. The suxamethonium-induced desensitizing block at the motor endplate produces the following electrophysiological events: a gradual decay of depolarization (Nojima et al., 1993), an enhanced decay of ACh-dependent potential, and an acceleration of the time-dependent decline in the opening frequency of ACh-activated channel currents measured by cell-attached patch-clamp techniques (Nojima et al., 1992). Suxamethonium produces biphasic effects on evoked ACh release at the neuromuscular junction (Kimura, I. et al., 1991c): it accelerates the depolarization-induced release of ACh at low concentrations and suppresses it at high concentrations. Bowman (1980) has proposed a feedback model of release in which ACh activates both postsynaptic and presynaptic receptors. The presynaptic effect is suggested by the fact that in humans, the paralyzing effect of a nondepolarizing blocker, such as pancuronium (Katz, 1971; Ono et al., 1989) is increased by pretreatment

186

with suxamethonium. In contrast, if suxamethonium is injected during the course of a nondepolarizing block (Bowman, 1964), it produces an obvious antagonism of the block, at least in animals. The question arises whether the suxamethonium-sensitive receptors, presumably coupled to Ca21 channels, are the same receptors postulated to function in the positive feedback mechanism that enhances presynaptic release during high-frequency stimulation of the nerve (Bowman et al., 1990; Wessler, 1992). Suxamethonium- and ACh-induced decreases in channel currents are strictly dependent on extracellular Ca21 concentrations. High extracellular Ca21 concentration decreases the single channel conductance and the channel opening frequency induced by suxamethonium and ACh (Kimura, I. et al., 1991b). Suxamethonium applied outside the patch pipette decreases both the channel conductance and the opening frequency of ACh-activated channel currents. These effects do not occur in a nominally Ca21-free medium (Kimura, I. et al., 1991b). The effective concentration ranges of suxamethonium on various aspects of the excitation-contraction events are shown in Fig. 1. Noncontractile Ca21 mobilization is inhibited at the lowest suxamethonium concentration. The sensitivities to (1)-tubocurarine, pancuronium, and a-BgTX are 10-times higher for noncontractile than for contractile Ca21 mobilization (Kimura et al., 1990b; Tsuneki et al., 1994). The different pharmacological sensitivities of contractile and noncontractile Ca21 mobilization strongly suggest (and led us to postulate) that they may depend on different nAChR subtypes. The noncontractile Ca21 mobilization elicited by nerve stimulation in the presence of anticholinesterase agents is depressed by mAb to the b2-subunit (Kimura et al., 1994). Patch-clamp experiments in the mouse flexor digitorum brevis (FDB) muscle indicate that ACh-induced channel currents are operated in part by a receptor that is not recognized by the anti-a1 mAb 210 (Kimura et al., 1994). Immu-

I. Kimura

nohistochemical data obtained with the anti-b2-subunit mAb 270 demonstrate that this subunit is located at the endplate in the mouse skeletal muscle (Tsuneki et al., 1995). Reverse transcription-polymerase chain reaction analysis also confirmed the presence of the b2-subunit in skeletal muscles (Sala et al., 1996). Several (a4, a5, a7, and b4) subunits of neuronal-type nAChR, as well as isoforms of the muscle a1- and g-subunits, have been shown to be expressed by skeletal muscle cells in vivo, in vitro, and in cell lines (Corriveau et al., 1995; Morris et al., 1991; Mileo et al., 1995). 2.3. Noncontractile Slow Ca21 (Receptor-Activity Modulating Intracellular Ca21) Mobilization: Relationship with Neuronal-Type Nicotinic Acetylcholine Receptor Located at the Neuromuscular Postsynapse When a mouse motor nerve is electrically stimulated in the presence of low concentrations of anticholinesterase agents, noncontractile Ca21 mobilization is observed at the neuromuscular junction using Ca21-aequorin luminescence (Kimura et al., 1990b). We have named this phase of the Ca21 mobilization RAMIC (receptor-activity modulating intracellular Ca21). Noncontractile Ca21 mobilization occurs following the contractile phase; is not accompanied by twitch tension; is long-lasting, with a duration of 700 msec; is extracellular Ca21-dependent and is more sensitive to competitive blockers of postsynaptic muscle-type nAChR than contractile Ca21 transients. The low concentrations of (1)-tubocurarine, pancuronium, and a-BgTX, which have no effects on contractile Ca21 transients, are effective on RAMIC (Kimura et al., 1990b; Tsuneki et al., 1994). The noncontractile slow Ca21 mobilization is completely abolished by low (0.6–1.25 mM) and is enhanced by high (5.0–20 mM) extracellular Ca21. The dependence on ex-

FIGURE 1. High sensitivity of suxamethonium to noncontractile slow Ca21 (RAMIC) mobilization compared with the inhibitory responses to indirectly stimulated twitch tension, nAChR channel conductance, opening frequency, contractile fast Ca21 mobilization, and ACh release from motor nerve at the neuromuscular junction. Data from Kimura, I. et al. (1991b,c) and Tsuneki et al. (1994).

Functions of Neuronal-Type Nicotinic ACh Receptors

tracellular Ca21 is clearly different between RAMIC and contractile Ca21 transients, suggesting different mechanisms of Ca21 mobilization. The total amount of Ca21 mobilization is far greater in RAMIC than in contractile transients, but RAMIC does not induce either twitch contraction or contracture. Nitrendipine, verapamil and diltiazem do not inhibit RAMIC, indicating that it is independent of Ca21 influx through L-type voltage-dependent Ca21 channels. RAMIC can be easily observed using neuromuscular preparations when the motor nerve is stimulated electrically in the presence of anticholinesterase agents. Its occurrence is not dependent on any direct effects of the anticholinesterase agents since the low concentrations of neostigmine used have no effects on the properties of the mouse endplate receptors (Chang et al., 1987). In the absence of anticholinesterase agents, RAMIC can also be elicited by local application of high concentrations of ACh to endplate regions of skeletal muscles (Dezaki and Kimura, 1998) and by bath application of ACh to single FDB muscle cells loaded with the fluorescent Ca21 indicator fluo-3 (Tsuneki et al., 1997). The time courses of the slow Ca21 mobilization are prolonged in the following order: nerve stimulation , ACh local application , ACh bath application (Fig. 2). The difference in time dependence may be caused by loss of enzymes during procedure for muscle preparation and by differences in the time required to attain effective ACh concentrations at endplate in the different experimental conditions. Anticholinesterase agents induce accumulation of ACh in the synaptic cleft, which in turn produces regenerative depolarization (Chang and Hong, 1986). Some properties are different between RAMIC and regenerative depolarization. Regenerative depolarization is initiated by tetanic stimulation of motor nerves, whereas RAMIC is evoked by a single pulse. Endplate potentials are not generated during

FIGURE 2. Common properties of biphasic Ca21 mobilization by nerve stimulation and by local application of ACh at the neuromuscular junction and by ACh bath application in isolated muscle cells. Neo, neostigmine. Data from Kimura et al. (1990b), Tsuneki et al. (1997) and Dezaki and Kimura (1998).

187

regenerative depolarization, whereas contractile Ca21 transients are elicited during RAMIC. The duration of regenerative depolarization is prolonged by low extracellular Ca21 (0.63 mM) and shortened by high extracellular Ca21 (5.4 mM), whereas the duration of RAMIC is unchanged in 1.25–5 mM extracellular Ca21. Regenerative depolarization, but not RAMIC, is blocked by voltage-dependent Ca21-channel blockers. RAMIC appears to occur independently of sarcoplasmic reticulum activation. Geographutoxin II, a muscle-selective Na1-channel blocker, depresses contractile Ca21 transients, but not RAMIC (Kimura et al., 1991). Caffeine (1–5 mM) enhances contractile Ca21 transients, but diminishes RAMIC (Kimura, I. et al., 1991a). The pharmacological sensitivity of RAMIC is compared with that of contractile Ca21 transients in Fig. 3. The specificities of responses by nAChR agonists (ACh, nicotine, 1,1-dimethyl-4-phenylpiperazinium, cytisine), nAChR antagonists ((1)-tubocurarine, a-BgTX, dihydro-b-erythroidine, k-BgTX, a-conotoxin ImI, methyllycaconitine, hexamethonium), the neuropeptide calcitonin gene-related peptide (CGRP) and its antagonist CGRP8-37, the protein kinase A inhibitor N-[2-(p-bromocinnamylamino) ethyl]5-isoquinoline sulfonamide (H-89), and the protein kinase C activator 12-O-tetradecanoyl phorbol 13-acetate (TPA) (described in Sections 4.1 and 4.2) have been investigated (Kimura et al., 1993b; Dezaki and Kimura, 1998). Cytisine has the highest affinity for the [3H]ACh binding site in rat brain (Romano and Goldstein, 1980; Schwartz et al., 1982). Methyllycaconitine is a neurotoxin used for the characterization of the neuronal, a-BgTX-sensitive nAChRs (Ward et al., 1990; Alkondon et al., 1992). The muscarinic AChR agonist pilocarpine and its antagonist atropine do not affect either RAMIC or contractile Ca21 mobilization. Type A botulinum toxin (intraperitoneal bolus injection of 100 mg into mice), which irreversibly blocks ACh release from motor nerve (Stanley and Drachman, 1983), completely de-

188

presses the generation of nerve-stimulated RAMIC. Indeed, RAMIC is triggered by accumulation of ACh released during nerve stimulation. The suggestion that RAMIC is elicited by activation of neuronal-type nAChR comes from the following experimental data: the slow noncontractile Ca21 mobilization is depressed by 10-times lower concentrations of (1)-tubocurarine, a-BgTX (competitive blockers), and by hexamethonium (Kimura et al., 1993b) and dihydro-b-erythroidine (ganglionic blockers), methyllycaconitine (a neuronal-type blocker) (Tsuneki et al., 1997), and by a mAb against the b2-subunit (Kimura et al., 1994), but not by a-conotoxin ImI. These antagonists do not affect contractile Ca21 transients at the concentrations used. a-Conotoxin ImI, which selectively binds to homomeric a7 neuronal-type nAChRs (McIntosh et al., 1994; Johnson et al., 1995), and a-conotoxin ImII, which blocks the response to ACh in oocyte expressing a3b2 nAChRs (Cartier et al., 1996), have not been used to characterize RAMIC yet.

