Muscle endplate cholinoreceptors

Pharmac. Ther. Vol. 38, pp. 331 to 385, 1988 Printed in Great Britain. All rights reserved

MUSCLE

0163-7258/88 $0.00 + 0.50 Copyright © 1988 Pergamon Press pie

ENDPLATE

CHOLINORECEPTORS

FRANCISCOJ.

BARRANTES

lnstituto de Investigaciones Bioquimicas, Consejo Nacional de Investigaciones Cientificas y Tecnicas/Universidad Nacional de! Sur, 8000 Bahia Blanca, Argentina

1. INTRODUCTION The nicotinic acetylcholine receptor (AChR) was the first chemoreceptor to be isolated, biochemically and physically characterized, reincorporated into artificial and heterologous biological membranes, cloned, sequenced and, most recently, to have its genes mutated. The muscle endplate AChR has been more elusive than its counterpart at the electromotor synapse of electric fish in terms of biochemical and molecular biology characterization, but has contributed an enormous wealth of information on the physiology, pharmacology, immunology and cell biology of the AChR molecule. This is mainly due to the natural abundance of the electric tissue AChR as opposed to the muscle tissue cholinoreceptors and, secondly, to the availability of appropriate cholinergic ligands which, used as biochemical tools, enabled the large-scale purification of AChRs, a prerequisite to many physical or biochemical descriptions of the molecule. The muscle endplate AChR remains, however, the landmark for the physiology and pharmacology of peripheral cholinergic transmission of the nicotinic type. Reviewing the subject of endplate AChRs necessarily requires reference to the electric tissue receptor, especially when the molecular aspects are to be discussed. Recent application of molecular genetics has shown that AChRs from these two species are fairly similar in terms of primary structure, and specific differences between the two revealed by hybridization and modern electrophysiological techniques are too tenuous not to justify resorting to the fish species when the comparison so invites. The present review discusses recent findings on the structure, molecular pharmacology, cell and molecular biology of the AChR and attempts bringing them together with parallel developments in recent years on the function of the acetylcholine (ACh)-operated ion channel. The reader is referred to several other more comprehensive overviews on general and topical aspects of the AChR, which are cited along the text.

2. THE ADULT NEUROMUSCULAR JUNCTION The site of contact between the nerve terminal and the muscle cell membrane (sarcolemma)--the neuromuscular junction (NMJ)---constitutes a specialized region in both neuron and muscle cell, comprising less than 0.1% of the total area of the adult muscle fibre. NMJs exhibit distinct anatomical features among species. Mammalian, reptilian, avian and fish NMJs can be described as round-shaped bouton-like structures sitting in shallow depressions. The clustering of postsynaptic nuclei and mitochondria, the nerve ending itself, and the associated Schwann cells in its vicinity, probably led early microscopists like Rouget (1862) to coin the word endplate ('plaque terminale') in relation to this type of junction. Amphibian NMJs, on the other hand, consist of long cylindrical terminals running in grooves or gutters, usually located near the middle of the muscle fibre in singly-innervated cells (Nudell and Grinnell, 1983). The above-mentioned clustering of organelles is not present in this type of NMJ, and Kfihne (1887) used the term Endbiischel instead of endplate to describe its morphology. J P T 38 3