3. Ca21-DEPENDENT FUNCTION OF NEURONAL-TYPE NICOTINIC ACETYLCHOLINE RECEPTOR An important feature of neuronal-type nAChR channels is the high permeability to calcium ions, which causes increases of intracellular Ca21 independent of activation of voltagedependent Ca21 channels (Mulle et al., 1992). The Ca21 permeability, as well as the sensitivity to a- and k-BgTX, of various nAChR subtypes are summarized in Fig. 4. Extracellular Ca21 enhances the stimulatory effects of nAChRs in the brain (Changeux, 1993). a-BgTX does not affect the electrophysiological responses to ACh of chick ciliary ganglion neurons (Ravdin and Berg, 1979; Halvorsen et al., 1991), but inhibits the rapidly desensitizing ACh-activated current in the same tissue (Zhang et al., 1994). A similar effect is also seen in hippocampus (Alkondon and Albuquerque, 1993). The hippocampal a-BgTXsensitive nAChR is a cation channel considerably selective to Ca21 and may mediate a fast rise in intracellular Ca21 (Castro and Albuquerque, 1995). In rat medial habenula and chick ciliary ganglion neurons, nAChR activation increases intracellular Ca21 independently of voltage-dependent Ca21 channels and opens Ca21-dependent chloride channels (Mulle et al., 1992). Ca21 influx through habenula nAChRs at hyperpolarized potentials is approximately 2-fold greater than that carried by voltage-gated Ca21 channels in the membrane potential range that activates those channels. Ca21 potentiates the physiological response of neuronaltype nAChRs to agonists by enhancing ionic current amplitudes, apparent agonist affinity and cooperativity. When a3b4 is expressed in oocytes, gating leads to sufficient increases in Ca21 to activate the endogenous Ca21-activated Cl2 conductance (Vernino et al., 1992). Neuronal-type nAChR channels in PC12 cells are more permeable to Ca21 than muscle-type nAChR channels, as inferred by the

I. Kimura

shift in reversal potentials (Sands and Barish, 1991). The process by which desensitization occurs appears to be dependent on leucine amino acids located near the center of the ion channel. The leucine ring locks the ion channel in closed configuration when the receptor is desensitized (Changeux, 1993). ACh can significantly increase intracellular Ca21 via a7 nAChRs. a7-Containing nAChRs are among the most Ca21-permeable ligand-gated ion channels, with a PCa/ PNa 5 20 (Seguela et al., 1993). This receptor actively conducts Ca21 at hyperpolarized potentials at which other ligand- or voltage-gated Ca21 permeant channels are inactive. The relative Ca21 permeability of a7 receptors is even greater than that of the N-methyl-d-aspartate glutamate receptors (Seguela et al., 1993). a7 nicotinic receptors in Xenopus oocytes are highly permeable to divalent cations (Sands et al., 1993). The a7 gene product can produce nAChRs in Xenopus oocytes capable of generating currents that are activated by nicotine, blocked by a-BgTX, and rapidly desensitized (Couturier et al., 1990). Choline is a full agonist for a7, but the hydroxy group is strongly nonpermissive for other receptor subtypes (a1b1gd, a3b4, a3b2 and a4b2). Physiological concentrations of choline desensitize a7 receptors to ACh, suggesting that in vivo, choline may regulate both the activation and inactivation of the receptor (Papke et al., 1996). Among the regulatory Ca21binding sites, the highly conserved (a7 162–172) domain may simultaneously contribute to Ca21 and agonist binding (Galzi et al., 1996). a8-Subunit of chick a-BgTX-sensitive neuronal-type nAChRs expressed in Xenopus oocytes form homomeric, ACh-gated, rapidly desensitizing, inwardly rectifying, Ca21-permeable cation channels similar to those formed by the a7 homomers (Gerzanich et al., 1994). By simultaneous whole cell recording and fura-2 measurement of intracellular Ca21, it has been shown that Ca21 influx contributes marginally to the total current carried by neuronaltype nAChRs in adrenal chromaffin cells (Zhou and Neher, 1993; Vernino et al., 1992, 1994). The nicotine-induced rise in intracellular Ca21 was blocked by a-BgTX, although specific changes in nicotine-induced currents were not detected by intracellular recording (Vijayaraghavan et al., 1992).

3.1. Desensitization of Muscle- by Neuronal-Type Nicotinic Acetylcholine Receptor Desensitization (loss of response) of the nAChR is triggered by prolonged or repeated exposure to agonists and results in inactivation of its ion channel. nAChR desensitization at the neuromuscular junction was first reported by Katz and Thesleff in 1957. The detailed molecular mechanism is not known with certainty. It readily develops as a result of accumulation of nerve-released ACh at the neuromuscular junction after cholinesterase inhibition (Magleby and Pallotta, 1981), or after repetitive and positive feedback stimulation to motor nerve terminals. The desensitization can be enhanced by exogenous and endogenous modulators, including noncompetitive blockers, Ca21, CGRP and recep-

Functions of Neuronal-Type Nicotinic ACh Receptors

189

FIGURE 3. Pharmacological spectra of RAMIC signals compared with contractile fast Ca21 mobilization, to competitive nAChR antagonists ((1)-tubocurarine and a-BgTX), neuronal nAChR antagonists (dihydro-b-erythroidine, k-BgTX, conotoxin ImI, methyllycaconitine, hexamethonium and mecamylamine), a muscarinic antagonist (atropine), CGRP, its competitive fragmental peptide (CGRP8–37), a muscle Na1 channel blocker (geographutoxin II), a Ca21 releaser from sarcoplasmic reticulum (caffeine), a protein kinase A inhibitor (H-89) and a protein kinase C activator (TPA). Data from Kimura, I. et al. (1990b, 1991a, 1993b), Kimura, M. et al. (1991) and Tsuneki et al. (1994).

tor phosphorylation. Desensitization may also play a role in transmission failure in neuromuscular diseases such as myasthenia gravis (Ochoa et al., 1989). 3.1.1. Ca21-dependent desensitization of muscle-type nicotinic acetylcholine receptor. The development of nAChR desensitization is enhanced by increasing extracellular Ca21

(Manthey, 1966, 1970; Nastuk and Parsons, 1970), a consequence of accumulation of Ca21 at the inner surface of the sarcolemmal membrane due to Ca21 influx via nAChR (Miledi, 1980; Chesnut, 1983). Patch-clamp experiments have shown that the time-dependent decline (desensitization) in the opening frequency of ACh-activated channels is dependent on external Ca21 concentrations (Nojima et

190

I. Kimura

FIGURE 4. The sensitivities of electrophysiological responses to a-BgTX and to k-BgTX, and Ca21 permeability by nAChR subtypes in the skeletal muscle and the autonomic and central nervous systems. Data from Anand et al. (1993), Castro and Albuquerque (1995), Conroy et al. (1992), Corriveau et al. (1995), Couturier et al. (1990), Decker and Dani (1990), Elgoyhen et al. (1994), Gerzanich et al. (1994), Papke et al. (1993), Vernallis et al. (1993), Vernino et al. (1992) and Vijayaraghavan et al. (1992).

al., 1992). The lack of effect of caffeine indicates that release of Ca21 from the sarcoplasmic reticulum does not play a role in this process (Magazanik and Vyskocil, 1970). 3.1.2. Desensitization by paired pulse. The contractile Ca21 transients elicited by the second pulse (S2) during the generation of noncontractile Ca21 mobilization are depressed at shorter pulse intervals, but are restored to the initial contractile response (S1) at longer pulse intervals (Kimura et al., 1995a), as shown in Fig. 5. The extent of the depression of S2, indicated by the decrease in S2/S1, was enhanced by increasing concentrations of an anticholinesterase agent neostigmine (Fig. 5a). The rapid depression of

contractile Ca21 transients, followed by gradual recovery to initial values, is a phenomenon quite similar to the time course of the nAChR desensitization, measured by paired pulses of ACh using the patch-clamp technique (Franke et al., 1992; Bufler et al., 1993). Noncontractile Ca21 mobilization may stabilize muscle-type nAChRs in refractory states, and may protect against over-excitation at the motor endplate, depressing contractile Ca21 transients. The depolarization of postsynaptic membranes is prolonged by the persistent action of ACh accumulated after cholinesterase inhibition. However, the depression of contractile Ca21 transients observed is not involved in the depolarizing blockade of nAChR (Burns and Paton, 1951) since the re-

Functions of Neuronal-Type Nicotinic ACh Receptors

covery time from the depression of contractile Ca21 transients based on the second pulse is 10- to 20-fold longer than the duration of the prolonged endplate potential in the presence of anticholinesterase agents. S2 is hardly depressed at all when S1 is increased by caffeine in the absence of neostigmine (Fig. 5b), although complete recovery of Ca21 content in the sarcoplasmic reticulum has been reported to require a few seconds (Somlyo et al., 1985). Therefore, the depression of the second contractile Ca21 transient by paired-pulse stimulation is not due to the inactivation or depletion of the sarcoplasmic reticulum caused by the first Ca21 release (Schneider and Simon, 1988). When noncontractile Ca21 mobilization is depressed either by low concentration (1.3 mM) of extracellular Ca21 (Fig. 5c) or by 0.05 mM pancuronium in the presence of neostigmine, the decrease in S2/S1 is greatly reduced. Part of the decrease still remains. This may reflect the direct desensitizing effect of ACh accumulated because of the cholinesterase inhibition since desensitization occurs also in the absence of external Ca21 (Nelson et al., 1980).

191

3.1.3. Desensitization by acetylcholine receptor channel blocker. nAChR channel blockers affect the peak height and the duration of noncontractile slow Ca21 (RAMIC) mobilization. The activation (rising phase) of the RAMIC signal is depressed by the open-channel blocker bupivacaine, while its desensitization (the falling phase) is accelerated by a closed-channel blocker chlorpromazine (Kimura, M. et al., 1991). Mixed-type channel blockers phencyclidine and b-eudesmol depress both phases. Competitive nAChR blockers (1)-tubocurarine, pancuronium and a-BgTX accelerate the falling phase; suxamethonium depresses the rising and accelerates the falling phase of the RAMIC signals (Tsuneki et al., 1994). In this respect, suxamethonium acts as a desensitizing blocker. Recordings at high concentrations of ACh in the presence of the glucocorticoid hydrocortisone showed a reduced number of openings per activation period and the long closed times typically observed in the desensitization phenomenon, suggesting that neurosteroids present at high concentrations in the brain could be active modulators of this receptor function (Bouzat and Barrantes, 1996). 3.2. Desensitization of Neuronal-Type Nicotinic Acetylcholine Receptors

FIGURE 5. Paired-pulse desensitization of contractile fast Ca 21 mobilization by RAMIC signal. a: Concentration-dependency of neostigmine (Neo); b: no desensitization in the absence of Neo, even though the enhancement of contractile fast Ca 21 mobilization by caffeine; c: external Ca 21 dependence; and d: depression of desensitization by a protein kinase C inhibitor (staurosporine [SS]), despite the existence of RAMIC. Data from Kimura et al. (1995a).

After a single nicotine pulse, receptor desensitization (calculated as a single exponential decay) was significantly slower for a4b2 than for either a3b2 or a2b2 (Vibat et al., 1995). 3,4,5-Trimethoxybenzoic acid 8-(diethylamino)octyl ester, an intracellular Ca21 antagonist, is a potent inhibitor of agonist-stimulated ion flux mediated by functional human muscle-type nAChR or ganglionic a3b4-nAChR subtypes expressed by TE671/RD or SH-SY5Y cells. 3,4,5Trimethoxybenzoic acid 8-(diethylamino)octyl ester might act like a local anesthetic to induce an nAChR open-channel block (Bencherif et al., 1995). Examination of the time course of the ACh-evoked currents of heterologously expressed a7 (and a8) homomeric channels, as well as the currents underlying specific nAChR subtypes in hippocampal neurons, revealed that both of these a-BgTX-sensitive currents have extremely rapid rates of desensitization (Seguela et al., 1993; Gerzanich et al., 1994). Incubation of neurons with low concentrations of nicotine (0.1–10 mM) causes a slowly developing, but pronounced, desensitization of nAChRs. A continuous exposure to 1 mM nicotine reduced the amplitude of the ACh (30 mM) response to more than 30% of its control value. With 1 mM or larger nicotine concentrations, the onset of the desensitization induced by 1 mM nicotine is biexponential, with fast and slow time constants of 15 sec and 1.7 min, respectively. Recovery from the desensitization induced by longer applications of nicotine is much slower than that observed with the brief pulses of high concentrations of nicotine (Lester and Dani, 1995). In cultured postnatal rat hippocampal neurons, nicotinic currents can be classified into two classes: (1) characterized by rapid and profound desensitization that is sensitive to a-BgTX and (2) a slowly activated, a-BgTX-insensitive

192

current that exhibits no desensitization, even during prolonged agonist applications (Zorumski et al., 1992). 3.3. Other Ca21-Dependent Functions of Muscle Nicotinic Acetylcholine Receptor Endplate channels are weakly permeable to Ca21 (Bregestovski et al., 1979; Lewis, 1979; Adam et al., 1980). The localized increase in intracellular Ca21 is observed during the endplate current (Miledi et al., 1980). Reversal potentials of endplate current in frog muscle indicate a PCa/PNa of 0.3 (Adam et al., 1980), comparable with the value for adult rat muscle endplates (Villarroel and Sakmann, 1996). Calcium contributes about 2% of the total inward current through a nAChR channel in physiological solution (Decker and Dani, 1990) from BC3H1 cells and 4% of the inward current through neuronal-type nAChRs from adrenal chromaffin cells (Vernino et al., 1994). ACh contractures in both normal and denervated muscle cells are external Ca21-dependent (Nojima et al., 1991). The 3-fold increase in Ca21 current through endplate channels during postnatal development is caused by replacement of the fetal g-subunit by the e-subunit in juvenile and adult muscle (Villarroel and Sakmann, 1996). Studies with g and e AChRs expressed in Xenopus oocytes show that the g- to e-subunit switch accelerates rates of desensitization and increases Ca21 permeability (Nishizaki and Sumikawa, 1994). 4. INTERACTION OF CALCITONIN GENE-RELATED PEPTIDE RECEPTOR WITH NEURONAL-TYPE NICOTINIC ACETYLCHOLINE RECEPTOR LOCATED AT THE NEUROMUSCULAR POSTSYNAPSE The neuropeptide CGRP has been identified in the spinal cord of several vertebrate species and in the motor nerve endings of the rodent neuromuscular junction (Takami et al., 1985b; Fontaine et al., 1986: New and Mudge, 1986; Matteoli et al., 1988). CGRP, the major neuropeptide coexisting with ACh at the motor nerve endings (Takami et al., 1985a,b; Matteoli et al., 1988), enhances the muscle contractions induced by nerve or direct electrical stimulation, and regulates neuromuscular transmission by modulation of nAChR phosphorylation and desensitization (Miles et al., 1989). Ten to 15 days after a peripheral nerve crush, about 80% of the motor nerve terminals reinnervating the soleus muscle contains detectable CGRP, but not secretogranin II (Sala et al., 1995). In the spinal cord, CGRP expression is higher than normal 1 day after a sciatic nerve crush and increases during the next few days. The CGRP binds to CGRP receptors located at the neuromuscular postsynapse (Jenning and Mudge, 1989; Popper and Micevych, 1989; Roa and Changeux, 1991). CGRP receptors are located on the surface of muscle cells (Jenning and Mudge, 1989; Popper and Micevych, 1989). This peptide stimulates the biosynthesis of nAChR (Fontaine et al., 1986; New and Mudge, 1986), and prolongs nAChR chan-

I. Kimura

nel open time (Lu et al., 1993; Owens and Kullberg, 1993) in cultured myotubes. Bath application of CGRP prolongs the duration of noncontractile slow Ca21 (RAMIC) mobilization via activation of protein kinase A, and CGRP8–37, a competitive CGRP antagonist, shortens its duration (Kimura et al., 1993b). It is likely that endogenous CGRP mobilizes RAMIC via the activation of protein kinase A, which in turn activates protein kinase C and enhances the desensitization of muscle-type nAChR at the neuromuscular junction (Dezaki et al., 1996). CGRP, which is released by electrical nerve stimulation or a cholinesterase inhibitor in neuromuscular preparations, negatively modulates nerveevoked acetylcholine release (Kimura et al., 1997). These effects on RAMIC indicate that CGRP also has modulatory activity on neuronal-type nAChR channels. 4.1. Calcitonin Gene-Related Peptide, Protein Kinase A, and Noncontractile Slow Ca21 (Receptor-Activity Modulating Intracellular Ca21) Mobilization Heterologous desensitization of nAChR by protein kinase A occurs in response to activation of the CGRP receptor by CGRP (Huganir and Greengard, 1987). CGRP activates adenylyl cyclase and induces localized increase in the concentration of intracellular cyclic AMP, leading to the activation of protein kinase A (cyclic AMP-dependent) in endplate-rich regions of skeletal muscle (Kobayashi et al., 1987; Matsumoto et al., 1992). CGRP enhances the nAChR desensitization via activation of protein kinase A (Mulle et al., 1988). The role of protein kinase A in nAChR desensitization has been questioned recently. Protein kinase A phosphorylates the g-, d- and a4-subunits of the nAChR (Huganir and Greengard, 1983, 1990); its activation accelerates the rate of nAChR desensitization (Albuquerque et al., 1986; Huganir et al., 1986; Middleton et al., 1988). Phosphorylated nAChRs reconstituted into liposomes show an increase in the rate of rapid desensitization (Huganir et al., 1986). However, negative (Wagoner and Pallotta, 1988), as well as opposite (increase rather than decrease in nAChR activity), results have also been reported (FerrerMontiel et al., 1991). nAChR purified from rat brain was phosphorylated in vitro by protein kinase A on the a4-subunit (Nakayama et al., 1993). H-89, a protein kinase A inhibitor, blocked noncontractile slow Ca21 (RAMIC) mobilization; in this condition, the depression of contractile Ca21 transients elicited by the second pulse is diminished (Dezaki et al., 1996). In contrast, the depression is enhanced by CGRP and 3-(29-hydroxy-49,59-diethoxybenzoyl) propionic acid, a protein kinase A activator that prolongs the duration of RAMIC. The enhancement of depression by CGRP is completely inhibited by H-89. Both CGRP and 3-(29-hydroxy-49,59-diethoxybenzoyl) propionic acid, however, fail to enhance the depression of contractile Ca21 transients elicited by the second pulse at low external Ca21 concentration (1.3 mM), a condition where

Functions of Neuronal-Type Nicotinic ACh Receptors

RAMIC is absent. Using the patch-clamp technique in adult mouse muscle cells, we have demonstrated that protein kinase A-induced acceleration of nAChR desensitization does not occur in external Ca21-free solution (Nojima et al., 1994), a condition where RAMIC mobilization does not occur (Kimura et al., 1990b), while receptor phosphorylation does (Huganir and Greengard, 1983). The extracellular Ca21 dependence of desensitization and RAMIC and the independence of phosphorylation suggest that the desensitizing effects of protein kinase A occur through RAMIC rather than through receptor phosphorylation. CGRP enhances RAMIC mobilization through protein kinase A activation within muscle cells (Kimura et al., 1993b). The CGRP-induced activation of protein kinase A may promote the bulk of nAChR desensitization via the mobilization of RAMIC; this does not exclude that phosphorylation by protein kinase A in part can contribute directly to nAChR desensitization (Huganir et al., 1986). 4.2. Receptor-Activity Modulating Intracellular Ca21, Protein Kinase C, and Desensitization by Paired Pulse Treatment of chick myotubes with phorbol esters or diacylglycerol analogs decreases the sensitivity of the nAChR to ACh and increases the rate of desensitization of the nAChR, providing evidence that protein kinase C (Ca21/ phospholipid-dependent)-induced phosphorylation of the nAChR regulates its rate of desensitization (Eusebi et al., 1985, 1987). Protein kinase C is translocated from the cytosol to the plasma membrane in a Ca21-dependent manner (Kraft and Anderson, 1983; Melloni et al., 1985). Activation of nAChR in skeletal muscle causes translocation of protein kinase C to postsynaptic membranes, thereby activating the protein kinase and enhancing receptor desensitization (Eusebi et al., 1987). Protein kinase C phosphorylates the d-subunit of Torpedo nAChR in both membrane-bound and solubilized forms (Safran et al., 1987, 1990), and b4-subunit (Huganir and Greengard, 1990). TPA, a protein kinase C activator, depresses contractile Ca21 transients elicited by single-pulse nerve stimulation in mouse diaphragm muscle (Kimura et al., 1993b). Noncontractile slow Ca21 (RAMIC)-induced depression of contractile Ca21 transients is diminished by staurosporine, a protein kinase C inhibitor (Kimura et al., 1995a). After pretreatment with protein kinase C inhibitors, even large amounts of RAMIC fail to depress the second contractile Ca21 transients elicited by paired-pulse stimulation (Fig. 5d). Therefore, nAChRoperated RAMIC mobilization may enhance nAChR desensitization through activation of protein kinase C. The depressant effect of protein kinase C on the second contractile Ca21 transients elicited by paired-pulse stimulation is not due to a decrease in ACh release from motor nerve terminals, which is actually increased by protein kinase C activators (Eusebi et al., 1986; Caratsch et al., 1988). No role is played in this process by Ca21 influx through the muscle dihydropyridine-sensitive Ca21 channels in the

193

transverse-tubule membranes, which are activated by protein kinase C phosphorylation (Chang et al., 1991). The agonist-induced phosphorylation of nAChR in rat myotubes is dependent on external Ca21 and is abolished by nAChR-channel blockers, suggesting that Ca21 influx via the nAChR channel is crucial to the activation of protein kinase C (Miles et al., 1994). There is also a possibility of Ca21 influx through Ca21 channels other than L type and activated by the AChR-induced depolarization. Tyrosine kinase phosphorylates the b-, g-, and d-subunits (Huganir et al., 1984; Hopfield et al., 1988; Qu et al., 1990). Desensitization of nAChR in reconstituted membranes is accelerated by the phosphorylation. Although the nAChR desensitization in skeletal muscle is enhanced by intracellular Ca21 (Miledi, 1980), protein kinase A (Huganir et al., 1986) and protein kinase C (Eusebi et al., 1987), their mutual relationships and the order of in-series phenomena in the process of desensitization has not been established yet. Based on observations on the effects of CGRP and a protein kinase C inhibitor on contractile and noncontractile Ca21 mobilization, we have defined the relationships between increase in local intracellular Ca21, protein kinase A activation, and protein kinase C activation in the process of nAChR desensitization. We proposed a model whereby the activation of protein kinase A mobilizes noncontractile Ca21, which in turn activates protein kinase C and stabilizes the postsynaptic muscle-type nAChR in refractory states, protecting it against over-excitation. Hence, nAChR desensitization may develop by “cross-talk” between the protein kinase A system and the protein kinase C system through RAMIC. A schematic representation of this sequence of events is shown in Fig. 6. From presently accumulated data, we proposed the scheme presenting the muscle-type nAChR-desensitizing mechanisms in which noncontractile slow Ca21 mobilized by CGRP receptor-neuronal-type nAChR interaction plays a main role. 5. ABNORMALITY INDUCED BY NEURONAL-TYPE NICOTINIC ACETYLCHOLINE RECEPTOR MUTATION The involvement of a particular neuronal-type nAChR subunit in pharmacology and behaviour has been examined using gene targeting to mutate the b2-subunit. Behavioral tests demonstrate that nicotine no longer augments the performance of b22/2 mice on passive avoidance, a test of associative memory (Picciotto et al., 1995). A spontaneous mutation of the b-subunit that interrupts the leucine ring of the nAChR channel gate causes an 8-fold increase in channel open time and a severe congenital myasthenic syndrome characterized by severe endplate myopathy and extensive remodeling of the postsynaptic membrane (Gomez et al., 1996). When the leucines in the ring are replaced with a smaller uncharged amino acid, the channel of mutated receptor is fixed in an open state, despite tight binding to ACh (Changeux, 1993). The mutation, located in the M2 segment, dramatically altered the single channel