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The anatomical characterization of vertebrate NMJs at the light-microscope level reached its climax with detailed descriptions like those of Couteaux (see review in Couteaux, 1960). These morphological differences probably reflect underlying physiological characteristics of neuromuscular transmission among species (see review by Peper et al., 1982). Efficient generation of postsynaptic currents requires membrane resistance to be low, and most likely the morphology optimizes the geometry needed to fulfill this requirement in a given species. Salpeter (1987) has recently produced a very comprehensive review of NMJ organization. The advent of electron microscope techniques in the 1950s led to the realization that the NMJ possesses the three morphological elements originally described for central nervous system (CNS) synapses: pre- and postsynaptic membranes and synaptic cleft. The latter is an electron-lucent region in between the two adjacent nerve and muscle membranes (Birks et al., 1960). In view of the differences in embryological origin, it is not surprising that certain specializations of the NMJ make it significantly distinct from CNS synapses. At the subcellular level the most characteristic of these are the folds present in the sarcolemmal (postsynaptic) membrane and the width of the primary synaptic cleft. The latter is about 50 nm wide (in contrast to the 10-20 nm width of most synapses) and joins up with the extracellular space at its lips. The sarcolemma invaginates deeply from the primary cleft to form the secondary clefts. These are present in mammalian, amphibian. reptilian but not in avian species. In the case of linear terminals, the secondary folds displa 3 a repeating pattern of slots running perpendicularly to the long axis of the cylinder. The5 can be seen at high magnification in the light microscope using, e.g. fluorescent, or-toxins After enzymic removal of the terminal they can also be visualized in unstained specimen! using Nomarski interference contrast and, alternatively, using scanning electron micro. scopy. In transmission electron microscopy freeze-fractured specimens in which th~ cleavage runs through the primary cleft itself make the secondary folds apparent. In th~ bouton-like NML the secondary folds are also parallel to each other, but because of the overall shape of the primary cleft the secondary clefts vary in length considerably. Botl the primary and secondary clefts are surrounded by the same basement membrane whicl completely envelopes the rest of the muscle cell. Here we also find another difference witl CNS synapses: there is a cellular boundary beyond the basement membrane or basa lamina constituted by a type of cell not found in the CNS, i.e. Schwann glial cells. The sarcolemma which directly faces the primary cleft and the top third of the secondar' cleft presents some distinctive features which make it different from the rest of the ce] membrane. This specialized portion of the sarcolemma has, in addition to the plasm~ membrane, a glycocalix and basal lamina. The plasma membrane itself is thicker thai non-junctional muscle plasmalemma. The basal lamina, 3 0 ~ 0 n m thick, presents a] amorphous appearance and is separated from the sarcolemma by a 10 nm-wide glycocali~ The reticular lamina is a collagen-rich reticulum and together with the basal lamin constitutes the basal membrane. The reticular lamina is interrupted at the synaptic fold, The junctional sarcolemma is thicker than non-junctional sarcolemma, and in freeze fractured specimens it displays a high density of intramembranous particles which protrud from the sarcolemmal membrane. The particles occur at a density of 10,000/#m 2, sho~ a central pit and, though not highly ordered, tend to form linear arrays (Heuser an Salpeter, 1979). The distribution of these particles is very similar to that of th ~-bungarotoxin binding sites, which occur at about twice this density (Matthews-Bellinge and Salpeter, 1978). The cytoplasmic region immediately in contact with AChR aggregates is densely packe with 4-10 nm intermediate-type filaments in between the postjunctional folds (Couteau and Pecot-Dechavasinne, 1968). Thinner 4-6 nm filaments are also present. A 5 nm thic amorphous layer is coextensive with the AChR-rich postsynaptic membrane. This layer restricted to a rather small (~0.1%) area of the total plasma membrane in the adu myofibril. The morphological specializations of the postsynaptic sarcolemma appear t have biochemical correlates. Thus, non-receptor proteins co-purify with AChR-ric membranes from electric tissue: the 43 K (Sobel et al., 1978) or v-protein (Hamilton et a,