194

I. Kimura

FIGURE 6. Schematic models of mechanisms generating RAMIC signals and linking desensitization at neuromuscular postsynapse. In series physiological responses are ACh accumulation, CGRP release, CGRP receptor (R) activation, protein kinase A (PK-A) activation, neuronal nAChR channel opening, Ca21 influx, RAMIC mobilization, protein kinase C (PK-C) activation, and muscular nAChR desensitization.

properties of the nAChR (Ohno et al., 1995). The inhibition of a3b4-containing receptors by k-BgTX has rapid kinetics of onset and recovery, which are in contrast to the slow and prolonged inhibition of a3b2-containing receptors, suggesting that the subtype of b-subunit determines the kinetics of the k-BgTX inhibition of a3 receptors (Papke et al., 1993). The Ca21 permeability, the apparent affinity for ACh, and the rates of activation and desensitization are also altered by mutations of two adjacent amino acids (Leu 254, Leu 255) located in proximity of the extracellular end of TM2 (Bertrand et al., 1993). 6. OTHER ABNORMALITIES IN DISEASES The receptor theory proposes that up-regulation of receptors is associated with increased sensitivity to agonists and decreased sensitivity to antagonists (Wonnacott, 1990). The classical pharmacological dogma regarding up-regulation (increased numbers) and down-regulation (decreased numbers) and its relationship to agonist and antagonist responses can be considered to explain the ACh-mediated abnormal responses to muscle relaxants (Martyn et al., 1992; Wonnacott, 1990). Up-regulation of AChRs (resistance to nondepolarizing muscle relaxants and hyperkalemia induced by agonists) is considered to occur in any neurologic motor defect, direct muscle trauma, thermal trauma, disuse atrophy, intensive care unit prolonged use of relaxants and severe infection. On the other hand, down-regulation (sensitivity to nondepolarizing muscle relaxants and resistance to agonists) may occur in myasthenia gravis, ex-

ercise conditioning, and organophosphorus poisoning (Martyn, 1995). Contrary to expectation, the up-regulation of nicotine-binding sites has been widely documented (reviewed by Wonnacott, 1990). In vitro assessment of the nicotinic modulation of transmitter release in brain preparations from animals chronically treated with nicotinic agonists has revealed both increased (Wonnacott et al., 1990) and decreased (Lapchak et al., 1989) nicotinic function. In vivo behavioral measurements have indicated increased (Ksir et al., 1987; Clarke et al., 1988) and decreased (Marks et al., 1985) responsiveness following chronic nicotine administration. Changes in nicotinic functions have been described in a number of neurologic and systemic diseases. Areas containing high-affinity nicotine binding, rather than those binding a-BgTX, primarily are affected in Alzheimer’s patients (Sugaya et al., 1990; Perry et al., 1995). Some of these changes have been detected using non-neuronal tissues as a source of material. For example, the a4 (and with rather lower intensity a3)-subunit genes of nAChRs are expressed in human lymphocytes (Hiemke et al., 1996). Changes in the expression of nAChRs on lymphocytes might reflect alterations of cholinergic neurotransmission in the brain, as suggested for senile dementia of the Alzheimer type and other diseases accompanied by cognitive dysfunction (Adem et al., 1986). Pathologic concentrations of hydrocortisone reduced the number of openings per activation period induced by high ACh concentration and caused the long closed times typically observed in the desensitization process (Bouzat and Barrantes, 1996). Hydrocortisone, a gluco-

Functions of Neuronal-Type Nicotinic ACh Receptors

corticoid, acts as a noncompetitive inhibitor of the nicotinic receptor. Neurosteroids present at high concentrations in the brain may be active modulators of receptor function. The diabetic state has great influences on nAChR function. In isolated skeletal muscles of spontaneously diabetic mice, the ACh potential amplitude was greater, and suxamethonium inhibited ACh potentials to a greater extent, than in nondiabetic mice (Kimura et al., 1986). Enhancement of desensitization, shown as accelerated decline of the responses to ACh delivered iontophoretically in trains of high frequency, and time-dependent decrease in the AChRchannel opening frequency, indicate the enhancement of nAChR desensitization by diabetes (Nojima et al., 1995). The diabetic state-induced shortening of the decay time may be caused by promotion of the inactivation process of nAChR. The diabetic effect also may be explained by a decrease in ACh release from the nerve terminal (Kimura et al., 1993a). The diabetic state also affects Ca21 transients in muscle: Ca21 mobilization evoked by direct stimulation

195

of the alloxan-diabetic diaphragm muscle is insensitive to the extracellular Ca21 changes, and the sensitivity to extracellular Ca21 is restored by intracellular injection of EGTA into the diabetic muscle cells (Kimura et al., 1990a). This raises the possibility that the resting intracellular Ca21 may be elevated in diabetic muscles. The amplitude and duration of slow Ca21 action potentials are decreased in the alloxan diabetic state (Kimura, I. et al., 1988). The slow Ca21 action potentials produced by repeated stimulation decay more rapidly in diabetic muscle, and unlike those in normal muscles, are insensitive to verapamil (Kimura, I. et al., 1988). The attenuation of contractile Ca21 transients in the diabetic state (Kimura et al., 1995b) may be related to the enhancement of the nAChR desensitization via the elevation of resting intracellular Ca21 (Nastuk and Parsons, 1970; Chang and Neumann, 1976; Miledi, 1980). These diabetic alterations of contractile Ca21 mobilization are not completely reverted to normal by insulin treatment, despite the recovery of blood glucose levels (Kimura et al., 1995b).

FIGURE 7. Generalized category on desensitizing role of neuronal-type nAChR to muscle-type nAChR or other types of receptor in the brain by (I) treatment with anticholinesterase agents or excessive amount of ACh caused by repetitive firing and by (II) de sensitizing nAChR channel blocker or in diseased state.

196

Abnormalities in resting membrane potentials and conductance also do not return to normal levels with insulin treatment (Kimura, M. et al., 1988). Therefore, a chronic diabetic state may cause muscle defects in the contractile Ca21 mobilization system, in addition to the influence of hyperglycemia. 7. CONCLUSION AND PERSPECTIVE The demonstration of the presence of neuronal-type nAChR in muscle poses the neuromuscular junction as a simplified model to study the sophisticated role of neuronal-type nAChRs elsewhere, including the brain. The abundance and variety of brain nAChRs had been uncovered by molecular and in situ studies (Boulter et al., 1986a; Sargent, 1993; McGehee and Role, 1995; Galzi and Changeux, 1995). The functional features of the nAChRs in the brain are still controversial because the usual electrophysiological techniques are not sufficient to elucidate in sufficient detail their exact functions in brain preparation and cultured neurons. Instead, by appropriately using the neuromuscular junction to these ends, we have been the first to propose that b2-containing neuronal-type nAChRs play an important role in modulation of Ca21-dependent functions of the synapse. Other studies, in particular those concerning the effects of anti-b2 antibodies on RAMIC, also provided new concepts about the functional significance of the neuronal b-subunits. One of the main functions of neuronal-type nAChRs is the Ca21-dependent desensitization caused by semi-heterogenous receptor-receptor interaction through localized noncontractile slow Ca21 mobilization beneath the postsynaptic membrane. The desensitizing interactions occur between neuronal- and muscle-type nAChRs; it is likely that similar mechanisms of receptor-receptor interaction involving neuronal AChRs also occur with other systems, such as muscarinic, dopaminergic and GABAergic transmission. The interaction between N-methyl-d-aspartate receptors and a-amino-3-hydroxy-5-methyl-isoxazole propionate/kainate receptors, which is relevant for longterm potentiation and depression, is an additional example of semi-heterologous receptor-receptor interaction, with the difference that in this case, potentiation and inhibition of function occur (Rang et al., 1995). The main results referred to in this review are summarized in Fig. 7, where the interactions occurring between neuronal- and muscle-type nAChRs are shown during exposure to excess ACh induced by (I) treatment with anticholinesterase agents and over-excitation of motor nerve and (II) by desensitizing nAChR channel blockers and in pathological conditions. Neuromuscular function may be regulated by a dual nAChR system to maintain the normal synaptic excitation. Neuronal-type nAChRs may be endowed with the same functional role in the CNS also. The terms “neuronal” and “muscle” applied to nicotinic AChRs were based on anatomical location. Since it has now been shown that certain subtypes that were formerly regarded as specifically of neuronal origin in fact also occur

I. Kimura

in muscle, the terms must be regarded as obsolete. The only appropriate description of a nAChR subtype is, therefore, its stoichiometry. Acknowledgements–The author is grateful to Professor Dr. M. Kimura (Emeritus Professor, Toyama Medical and Pharmaceutical University, Toyama, Japan; Adjunct Professor, University of Texas Health & Science Center, San Antonio, TX, USA) for his continuous encouragement, Drs. H. Nojima, H. Tsuneki and K. Dezaki (Department of Chemical Pharmacology, Toyama Medical and Pharmaceutical University) for their skillful experiments, and Professor G. Fumagalli (Universita degli Studi di Verona, Verona, Italy) for his critical reading of this manuscript. This work has been supported in part by Monbusho short-term fellowship (1992) (Professor F. Eusebi, Instituto Regina Elena, Centro Ricerca Sperimentale, Rome, Italy), JSPS fellowship (1994) (Professor F. Clementi, Universita degli Studi di Milano, Milano, Italy), Grant-in-Aid from the Ministry of Education, Science and Culture for Monbusho International Joint Research (No. 09044276) (1997–1998) (with Professor J.-P. Changeux, Institut Pasteur, Paris, France).

References Adam, D. J., Dwyer, T. M. and Hille, B. (1980) The permeability of endplate channels to monovalent and divalent metal cations. J. Gen. Physiol. 75: 493–510. Adem, A., Nordberg, A., Bucht, G. and Winblad, A. (1986) Extraneural cholinergic markers in Alzheimer’s and Parkinson’s disease. Prog. Neuropsychopharmacol. Biol. Psychiatry 10: 247– 257. Albuquerque, E. X., Deshpande, S. S., Aracava, Y., Alkondon, M. and Daly, J. W. (1986) A possible involvement of cyclic AMP in the expression of desensitizatin of the nicotinic acetylcholine receptor. FEBS Lett. 199: 113–120. Alkondon, M. and Albuquerque, E. X. (1993) Diversity of nicotinic acetylcholine receptors in rat hippocampal neurons. I. Pharmacological and functional evidence for distinct structural subtypes. J. Pharmacol. Exp. Ther. 265: 1455–1473. Alkondon, M., Pereira, E. F. R., Wonnacott, S. and Albuquerque, E. X. (1992) Blockade of nicotinic currents in hippocampal neurons defines methyllycaconitine as a potent and specific receptor antagonist. Mol. Pharmacol. 41: 802–808. Anand, R., Peng, X., Ballesta, J. J. and Lindstrom, J. (1993) Pharmacological characterization of a-bungarotoxin-sensitive acetylcholine receptors immunoisolated from chick retina: contrasting properties of a7 and a8 subunit-containing subtypes. Mol. Pharmacol. 44: 1046–1050. Bencherif, M., Eisenhour, C. M., Prince, R. J., Lippielo, P. M. and Lukas, R. J. (1995) The “calcium antagonist” TMB-8 [3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester] is a potent, non-competitive, functional antagonist at diverse nicotinic acetylcholine receptor subtypes. J. Pharmacol. Exp. Ther. 275: 1418–1426. Bertrand, D., Galzi, J. H., Devillers-Thiery, A., Bertrand, S. and Changeux, J.-P. (1993) Mutations at two distinct sites within the channel domain M2 alter calcium permeability of neuronal a7 nicotinic receptor. Proc. Natl. Acad. Sci. USA 90: 6971–6975. Bessis, A., Salmon, A.-M., Zoli, M., Le Novere, N., Picciotto, M. and Changeux, J.-P. (1995) Promotor elements conferring neuron-specific expression of the b2-subunit of the neuronal nicotinic acetylcholine receptor studied in vitro and in transgenic mice. Neuroscience 69: 807–819. Boulter, J., Luyten, W., Evans, K., Mason, P., Ballivet, M., Goldman, D., Stengelin, S., Martin, G., Heinemann, S. and Patrick, J. (1985) Isolation of a clone coding for the alpha-subunit of a mouse acetylcholine receptor. J. Neurosci. 5: 2545–2552.

Functions of Neuronal-Type Nicotinic ACh Receptors Boulter, J., Evans, K., Goldman, D., Martin, G., Treco, D., Heinemann, S. and Patrick, J. (1986a) Isolation of a cDNA clone coding for a possible neural nicotinic acetylcholine receptor a-subunit. Nature 319: 368–374. Boulter, J., Evans, K., Martin, G., Mason, P., Stengelin, S., Goldman, D., Heinemann, S. and Patrick, J. (1986b) Isolation and sequence or cDNA clones coding for the precursor to the gamma subunit of mouse muscle nicotinic acetylcholine receptor. J. Neurosci. Res. 16: 37–49. Boulter, J., Connolly, J., Denneris, E., Goldman, D., Heinemann, S. and Patrick, J. (1987) Functional expression of two neuronal nicotinic acetylcholine receptors from cDNA clones identifies a gene family. Proc. Natl. Acad. Sci. USA 84: 7763–7767. Boulter, J., O’Shea-Greenfield, A., Duvoisin, R. M., Connoly, J. G., Wada, E., Jensen, A., Gardner, P. D., Ballvivet, M., Deneris, E. S., McKinnon, D., Heinemann, S. and Patrick, J. (1990) a3, a5, and b4: three members of the rat neuronal nicotinic acetylcholine receptor-related gene family form a gene cluster. J. Biol. Chem. 265: 4472–4482. Bouzat, C. and Barrantes, F. J. (1996) Modulation of muscle nicotinic acetylcholine receptors by the glucocorticoid hydrocortisone. Possible allosteric mechanism of channel blockade. J. Biol. Chem. 271: 25835–25841. Bowman, W. C. (1964) Neuromuscular blocking agents. In: Evaluation of Drug Activities, Vol. 1, Ch. 16, pp. 325–351, Laurence, D. R. and Bacharach, A. L. (eds.) Academic Press, London. Bowman, W. C. (1980) Prejunctional and postjunctional cholinoceptors at the neuromuscular junction. Anesth. Analg. 59: 935–948. Bowman, W. C., Prior, C. and Marshall, I. G. (1990) Presynaptic receptors in the neuromuscular junction. Ann. NY Acad. Sci. 604: 69–81. Bregestovski, P. D., Miledi, R. and Parker, I. (1979) Calcium conductance of acetylcholine-induced endplate channels. Nature 279: 638–639. Brenner, H. R., Witzemann, V. and Sakmann, B. (1990) Imprinting of acetylcholine receptor messenger RNA accumulation in mammalian neuromuscular synapses. Nature 344: 544–547. Bufler, J., Franke, C., Witzemann, V., Ruppersberg, J. P., Merlitze, S. and Dudel, J. (1993) Desensitization of embryonic nicotinic acetylcholine receptors expressed in Xenopus oocytes. Neurosci. Lett. 152: 77–80. Burns, B. D. and Paton, W. D. M. (1951) Depolarization of the motor end-plate by decamethonium and acetylcholine. J. Physiol. 115: 41–73. Caratsch, C. G., Schumacher, S., Grassi, F. and Eusebi, F. (1988) Influence of protein kinase C-stimulation by a phorbor ester on the neurotransmitter release at frog end-plates. Naunyn Schmiedebergs Arch. Pharmacol. 337: 9–12. Cartier, G. E., Yoshikami, D., Gray, W. R., Luo, S., Olivera, B. M. and McIntosh, J. M. (1996) A new alpha-conotoxin which targets alpha3beta2 nicotinic acetylcholine receptors. J. Biol. Chem. 271: 7522–7528. Castro, N. G. and Albuquerque, E. X. (1995) alpha-Bungarotoxinsensitive hippocampal nicotinic receptor channel has a high calcium permeability. Biophys. J. 68: 516–524. Chang, C. C. and Hong, S. J. (1986) A generative release of acetylcholine from motor nerve terminals treated with anticholinesterase agents. Neurosci. Lett. 69: 203–207. Chang, C. C., Chen, S. M. and Hong, S. J. (1987) Concentration dependence of the effects of neostigmine on the endplate

197 potential and neuromuscular transmission in a mammalian muscle. Asia Pac. J. Pharmacol. 2: 87–92. Chang, C. F., Gutierrez, L. M., Mundina-Weilenmann, C. and Hosey, M. M. (1991) Dihydropyridine-sensitive calcium channels from skeletal muscle. II. Functional effects of differential phosphorylation of channel subunits. J. Biol. Chem. 266: 16395–16400. Chang, H. W. and Neumann, E. (1976) Dynamic properties of isolated acetylcholine receptor proteins: release of calcium ions caused by acetylcholine binding. Proc. Natl. Acad. Sci. USA 73: 3364–3368. Changeux, J.-P. (1993) Chemical signaling in the brain: studies of acetylcholine receptors in the electric organs of fish have generated critical insights into how neurons in the human brain communicate with one another. Sci. Am. 269: 30–37. Chesnut, T. J. (1983) Two-component desensitization at the neuromuscular junction of the frog. J. Physiol. 336: 229–241. Clarke, P. B. S. (1992) The fall and rise of neuronal a-bugarotoxin binding proteins. Trends Pharmacol. Sci. 13: 407–413. Clarke, P. B. S., Fu, D. S., Jakubovic, A. and Fibiger, H. C. (1988) Evidence that mesolimbic dopaminergic activation underlies the locomotor stimulant action of nicotine in rats. J. Pharmacol. Exp. Ther. 246: 701–708. Colquhoun, D. and Sakmann, B. (1985) Fast events in singlechannel currents activated by acetylcholine and its analogues at the frog muscle end-plate. J. Physiol. 369: 501–557. Conroy, W. G. and Berg, D. K. (1995) Neurons can maintain multiple classes of nicotinic acetylcholine receptors distinguished by different subunit compositions. J. Biol. Chem. 270: 4424– 4431. Conroy, W. G., Vernallis, A. B. and Berg, D. K. (1992) The a5 gene product assembles with multiple acetylcholine subunits to form distinctive receptor subtypes in brain. Neuron 9: 679–691. Corriveau, R. A., Romano, S. J., Conroy, W. G., Oliva, L. and Berg, D. K. (1995) Expression of neuronal acetylcholine receptor genes in vertebrate skeletal muscle during development. J. Neurosci. 15: 1372–1383. Couturier, S., Bertrand, D., Matter, J.-M., Hernandez, M.-C., Bertrand, S., Millar, N., Valera, S., Barkas, T. and Ballivet, M. (1990) A neuronal nicotinic acetylcholine receptor subunit (a7) is developmentally regulated and forms a homo-oligomeric channel blocked by a-BuTX. Neuron 5: 847–856. Decker, E. R. and Dani, J. A. (1990) Calcium permeability of the nicotinic acetylcholine receptor: the single-channel calcium influx is significant. J. Neurosci. 10: 3413–3420. Deneris, E. S., Connolly, J., Boulter, J., Wada, E., Wada, K., Swanson, L. W., Patrick, J. and Heinemann, S. (1988) Primary structure and expression of b2: a novel subunit of neuronal nicotinic acetylcholine receptors. Neuron 1: 45–54. Deneris, E. S., Boulter, J., Swanson, L. W., Patrick, J. and Heinemann, S. (1989) b3: a new member of nicotinic acetylcholine receptor gene family is expressed in brain. J. Biol. Chem. 264: 6268–6272. Dezaki, K., Kimura, I., Tsuneki, H. and Kimura, M. (1996) Enhancement by calcitonin gene-related peptide of noncontractile Ca21-induced nicotinic receptor desensitization at the mouse neuromuscular junction. Br. J. Pharmacol. 118: 1971– 1976. Dezaki, K. and Kimura, I. (1998) Acetylcholine sensitivity of biphasic Ca21 mobilization induced by nicotinic receptor activation at the mouse skeletal muscle endplate. Br. J. Pharmacol. 123: in press.

198 Duvoisin, R. M., Deneris, E. S., Patrick, J. and Heinemann, S. (1989) The functional diversity of the neuronal nicotinic acetylcholine receptors is increased by a novel subunit: b4. Neuron 3: 487–496. Elgoyhen, A. B., Johnson, D. S., Boulter, J., Vetter, D. E. and Heinemann, S. (1994) a9: an acetylcholine receptor with novel pharmacological properties expressed in rat cochlear hair cells. Cell 79: 705–715. Eusebi, F., Molinaro, M. and Zani, B. M. (1985) Agents that activate protein kinase C reduced acetylcholine sensitivity in cultured myotubes. J. Cell Biol. 100: 1339–1342. Eusebi, F., Molinaro, M. and Caratsh, C. G. (1986) Effects of phorbor ester on spontaneous transmitter release at frog neuromuscular junction. Pflügers Arch. 406: 181–183. Eusebi, F., Grassi, F., Nervi, C., Caporale, C., Adamo, S., Zani, B. M. and Molinaro, M. (1987) Acetylcholine may regulate its own nicotinic receptor-channel through the C-kinase system. Proc. R. Soc. Lond. B 230: 355–365. Ferrer-Montiel, A. V., Montal, M. S., Diaz-Munoz, M. and Montal, M. (1991) Agonist-independent activation of acetylcholine receptor channels by protein kinase A phosphorylation. Proc. Natl. Acad. Sci. USA 88: 10213–10217. Flores, C., Rogers, S., Pabreza, L., Wolfe, B. and Kellar, K. (1992) A subtype of nicotinic cholinergic receptor in rat brain is composed of a4 and b2 subunits and is upregulated by chronic nicotine treatment. Mol. Pharmacol. 41: 31–37. Fontaine, B., Klarsfeld, A., Hokfelt, T. and Changeux, J.-P. (1986) Calcitonin gene-related peptide, a peptide present in spinal cord motoneurons, increases the number of acetylcholine receptors in primary cultures of chick embryo myotubes. Neurosci. Lett. 71: 59–65. Franke, C., Hatt, H., Parnas, H. and Dudel, J. (1992) Recovery from the rapid desensitization of nicotinic acetylcholine receptor channels on mouse muscle. Neurosci. Lett. 140: 169–172. Galzi, J.-L. and Changeux, J.-P. (1995) Neurotransmitter receptors VI: molecular organization and regulations. Neuropharmacology 34: 563–582. Galzi, J.-L., Bertrand, S., Corringer, P.-L., Changeux, J.-P. and Bertrand, D. (1996) Identification of calcium binding sites that regulate potentiation of a neuronal nicotinic acetylcholine receptor. EMBO J. 15: 5824–5832. Gerzanich, V., Anand, R. and Lindstrom, J. (1994) Homomers of alpha 8 and alpha 7 subunits of nicotinic receptors exhibit similar channel but contrasting binding site properties. Mol. Pharmacol. 45: 212–220. Goldman, D. and Staple, J. (1989) Spatial and temporal expression of acetylcholine receptor RNAs in innervated and denervated rat soleus muscle. Neuron 3: 219–228. Goldman, D., Denneris, E., Luyten, W., Kochhar, A., Patrick, J. and Heinemann, S. (1987) Members of a nicotinic acetylcholine receptor gene family are expressed in different regions of the mammalian central nervous system. Cell 48: 965–973. Gomez, C. M., Maselli, R., Gammack, J., Lasaide, J., Tamamizu, S., Cornblath, D. R., Lehar, M., McNamee, M. and Kuncl, R. W. (1996) A b-subunit mutation in the acetylcholine receptor channel gate causes severe slow-channel syndrome. Ann. Neurol. 39: 712–723. Grady, S., Marks, M. J., Wonnaott, S. and Collins, A. C. (1992) Characterization of nicotinic receptor-mediated [3H] dopamine release from synaptosomes prepared from mouse striatum. J. Neurochem. 59: 848–856. Gu, Y. and Hall, Z. W. (1988) Immunological evidence for a