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1979) or v-doublet (Barrantes et al., 1980), present in a 1:1 stoichiometry with the AChR (Burden et al., 1983), an Mr 270,000 protein (Burden et al., 1983), and an Mr 58,000 protein (Froehner, 1984). Immunocytochemical experiments have revealed that the 43 K proteins are concentrated at the cytoplasmic face of the postsynaptic sarcolemma (Froehner et al., 1981; Froehner, 1984; Sealock et al., 1984; Burden, 1985) as is the Mr 58,000 protein (Froehner et al., 1987). Another protein of identical apparent molecular weight with the 43 K but different pI, named v2 by Gysin et al. (1981), was found to be a form of creatine kinase (Barrantes et al., 1983a,b). Although the major isoenzyme of creatine kinase in electric tissue is of the MM (muscle) type (Gysin et al., 1983; West et al., 1984; Giraudat et al., 1984; Perryman et al., 1985), a BB (brain)-like isoform is also present in the electrocyte and in AChR-rich membranes (Barrantes et al., 1983a; 1985; Wallimann et al., 1985). Other proteins present in the vicinity of the AChR at the postsynaptic membrane are discussed in Section 4 in relation to receptor development. Although the wealth of information on the anatomy of the cholinergic synapse has been gained from mammalian or amphibian neuromuscular junction, there is another nicotinic synapse which has contributed to understanding both the structure and function of cholinergic systems: this is the electromotor synapse at the electrocyte (electric fish electroplaque) (Fig. 1). The pioneer study of Heuser and Salpeter (1979) using quick-freezing, freeze-fracture, deep-etching and rotary-replication revealed the threedimensional organization of this cholinergic synapse in great detail. The characteristic preand post-synaptic elements correspond closely to those of the vertebrate neuromuscular junction. Each electroplaque is innervated by thousands of terminals at its ventral face. The postsynaptic membrane, which has few secondary folds, is again studded with 8 9 nm particles often paired in linear rows (Heuser and Salpeter, 1979). These can also be seen in electron micrographs of AChR-enriched membranes (Barrantes, 1982a,b; Cartaud et al., 1981). 3. THE DEVELOPING N E U R O M U S C U L A R JUNCTION Development of skeletal muscle fibers involves an initial proliferative phase in which mononucleated cells called myoblasts undergo cellular division. Later in development, in the period of differentiation, myoblasts fuse to form larger, elongated multinucleated cells (myotubes), which cease to divide further. The myotomes are specialized areas where these processes occur in the embryo. Already during the proliferative phase undifferentiated nerves invade the myotomes, but they do not establish contact with the developing cells. In the differentiation phase myotubes already synthesize contractile proteins and myofibrils can be seen in their cytoplasm. Nerves begin to establish imperfect contact with myotubes and in some cases electrical coupling takes place. At a later stage axon terminals are often found in contact with myotube sarcolemma immediately above the muscle cell nuclei, separated by an intersynaptic space of 50-100 nm filled with amorphous material, probably a precusor of the basal lamina. Chemical transmission begins to occur. The morphological differentiation described is accompanied by discrete events at the molecular level. At a given time along ontogenetic development, myotubes possess two distinct types of AChR molecules, differing in their functional characteristics (e.g. gating behaviour), their morphological distribution (diffuse and localized, respectively), their physical properties (mobile and immobilized) and, as has recently been elucidated, their ultimate molecular structure. The meaning of these differences is now beginning to be unravelled. 3.1. THE FORMATION OF A C H R CLUSTERS

At early stages of muscle tissue development the AChR molecules are diffusely distributed over the entire sarcolemma and occur at low densities. In the process of innervation the tips of motoneuronal axons (termed growth cones) contacting the muscle cell induce major changes in the distribution of AChR molecules, one of which is their

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FIG. I. The now classical view of the cholinergic synapse of the Torpedo electrocyte revealed by deep etching and replication by rotary deposition of platinum. To the left, the lace-like basal lamina. At the bottom centre of the figure the basal lamina assumes a ring-like appearance as it dips down into a postsynaptic invagination. In the middle of the figure, the basal lamina has been fractured away to reveal the true external surface of the postsynaptie membrane, characterized by clusters and linear arrays of 8 9 nm protrusions--the acetylcholine receptor particles. To the right, the postsynaptic membrane has broken through to reveal the underlying meshwork of cytoplasmic microtrabeculae which gird it from beneath, x 175,000. From Heuser and Salpeter [J. Cell Biol. 82:150 173 (1979)].