I. Kimura change in subunits of the acetylcholine receptor in developing and denervated rat muscle. Neuron 1: 117–125. Halvorsen, S. W., Schmid, H. A., McEachern, A. E. and Berg, D. K. (1991) Regulation of acetylcholine receptors on chick ciliary ganglion neurons by components from the synaptic target tissue. J. Neurosci. 11: 2177–2186. Herlitze, S., Villarroel, A., Witzemann, V., Koenen, M. and Sakmann, B. (1996) Structural determinants of channel conductance in fetal and adult rat muscle acetylcholine receptor. J. Physiol. 492: 775–787. Hiemke, C., Stolp, M., Reuss, S., Wevers, A., Reinhardt, S., Maelickes, A., Schlegel, S. and Schroder, H. (1996) Expression of alpha subunit genes of nicotinic acetylcholine receptors in human lymphocytes. Neurosci. Lett. 214: 171–174. Hopfield, J. F., Tank, D. W., Greengard, P. and Huganir, R. L. (1988) Functional modulation of the nicotinic acetylcholine receptor by tyrosine phosphorylation. Nature 336: 677–680. Hucho, F., Tsetlin, V. I. and Machold, J. (1996) The emerging three-dimensional structure of a receptor: the nicotinic acetylcholine receptor. Eur. J. Biochem. 239: 539–557. Huganir, R. L. and Greengard, P. (1983) cAMP-dependent protein kinase phosphorylates the nicotinic acetylcholine receptor. Proc. Natl. Acad. Sci. USA 80: 1130–1134. Huganir, R. L. and Greengard, P. (1987) Regulation of receptor function by protein phosphorylation. Trends Pharmacol. Sci. 8: 472–477. Huganir, R. L. and Greengard, P. (1990) Regulation of neurotransmitter receptor desensitization by phosphorylation. Neuron 5: 555–567. Huganir, R. L., Miles, K. and Greengard, P. (1984) Phosphorylation of the nicotinic acetylcholine receptor by an endogenous tyrosine-specific protein kinase. Proc. Natl. Acad. Sci. USA 81: 6963–6972. Huganir, R. L., Delcour, A. H., Greengard, P. and Hess, G. P. (1986) Phosphorylation of the nicotinic acetylcholine receptor regulates its rate of desensitization. Nature 321: 774–776. Imoto, K., Busch, C., Sakmann, B., Mishina, M., Konno, T., Nakai, J., Bujo, H., Mori, Y., Fukuda, K. and Numa, S. (1988) Rings of negatively charged amino acids determine the acetylcholine receptor channel conductance. Nature 335: 645–648. Jenning, C. G. B. and Mudge, A. W. (1989) Chick myotubes in culture express high-affinity receptors for calcitonin generelated peptide. Brain Res. 504: 199–205. Johnson, D. S., Martinez, J., Elgoyhen, A. B., Heinemann, S. and McIntosh, J. M. (1995) a-Conotoxin ImI exhibits subtypespecific nicotinic AChR blockade: preferential inhibition of homomeric a7 and a9 receptors. Mol. Pharmacol. 48: 194–199. Katz, B. and Thesleff, S. (1957) A study of the “desensitization” produced by acetylcholine at the motor end-plate. J. Physiol. 138: 63–80. Katz, R. L. (1971) Modification of the action of pancronium by succinylcholine and halothane. Anesthesiology 35: 602–606. Kerr, L. M. and Sperelakis, N. (1982) Effects of calcium antagonists bepridil and verapamil on Ca21-dependent slow potentials in frog skeletal muscle. J. Pharmacol. Exp. Ther. 222: 80–86. Kimura, I., Nakamura, T. and Kimura, M. (1988) Alloxan-diabetic state-induced suppression of Ca21-dependent slow action potentials in mouse diaphragm muscle. Jpn. J. Pharmacol. 47: 197–199. Kimura, I., Kimura, M. and Kimura, M. (1990a) Increase in electrically-stimulated Ca21-release and suppression of caffeine response in diaphragm muscle of alloxan-diabetic mice compared with the denervation effect. Diabetologia 33: 72–76.

Functions of Neuronal-Type Nicotinic ACh Receptors Kimura, I., Kondoh, T. and Kimura, M. (1990b) Postsynaptic nicotinic ACh receptor-operated Ca21 transients with neostigmine in phrenic nerve-diaphragm muscles of mice. Brain Res. 507: 309–311. Kimura, I., Kondoh, T., Tsuneki, H. and Kimura, M. (1991a) Reversed effect of caffeine on non-contractile and contractile Ca21 mobilization operated by acetylcholine receptor in mouse diaphragm muscle. Neurosci. Lett. 127: 28–30. Kimura, I., Nojima, H. and Kimura, M. (1991b) External Ca21dependence of acetylcholine- and succinylcholine-induced changes in channel conductance, open time and frequency at endplates of single muscle cells of adult mice. Neuropharmacology 30: 1211–1217. Kimura, I., Okazaki, M., Uwano, T., Kobayashi, S. and Kimura, M. (1991c) Succinylcholine-induced acceleration and suppression of electrically evoked acetylcholine release from mouse phrenic nerve-hemidiaphragm muscle preparation. Jpn. J. Pharmacol. 57: 397–403. Kimura, I., Okazaki, M. and Kimura, M. (1993a) Streptozocin-diabetes modifies acetylcholine release from mouse phrenic nerve terminal and presynaptic sensitivity to succinylcholine. Jpn. J. Pharmacol. 62: 35–41. Kimura, I., Tsuneki, H., Dezaki, K. and Kimura, M. (1993b) Enhancement by calcitonin gene-related peptide of nicotinic receptor-operated noncontractile Ca21 mobilization at the mouse neuromuscular junction. Br. J. Pharmacol. 110: 639–644. Kimura, I., Tsuneki, H., Dezaki, K., Nojima, H. and Kimura, M. (1994) Monoclonal antibody to b2 subunit of neuronal nicotinic receptor depresses the postjunctional non-contractile Ca21 mobilization in the mouse diaphragm muscle. Neurosci. Lett. 180: 101–104. Kimura, I., Dezaki, K., Tsuneki, H. and Kimura, M. (1995a) Postsynaptic nicotinic receptor desensitized by non-contractile Ca21 mobilization via protein kinase-C activation at the mouse neuromuscular junction. Br. J. Pharmacol. 114: 461–467. Kimura, I., Tsuneki, H., Dezaki, K. and Kimura, M. (1995b) Diabetic state-induced rapid inactivation of noncontractile Ca21 mobilization operated by nicotinic acetylcholine receptor in mouse diaphragm muscle. Br. J. Pharmacol. 116: 2685–2690. Kimura, I., Okazaki, M. and Nojima, H. (1997) Mutual dependence of calcitonin-gene related peptide and acetylcholine release in neuromuscular preparations. Eur. J. Pharmacol. 330: 123–128. Kimura, M., Kimura, I., Nojima, H. and Muroi, M. (1986) Diabetes mellitus-induced hypersensitivity of mouse skeletal muscles to acetylcholine and succinylcholine. Jpn. J. Pharmacol. 40: 251–258. Kimura, M., Kimura, I., Nakamura, T. and Nojima, H. (1988) Diabetic state-induced modification of resting membrane potential and conductance in diaphragm muscle of alloxan and diabetic KK-CAy mice. Diabetologia 31: 103–107. Kimura, M., Kimura, I., Kondoh, T. and Tsuneki, H. (1991) Noncontractile acetylcholine receptor-operated Ca11 mobilization: suppression of activation by open channel blockers and acceleration of desensitization by closed channel blockers in mouse diaphragm muscle. J. Pharmacol. Exp. Ther. 256: 18–23. Kobayashi, H., Hashimoto, K., Uchida, S., Sakuma, J., Takami, K., Tohyama, M., Izumi, F. and Yoshida, H. (1987) Calcitonin gene-related peptide stimulates adenylate cyclase activity in rat striated mouse. Experientia 43: 314–316. Kraft, A. S. and Anderson, W. B. (1983) Phorbol esters increase the amount of Ca21, phospholipid-dependent protein kinase associated with plasma membrane. Nature 301: 621–623.

199 Ksir, C., Haken, R. L. and Kellar, K. J. (1987) Chronic nicotine and locomotor activity: influences of exposure dose and test dose. Psychopharmacology 92: 25–29. Lamar, E., Miller, K. and Patrick, J. (1990) Amplification of genomic sequences identifies a new gene, a6, in the nicotinic acetylcholine receptor gene family. Soc. Neurosci. Abstr. 16: 681. Lapchak, P. A., Araujo, D. M., Quirion, R. and Collier, B. (1989) Effect of chronic nicotine treatment on nicotinic autoreceptor function and N-[3H]methylcarbamylcholine binding sites in the rat brain. J. Neurochem. 52: 483–491. Lena, C., Changeux, J.-P. and Mulle, C. (1993) Evidence for “preterminal” nicotinic receptors of GABAergic axons in the rat interpeduncular nucleus. J. Neurosci. 13: 2680–2688. Le Novere, N. and Changeux, J. P. (1995) Molecular evolution of the nicotinic acetylcholine receptor: an example of multigene family in excitable cells. J. Mol. Evol. 40: 155–172. Lester, R. A. J. and Dani, J. A. (1995) Acetylcholine receptor desensitization induced by nicotine in rat medial habenula neurons. J. Neurophysiol. 74: 195–206. Lewis, C. (1979) Ion-concentration dependence of the reversal potential and the single channel conductance of ion channels at the frog neuromuscular junction. J. Physiol. 286: 417–445. Lu, B., Fu, W., Greengard, P. and Poo, M.-M. (1993) Calcitonin gene-related peptide potentiates synaptic responses at developing neuromuscular junction. Nature 363: 76–79. Machold, J., Weise, C., Utkin, Y., Tsetlin, V. and Hucho, F. (1995) The handedness of the subunit arrangement of the nicotinic acetylcholine receptor from Torpedo californica. Eur. J. Biochem. 234: 427–430. Magazanik, L. G. and Vyskocil, F. (1970) Dependence of acetylcholine desensitization on the membrane potential of frog muscle fibre and the ionic changes in the medium. J. Physiol. 210: 507–518. Magleby, K. L. and Pallotta, B. S. (1981) A study of desensitization of acetylcholine receptors using nerve-released transmitter in the frog. J. Physiol. 316: 225–250. Manthey, A. A. (1966) The effect of calcium on the desensitization of membrane receptors at the neuromuscular junction. J. Gen. Physiol. 49: 963–976. Manthey, A. A. (1970) Further studies of the effect of calcium on the time course of action of carbamylcholine at the neuromuscular junction. J. Gen. Physiol. 56: 407–419. Marks, M. J., Stitzel, J. A. and Collins, A. C. (1985) Time course study of the effects of chronic nicotine infusion on drug response and brain receptors. J. Pharmacol. Exp. Ther. 235: 619–628. Martinou, J. C. and Merlie, J. P. (1991) Nerve-dependent modulation of acetylcholine receptor epsilon-subunit gene expression. J. Neurosci. 11: 1291–1299. Martyn, J. A. J. (1995) The neuromuscular junction-basic receptor pharmacology. In: Muscle relaxants. Physiologic and Pharmacologic Aspects, pp. 37–47, Fukushima, K. and Ochiai, R. (eds.) Springer, Tokyo. Martyn, J. A. J., White, D. A., Gronert, G. A., Jaffe, R. S. and Ward, J. M. (1992) Up-and-down regulation of skeletal muscle acetylcholine receptors. Anesthesiology 76: 822–843. Matsumoto, N., Wang, X.-B. and Uchida, S. (1992) Different natures of supersensitivity of adenylate cyclase stimulated by calcitonin gene-related peptide and isoproterenol in rat diaphragm after denervation and reserpine treatment. J. Neurochem. 58: 357–361.

200 Matteoli, M., Haimann, C., Torri-Tarelli, F., Polak, J. M., Ceccarelli, B. and De Camilli, P. (1988) Differential effect of a-latrotoxin on exocytosis from small synaptic vesicles and from large dense-core vesicles containing calcitonin generelated peptide at the frog neuromuscular junction. Proc. Natl. Acad. Sci. USA 85: 7366–7370. McGehee, D. S. and Role, L. W. (1995) Physiological diversity of nicotinic acetylcholine receptors expressed by vertebrate neurons. Annu. Rev. Physiol. 57: 521–546. McGehee, D. S., Heath, M. J. S., Gelber, S., Devay, P. and Role, L. W. (1995) Nicotine enhancement of fast excitatory synaptic transmission in CNS by presynaptic receptors. Science 269: 1692–1696. McIntosh, J. M., Yoshikami, D., Mahe, E., Nielsen, D. B., Rivier, J. E., Gray, W. R. and Olivera, B. M. (1994) A nicotinic acetylcholine receptor ligand of unique specificity, alpha-conotoxin ImI. J. Biol. Chem. 269: 16733–16739. McMahon, L. L., Yoon, K.-W. and Chiappinelli, V. A. (1994) Electrophysiological evidence for presynaptic nicotinic receptors in the avian ventral lateral geniculate nucleus. J. Neurophysiol. 71: 826–829. Melloni, E., Pontremoli, S., Michetti, M., Sacco, O., Sparatore, B., Salamino, F. and Horecker, B. L. (1985) Binding of protein kinase C to neutrophil membranes in the presence of Ca21 and its activation by a Ca21-requiring proteinase. Proc. Natl. Acad. Sci. USA 62: 6435–6439. Middleton, P., Rubin, L. L. and Schuetze, S. M. (1988) Desensitization of acetylcholine receptors in rat myotubes is enhanced by agents that elevate intracellular cAMP. J. Neurosci. 8: 3405– 3412. Miledi, R. (1980) Intracellular calcium and desensitization of acetylcholine receptors. Proc. R. Soc. Lond. B. 209: 447–452. Miledi, R., Parker, I. and Schalow, G. (1980) Transmitter induced calcium entry across the post-synaptic membrane at frog endplates measured using arsenazo III. J. Physiol. 200: 197–212. Mileo, A. M., Monaco, L., Palma, M., Grassi, F., Miledi, R. and Eusebi, F. (1995) Two forms of acetylcholine receptor g subunit in mouse muscle. Proc. Natl. Acad. Sci. USA 92: 2686–2690. Miles, K., Greengard, P. and Huganir, R. L. (1989) Calcitonin gene-related peptide regulates phosphorylation of the nicotinic acetylcholine receptor in rat myotubes. Neuron 2: 1517–1524. Miles, K., Audigier, S. S. M., Greengard, P. and Huganir, R. L. (1994) Autoregulation of phosphorylation of the nicotinic acetylcholine receptor. J. Neurosci. 14: 3271–3279. Mishina, M., Takai, T., Imoto, K., Noda, M., Takahashi, T., Numa, S., Methefessel, C. and Sakmann, B. (1986) Molecular distinction between fetal and adult forms of muscle acetylcholine receptor. Nature 321: 406–411. Morris, A., Beeson, D., Jacobson, L., Baggi, F., Vincent, A. and Newsom-Davis, J. (1991) Two isoforms of the muscle acetylcholine receptor a-subunit are translated in the human cell line TE671. FEBS Lett. 295: 116–118. Mulle, C., Benoit, P., Pinset, C., Roa, M. and Changeux, J.-P. (1988) Calcitonin gene-related peptide enhances the rate of desensitization of the nicotinic acetylcholine receptor in cultured mouse mucle cells. Proc. Natl. Acad. Sci. USA 85: 5728– 5732. Mulle, C., Choquet, D., Korn, H. and Changeux, J.-P. (1992) Calcium influx through nicotinic receptor in rat central neurons: its relevance to cellular regulation. Neuron 8: 135–143. Nakayama, H., Okuda, H. and Nakashima, T. (1993) Phosphorylation of rat brain nicotinic acetylcholine receptor by cAMP-

I. Kimura dependent protein kinase in vitro. Mol. Brain. Res. 20: 171– 177. Nastuk, W. L. and Parsons, R. L. (1970) Factors in the inactivation of postjunctional membrane receptors of frog skeletal muscle. J. Gen. Physiol. 56: 218–249. Nelson, N., Anholt, R., Lindstrom, J. and Montal, M. (1980) Reconstitution of purified acetylcholine receptors with functional ion channels in planar lipid bilayers. Proc. Natl. Acad. Sci. USA 77: 3057–3061. New, H. V. and Mudge, A. W. (1986) Calcitonin gene-related peptide regulates muscle acetylcholine receptor synthesis. Nature 323: 809–811. Nishizaki, T. and Sumikawa, K. (1994) A cAMP-dependent Ca21 signaling pathway at the endplate provided by the gamma to epsilon subunit switch in ACh receptors. Mol. Brain Res. 24: 341–345. Noda, M., Takahashi, H., Tanabe, T., Toyosato, M., Kikyotani, S., Hirose, T., Asai, M., Takashima, H., Inayama, S., Miyata, T. and Numa, S. (1983) Primary structures of b- and g-subunit precursors of Torpedo californica acetylcholine receptor deduced from cDNA sequences. Nature 301: 251–255. Nojima, H., Kimura, I. and Kimura, M. (1991) The external Ca21dependence of acetylcholine-induced contracture in single innervated and denervated skeletal muscle cells in mice. Jpn. J. Pharmacol. 56: 109–112. Nojima, H., Muroi, M., Kimura, I. and Kimura, M. (1992) Indirect inhibitory effect of succinylcholine on acetylcholine-activated channel activities and its modulation by external Ca21 in mouse skeletal muscles. Br. J. Pharmacol. 105: 23–26. Nojima, H., Kimura, I., Muroi, M. and Kimura, M. (1993) Different modes of blockade by p-phenylene-polymethylene bisammonium compounds of the nicotinic acetylcholine receptor channel in skeletal muscle cells of mice. J. Pharm. Pharmacol. 45: 309–314. Nojima, H., Kimura, I. and Kimura, M. (1994) The evidence of accelerative interaction between cAMP-dependent protein kinase and external calcium for the desensitization of nicotinic acetylcholine receptor channel in mouse skeletal muscle cells. Neurosci. Lett. 167: 113–116. Nojima, H., Tsuneki, H., Kimura, I. and Kimura, M. (1995) Accelerated desensitization of nicotinic receptor channels and its dependence on extracellular calcium in isolated skeletal muscles of streptozotocin-diabetic mice. Br. J. Pharmacol. 116: 1680–1684. Ochoa, E. L., Chattopadhyay, A. and McNamee, M. G. (1989) Desensitization of the nicotinic acetylcholine receptor: molecular mechanisms and effect of modulators. Cell. Mol. Neurobiol. 9: 141–178. Ohno, K., Huchinson, D. O., Milone, M., Brengman, J. M., Bouzat, C., Sine, S. and Engel, A. (1995) Congenital myasthenic syndrome caused by prolonged acetylcholine receptor channel openings due to a mutation in the M2 domain of the e subunit. Proc. Natl. Acad. Sci. USA 92: 758–762. Ono, K., Manabe, N., Ohta, Y., Morita, K. and Kosaka, F. (1989) Influence of suxamethonium on the action of subsequently administered vecronium or pancuronium. Br. J. Anaesth. 62: 324–326. Ortells, M. O. and Lunt, G. G. (1995) Evolutionary history of the ligand-gated ion-channel superfamily of receptors. Trends Neurosci. 18: 121–127. Owens, J. L. and Kullberg, R. W. (1993) Calcitonin gene-related peptide lengthens acetylcholine receptor channel open time in developing muscle. Recept. Channels 1: 165–171.