s u p r a m o l e c u l a r o r g a n i z a t i o n into clusters ( A n d e r s o n a n d Cohen, 1977). This is a hig] specific process which c a n n o t be m i m i c k e d by other cholinergic n e u r o n s like ' sympathetic n e u r o n s ( C o h e n a n d W e l d o n , 1980), a n d restricted to only certain areas the neurite ( A n d e r s o n a n d C o h e n , 1977), a l t h o u g h the clustering of A C h R can take pl~ in muscle cell cultures in the absence of nerve contacts (Fischbach a n d C o h e n , 1973). this case the A C h R clusters have been coined 'hot-spots' a n d m a y n o t necessm c o r r e s p o n d to the future nerve-induced endplates. Physical a n d chemical signals, such as a D C electric field (Orida a n d Poo, 1978) extracts from b r a i n or spinal cord (see below), can also induce the f o r m a t i o n of clust or e n h a n c e their n u m b e r . The clustering process can occur in the presence of s a t u r a t c o n c e n t r a t i o n s o f competitive a n t a g o n i s t s such as c~-bungarotoxin (Steinbach et al., 19

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FIG.2. Deep-etchedpostsynapticmembraneshowinglinear arraysand clustersof AChR particles. x 300,000. From Heuser and Salpeter (1979). or curare (Cohen, 1972). The signal for clustering is not likely to depend on the AChR molecule itself, nor on the channel activity which underlies synaptic functioning. The redistribution of AChR molecules (migration) now seems to be the most important source of AChR cluster formation. The second source is the insertion of newly synthesized receptors. Before innervation the myoblast sarcolemma has, on average, a higher density of AChRs than the extrajunctional regions of the adult myocyte after innervation. Early work showed that the diffusely located AChRs migrate to the cluster region where nerve cells establish contact with the developing myoblast (Anderson and Cohen, 1977; Stya and Axelrod, 1983). Thus, pre-existing receptors constitute the bulk of a cluster and randomly inserted, newly synthesized AChR molecules only contribute secondarily. The simplest hypothesis to account for the available experimental evidence on the formation of clusters is that of the 'diffusion-mediated trapping' (Poo, 1985). This theory weighs heavily on the observation that diffusely distributed AChRsexhibit a higher mobility, i.e. higher lateral diffusion coefficient (see review in Barrantes, 1988), than junctional AChRs, thus being able to move in the plane of the membrane towards a trap. The trap for freely diffusing AChR molecules can be envisaged as an area in the sarcolemma or the underlying sarcoplasma where certain molecules having receptor-trapping abilities occur. The trap could also involve extracellular molecules present in the synaptic cleft or the basal lamina. In fact, a variety of molecules have been shown to co-localize in discrete regions overlapping the distribution of AChR molecules (Poo, 1985, and see below), but none as yet has been firmly demonstrated to be 'the' trap. The formation of clusters by passive diffusion, independently of other molecules, has also been discussed (see Barrantes, 1979). Another hypothesis recently discussed is that of an energy-dependent aggregation process, in analogy to the phenomenon of receptor capping. No evidence supports this hypothesis, although other phenomena linked to AChR metabolism, such as AChR internalization (see review by Salpeter and Loring, 1985) are energy-dependent. Furthermore, several cytoskeletal proteins co-localize with AChR aggregates: ~-actinin (Bloch and Hall, 1983), a form of actin (Hall et al., 1981; Bloch, 1986), vinculin (Hall et al., 1981), filamin (Bloch and Hall, 1983), an intermediate filament protein (Burden, 1982), fodrin (Walker et al., 1985), talin (Sealock et al., 1986), an Mr 58,000 protein (Froehner, 1984; Tobler et al., 1986), myosin (Luther and Bloch, 1986) and, most conspicuously, the Mr 43,000 protein (43 K) (Sealock et al., 1984a; Froehner, 1984; Peng and Froehner, 1985;

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FIG. 3. The current model system in the study of the cholinergicsynapse: the electromuw, ~y,,,pse in electrictissue of Torpedinidae. The electronmicrograph shows three electrocytes(electroplaques) of Discopyge tschudii electric tissue, a highly polarised cell with a non-innervated dorsal face and a ventral face patched with cholinergicterminal (arrowheads). Magnification: × 6,345. (Maria Prado-Figueroa and F.J.R. unpublished). Kordeli et al., 1986; LaRochelle and Froehner, 1986a; Bloch and Froehner, 1987). Son of these cytoskeletal proteins undergo energy-dependent processes, This coincidence , spatial localization does not necessarily imply that the cytoskeletal proteins act as tral for the AChR, since such co-localization could well develop simultaneously and subs quently serve for the stabilization of the aggregates, although there is a report on tl appearance of actin filament meshworks about 2 hr prior to the formation of ACh clusters (Peng and Phelan, 1984). In rat, actin is already present at embryonic day 18 (H~ et al., 198 l), by which time AChRs accumulate at synaptic regions but postjunctional fob have not formed. In fact, an elegant recent study has precisely shown the temporari coincidental development of the AChR and a cytoskeletal protein and casts doubts on tl participation of one of the most firm candidates, the 43 K protein, in AChR anchorin LaRochelle and Froehner (1986b) used two different muscle cell lines, the C2 and BC3Honly the first one exhibits AChR clustering. However, both AChR and 43 K are expressc coincidentally in the two cell lines and in approximately equimolar quantities. Thus, tl lack of AChR clustering in the BC3H-i line is not related to the 43 K protein, whi~ co-localizes with AChR.

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AChR clusters in rat and chick myotubes have been found to be localized preferentially in membrane regions above myonuclei. The latter appear stationary in contrast to other nuclei in non-cluster regions, which are able to translocate throughout the myotube (Englander and Rubin, 1987). 3.2. STABILIZATIONOF CLUSTERS In the adult NMJ cytoskeletal elements may play a role in the anchoring of AChR molecules. In T. m a r m o r a t a electromotor synapses, 100-120 nm intermediate-sized filaments terminate onto the cytoplasmic face of the postsynaptic membrane (Heuser and Salpeter, 1979). These filaments are decorated with anti-43 K protein antibodies, suggesting that the latter may link the filaments to the postsynaptic membrane (Kordeli et al., 1986). Vertebrate NMJs possess proteins immunologically related to the Torpedo 43 K (Froehner, 1984; Froehner et al., 1981; Burden, 1985; LaRochelle and Froehner, 1986a; Bloch and Froehner, 1987) and an Mr 51,000 protein related to intermediate filaments (Burden, 1982), but the pattern in the adult NMJ may differ from that of the simpler electromotor synapse (Kordeli et al., 1986). Clustered AChR in rat myotubes redistributes more homogeneously on the plane of the membrane upon extraction of non-receptor peripheral proteins, the 43 K included, and they become more susceptible to chymotrypsin degradation. These results suggest that peripheral proteins cover part of the AChR molecule in the clusters (Bloch and Froehner, 1987). Most recently, phorbol esters (agents which activate the phospholipid/Ca2+-dependent protein kinase C) have been found to disrupt intermediate filaments and AChR clusters in cultured skeletal myotubes (Bursztajn, 1986). As discussed in Section 7, phosphorylation is now known to exert an influence on other properties of the AChR such as desensitization. When AChR clusters in rat myotubes are disrupted by addition of azide, intramembranous particles which are originally evenly distributed within the cluster domains become unevenly scattered, with some microaggregation. Upon removal of azide, clusters reform, and intramembranous particles adopt again the evenly spaced distribution within the clusters after passing through a microaggregation step. Cluster disruption/reformation appears therefore to be a reversible process, clustering itself being a two-step event in which the concentration of intramembranous particles into small patches precedes their arrangement into evenly spaced sites (Pumplin and Bloch, 1987). 3.3. METABOLIC STABILITYOF THE A C H R MOLECULE ITSELF The process of AChR biosynthesis and maturation involves various stages during which the molecule undergoes modifications to become a fully functional cell surface receptor; newly synthesized AChRs do not even recognize s-bungarotoxin. Operationally, four forms of AChR can be distinguished: (1) the primary translation product, which Merlie and co-workers call s0; (2) the s-subunit that has acquired the ligand-recognition ability, identified by s-bungarotoxin binding (STx); (3) the stage at which the s chain has assembled with the other subunits; and (4) the final, cell surface-expressed AChR. It is not as yet known which posttranslational covalent modifications, if any, bear relationship to the transitions between these stages, nor is it known whether these changes occur in the endoplasmic reticulum or the Golgi apparatus. Merlie and Lindstrom (1983) and Carlin et al. (1986) have shown that the s-chain acquires high-affinity s-bungarotoxin binding ability after translation but before subunit assembly. Given the fact that the in vitro translated s-chain does not bind s-bungarotoxin with high affinity (Anderson and Blobel, 1981; Sebbane et al., 1983) and that it does not acquire such ability for several minutes (t~/2 ~ 40min) after translation in vivo (Merlie and Sebbane, 1981; Smith et al., 1987), the primary translation product s 0 must be subjected to additional posttranslational modification(s) in order to reach the STx stage. Assembly of the AChR takes about 80 min (Smith et al., 1987). Since the pulse-labelled s-subunit co-migrates with glucose-6phosphatase (a marker of the endoplasmic reticulum) activity in sucrose gradients, these