Functions of Neuronal-Type Nicotinic ACh Receptors Papke, R. L., Duvoisin, R. M. and Heinemann, S. F. (1993) The amino terminal half of the nicotinic b-subunit extracellular domain regulates the kinetics of inhibition by neuronal bungarotoxin. Proc. R. Soc. Lond. B 252: 141–148. Papke, R. L., Bencherif, M. and Lippiello, P. (1996) An evaluation of neuronal nicotinic acetylcholine receptor activation by quaternary nitrogen compounds indicates that choline is selective for the a7 subtype. Neurosci. Lett. 213: 201–204. Perry, E. K., Morris, C. M., Court, J. A., Cheng, A., Fairbairn, A. F., Mckeith, I. G., Irving, D., Brown, A. and Perry, R. H. (1995) Alteration in nicotine binding sites in Parkinson’s disease, Lewy body dementia and Alzheimer’s disease: possible index of early neuropathology. Neuroscience 64: 385–395. Picciotto, M. R., Zoll, M., Lena, C., Bessis, A., Lallemand, Y., LeNovere, N., Vincent, P., Pitch, E. M., Brulet, P. and Changeux, J.-P. (1995) Abnormal avoidance learning in mice lacking functional high-affinity nicotine receptor in the brain. Nature 374: 65–67. Popper, P. and Micevych, P. E. (1989) Localization of calcitonin gene-related peptide and its receptors in the striated muscle. Brain Res. 496: 180–186. Qu, Z., Moritz, E. and Huganir, R. L. (1990) Regulation of tyrosine phosphorylation of the nicotinic acetylcholine receptor at the rat neuromuscular junction. Neuron 4: 367–378. Ramirez-Latorre, J., Yu, C. R., Qu, X., Perin, F., Karlin, A. and Role, L. (1996) Functional contributions of a5 subunit to neuronal acetylcholine receptor channels. Nature 380: 347–351. Rapier, C., Lunt, G. G. and Wonnacott, S. (1990) Nicotinic modulation of [3H] dopamine release from striatal synaptosomes: pharmacological characterization. J. Neurochem. 54: 937–945. Ravdin, P. M. and Berg, D. K. (1979) Inhibition of neuronal acetylcholine sensitivity by a-toxins from Bungarus multicinctus venom. Proc. Natl. Acad. Sci. USA 76: 2072–2076. Roa, M. and Changeux, J.-P. (1991) Characterization and developmental evolution of a high-affinity binding site for calcitonin gene-related peptide on chick skeletal muscle membrane. Neuroscience 41: 563–570. Romano, C. and Goldstein, A. (1980) Stereospecific nicotine receptors on rat brain membranes. Science 210: 647–650. Safran, A., Sagi-Eisenberg, R., Neumann, D. and Fuchs, S. (1987) Phosphorylation of the acetylcholine receptor by protein kinase C and identification of the phosphorylation site within the receptor delta subunit. J. Biol. Chem. 262: 10506–10510. Safran, A., Provenzano, C., Sagi-Eisenberg, R. and Fuchs, S. (1990) Phosphorylation of membrane-bound acetylcholine receptor by protein kinase C: characterization and subunit specificity. Biochemistry 29: 6730–6734. Sala, C., Andreose, J. S., Fumagalli, G. and Lomo, T. (1995) Calcitonin gene-related peptide: possible role in formation and maintenance of neuromuscular junctions. J. Neurosci. 15: 520–528. Sala, C., Kimura, I., Santoro, G., Kimura, M. and Fumagalli, G. (1996) Expression of two neuronal nicotinic receptor subunits in innervated and denervated rat muscle. Neurosci. Lett. 214: 71–74. Sands, S. B. and Barish, M. E. (1991) Calcium permeability of neuronal nicotinic acetylcholine receptor channels in PC12 cells. Brain Res. 560: 38–42. Sands, S. B., Costa, A. C. and Patrick, J. W. (1993) Barium permeability of neuronal nicotinic receptor alpha 7 expressed in Xenopus oocytes. Biophys. J. 65: 2614–2621. Sargent, P. B. (1993) The diversity of neuronal nicotinic acetylcholine receptors. Annu. Rev. Neurosci. 16: 403–443.

201 Schneider, M. F. and Simon, B. J. (1988) Inactivation of calcium release from the sarcoplasmic reticulum in frog skeletal muscle. J. Physiol. 405: 727–745. Schoepfer, R., Whiting, P., Esch, F., Blacher, R., Shimasaki, S. and Lindstrom, J. (1988) cDNA clones coding for the structural subunit of a chicken brain nicotinic acetylcholine receptor. Neuron 1: 241–248. Schoepfer, R., Conroy, W. G., Whiting, P., Gore, M. and Lindstrom, J. (1990) Brain a-bungarotoxin binding protein cDNAs and mAbs reveal subtypes of this branch of the ligand-gated ion channel gene family. Neuron 5: 35–48. Schwartz, R. D., McGee, R., Jr. and Keller, K. J. (1982) Nicotinic cholinergic receptors labeled by [3H] acetylcholine in rat brain. Mol. Pharmacol. 22: 56–62. Seguela, P., Wadiche, J., Dineley-Miller, K., Dani, J. A. and Patrick, J. W. (1993) Molecular cloning, functional properties, and distribution of rat brain alpha 7: a nicotinic cation channel highly permeable to calcium. J. Neurosci. 13: 596–604. Sivilotti, L. and Colquhoun, D. (1995) Acetylcholine receptors: too many channels, too few functions. Science 269: 1681–1682. Somlyo, A. V., McCellan, G., Gonzalez-Serratos, H. and Somlyo, A. P. (1985) Electron probe X-ray microanalysis of posttetanic Ca21 and Mg2+ movement across the sarcoplasmic reticulum in situ. J. Biol. Chem. 260: 6801–6807. Stanley, E. F. and Drachman, D. B. (1983) Botulinum toxin blocks quantal but not non-quantal release of ACh at the neuromuscular junction. Brain Res. 261: 172–175. Sugaya, K., Giacobini, E. and Chiappinelii, V. A. (1990) Nicotinic acetylcholine receptor subtypes in human frontal cortex: changes in Alzheimer’s disease. J. Neurosci. Res. 27: 349–359. Takami, K., Kawai, Y., Shosaka, S., Lee, Y., Girgis, S., Hillyard, C. J., Nacintyre, I., Emson, P. C. and Tohyama, M. (1985a) Immunohistochemical evidence for the coexistence of calcitonin gene-related peptide- and choline acetyltransferase-like immunoreactivity in neurons of the rat hypoglossal, facial and ambiguus nuclei. Brain Res. 328: 386–389. Takami, K., Kawai, Y., Uchida, S., Tohyama, M., Shiotani, Y., Yoshida, H., Emson, P. C., Girgis, S., Hillyard, C. J. and MacIntire, I. (1985b) Effect of calcitonin gene-related peptide on contraction of striated muscle in the mouse. Neurosci. Lett. 60: 227–230. Thesleff, S. (1955a) The mode of neuromuscular block caused by acetylcholine, nicotine, decamethonium and succinylcholine. Acta Physiol. Scand. 34: 218–231. Thesleff, S. (1955b) The effects of acetylcholine, decamethonium and succinylcholine on neuromuscular transmission in the rat. Acta Physiol. Scand. 34: 386–392. Tsuneki, H., Kimura, I. and Kimura, M. (1994) Independent regulation of activation and inactivation phases in non-contractile Ca21-transients by nicotinic receptor at the mouse neuromuscular junction. Brain Res. 650: 299–304. Tsuneki, H., Kimura, I., Dezaki, K., Kimura, M., Sala, C. and Fumagalli, G. (1995) Immunohistochemical localization of neuronal nicotinic receptor subtypes at the pre- and postjunctional sites in mouse diaphragm muscle. Neurosci. Lett. 196: 13–16. Tsuneki, H., Dezaki, K. and Kimura, I. (1997) Neuronal nicotinic receptor operates slow Ca21 mobilization at mouse muscle endplate. Neurosci. Lett. 225: 185–188. Unwin, N. (1993) Nicotinic acetylcholine receptor at 9 Å resolution. J. Mol. Biol. 229: 1101–1124. Unwin, N. (1995) Acetylcholine receptor channel imaged in the open state. Nature 373: 37–43.

202 Vernallis, A. B., Conroy, W. G. and Berg, D. W. (1993) Neurons assemble acetylcholine receptors with as many as three kinds of subunits while maintaining subunit segregation among receptor subtypes. Neuron 10: 451–464. Vernino, S., Amador, M., Luetje, C. W., Patrick, J. and Dani, J. A. (1992) Calcium modulation and high calcium permeability of neuronal nicotinic acetylcholine receptors. Neuron 8: 127–134. Vernino, S., Rogers, M., Radcliff, K. A. and Dani, J. A. (1994) Quantitative measurement of calcium flux through muscle and neuronal nicotinic acetylcholine receptors. J. Neurosci. 14: 5514–5524. Vibat, C. R. T., Lasalde, J. A., McNamee, M. G. and Ochoa, E. L. M. (1995) Differential desensitization properties of rat neuronal nicotinic acetylcholine receptor subunit combination expressed in Xenopus laevis oocytes. Cell. Mol. Neurobiol. 15: 411–425. Vidal, C. and Changeux, J.-P. (1993) Nicotinic and muscarinic modulations of excitatory synaptic transmission in the rat prefrontal cortex in vitro. Neuroscience 56: 23–32. Vijayaraghavan, S., Pugh, P. C., Zhang, Z.-W., Rathouz, M. M. and Berg, D. K. (1992) Nicotinic receptors that bind a-bungarotoxin on neurons raise intracellular free Ca21. Neuron 8: 353–362. Villarroel, A. and Sakmann, B. (1996) Calcium permeability increase of endplate channels in rat muscle during postnatal development. J. Physiol. 496: 331–338. Vizi, E. S., Somogyi, G. T., Nagashima, H., Duncalf, D., Chaudhry, I. A., Kobayashi, O. and Foldes, F. F. (1987) d-Tubocurarine and pancuronium inhibit evoked release of acetylcholine from the mouse hemidiaphragm preparations. Br. J. Anaesth. 59: 226–231. Vizi, E. S., Sershen, H., Balla, A., Mike, A., Windisch, K., Juranyi, Zs. and Lajtha, A. (1994) Neurochemical evidence of heterogeneity of presynaptic and somatodendric nicotinic acetylcholine receptors. Ann. NY Acad. Sci. 757: 84–99. Wada, K., Ballivet, M., Boulter, J., Connolly, J., Wada, E., Deneris, E. S., Swanson, L. W., Heinenmann, S. and Patrick, J. (1988) Functional expression of a new pharmacological subtype of brain nicotinic acetylcholine receptor. Science 240: 330–334.

I. Kimura Wagoner, P. K. and Pallotta, B. S. (1988) Modulation of acetylcholine receptor desensitization by forskolin is independent of cAMP. Science 240: 1655–1657. Walsh, K. B., Shirley, H. and Schwartz, A. (1986) Effect of calcium antagonist drugs on calcium currents in mammalian skeletal muscle fibers. J. Pharmacol. Exp. Ther. 236: 403–407. Ward, J. M., Cockcroft, V. B., Lunt, G. G., Smillie, F. S. and Wonnacott, S. (1990) Methyllycaconitine: a selective probe for neuronal a-bungarotoxin binding sites. FEBS Lett. 270: 45–48. Wessler, I. (1992) Acetylcholine at motor nerves: storage, release and presynaptic modulation by autoreceptors and adrenoceptors. Int. Rev. Neurophysiol. 34: 283–284. Whiting, P. J., Esch, F., Shimasaki, S. and Lindstrom, J. (1987a) Neuronal nicotinic acetylcholine receptor beta-subunit is coded by the cDNA clone alpha 4. FEBS Lett. 219: 459–463. Whiting, P. J., Liu, R., Morley, B. J. and Lindstrom, J. M. (1987b) Structurally different neuronal nicotinic acetylcholine receptor subtypes purified and characterized using monoclonal antibodies. J. Neurosci. 7: 4005–4016. Witzemann, V., Berg, B., Nishikawa, Y., Sakmann, B. and Numa, S. (1987) Differential regulation of muscle acetylcholine receptor gamma- and epsilon-subunit mRNA. FEBS Lett. 223: 104–112. Wonnacott, S. (1990) The paradox of nicotinic acetylcholine receptor upregulation by nicotine. Trends Pharmacol. Sci. 11: 216–219. Wonnacott, S., Drasdo, A., Sanderson, E. and Rowell, P. (1990) Presynaptic nicotinic receptors and the modulation of transmitter release. Ciba Found. Symp. 152: 87–105. Zhang, Z. W., Vijayaraghavan, S. and Berg, D. K. (1994) Neuronal acetylcholine receptors that bind a-bungarotoxin with high affinity function as ligand-gated ion channels. Neuron 12: 167–177. Zhou, Z. and Neher, E. (1993) Calcium permeability of nicotinic acetylcholine receptor channels in bovine adrenal chromaffin cells. Pflügers Arch. 425: 511–517. Zorumski, C. F., Thio, L. L., Isenberg, K. E. and Clifford, D. B. (1992) Nicotinic acetylcholine currents in cultured postnatal rat hippocampal neurons. Mol. Pharmacol. 41: 931–836.