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authors concluded that g-chains acquired the capacity to bind g-toxin while at the endoplasmic reticulum compartment. A rapid transport to the Golgi apparatus follows. There, Golgi-mediated posttranslational modifications occur: oligosaccharides of the complex type (Nomoto et al., 1986) are known to be attached at least to the 7- and -AChR chains. Mature AChR can then be inserted with the correct vectorial orientation into its final membrane environment, i.e. the cell surface membrane. The clustering process reviewed above is accompanied by changes in the metabolism of AChR molecules, leading to their stabilization. AChR clusters are relatively stable for several days before innervation occurs (Moody-Corbett and Cohen, 1982). Upon innervation, the stability of AChR clusters is not immediately acquired: denervation in 1-day-old nerve-muscle co-cultures results in their dispersal, whereas after 3 days they are more resistant to denervation. Establishment of definitive synaptic contacts is also preceded by dispersal of pre-existing clusters, without significant changes in the density of diffuse AChRs. The AChRs at relatively new endplates are therefore metabolically unstable. Their half-life is about 24 hr. Once the neuromuscular junction commences to work, its activity may contribute to the maintained dispersal of extrasynaptic AChR clusters. The nature of this phenomenon is still not understood (Peng and Poo, 1986). Within days of innervation in rat endplates, or weeks in the chicken, there is a marked increase (about an order of magnitude) in the metabolic stability of junctional AChRs. Current hypotheses favour the association of the AChR with non-receptor proteins at the synapse to account for this phenomenon. The alternative view is that replacement of metabolically unstable AChRs by newly synthesized, metabolically stable ones takes place. Since AChRs in extrasynaptic regions are metabolically unstable, and are not replaced under normal circumstances by metabolically stable ones, the fate of non-junctional AChRs is clear: when their synthesis decreases after innervation they are outnumbered by the stabilized junctional receptors, and the adult muscle practically concentrates its endowment of AChRs at the endplate region. Whether covalent posttranslational modifications other than glycosylation (methylation, phosphorylation, fatty acid acylation) are involved in the acquisition of metabolic stability of the AChR molecule is still not known. The lower pI of the adult AChR as compared to the embryonic AChR (Brockes and Hall, 1975) has been equated with a higher degree of phosphorylation (Saitoh and Changeux, 1981). The immunological differences between the two types of AChR, which appear to have consequences on the channel gating behaviour of the receptor (see Section 3.5), may help to solve this query. See section 9 for other molecular changes of the AChR along development. 3.4. FACTORS AFFECTING A C H R NUMBER AND DISTRIBUTION ALONG DEVELOPMENT

In addition to some physical factors capable of inducing redistribution of AChR molecules an increasing number of chemical signals are beginning to be identified. The general strategy in this field of research has been to test tissue extracts of varying degrees of purity on uninnervated muscle cells in culture and subsequently to measure the number and distribution of AChRs induced by such extracts. Early reports described the increase in AChR number and induction of AChR clusters by low molecular weight fractions from chick brain (Jessell et al., 1979; Buc-Caron et al., 1983). Sciatin, a myotrophic Mr 84,000 protein from sciatic nerves, promotes proliferation of mononucleated muscle precursor cells, the survival of myotubes (Markelonis and Oh, 1979) and a marked enhancement in protein synthesis (Markeionis et al., 1980). The latter effect involves small increase in AChR synthesis, which develops within days. It has subsequently beer found that sciatin is virtually identical to transferrin (Ii et al., 1982). An Mr 80,000 proteir extracted from basement-membrane enriched fractions from Torpedo electric orgar induces AChR clustering without apparently affecting the number of receptors (Nitkir et al., 1983). Non-protein low molecular weight components have also been reported to increas~ the number of AChRs, e.g. ascorbic acid in L5 rat myotubes (Knaak and Podleski, 1985)

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One particularly well characterized glygoprotein of Mr 42,000 has recently been shown to selectively increase from two- to six-fold the rate of incorporation of AChRs into chick myotube membranes (Usdin and Fischbach, 1986). Another factor recently discovered is a molecule immunologically related to the calcitonin gene-related peptide (CGRP-1), a trophic factor which occurs in mammalian motoneurons, including man (New and Mudge, 1986). This factor increases AChR synthesis at vertebrate NMJs. The rate of membrane insertion of newly synthesized receptors augments by about 45% in CGRP-treated cultures of rat myotubes without significant changes in their degradation rate. We thus find three types of factors affecting AChR occurrence: (1) those which mainly increase their number, via augmented synthesis and rate of membrane insertion; (2) those which mainly affect their distribution, e.g. clustering, without changing their number; and (3) those influencing both number and distribution of receptor molecules. Examples of the first type of factors are the CGRP-1 calcitonin gene-related peptide and the Mr 42,000 protein described above: type 2 factors include the Torpedo basement-membrane Mr 80,000 protein, and among factors affecting both number of AChRs and the clustering process are the ascorbic acid-like molecule, which only induces clusters on cultured primary muscle cells (Kalcheim et al., 1982) and both clusters and increased synthesis on muscle cell lines (Knaak et al., 1986) and the brain extract of Buc-Caron et al. (1983). 3.5. DEVELOPMENTOF CHANNEL PROPERTIESOF THE ACHR In addition to the changes in AChR number, distribution and metabolic stability, developmental changes in channel gating characteristics occur during endplate formation. Some of these changes have been measured with conventional electrophysiological techniques (e.g. Fischbach and Schuetze, 1980). Improvements in the patch-clamp methods (Hamill et al., 1981) have lead to the description of fine structural details (see Section 8) within single-channel events and the observation of different classes of current amplitudes. Two different types of channel were observed in muscle cells, the junctional (J) and extrajunctional (E) channels found in adult innervated and denervated muscle fibres. Extrajunctionai AChR channels were known for some time to have smaller conductances and longer open times than junctional channels (Neher and Sakmann, 1976a). Analogously, E- and J-type AChR channels were described in developing muscle in culture (Hamill and Sakmann, 1981; Clark and Adams, 1981a; Brehm et al., 1984; Siegelbaum et al., 1984) and in developing rat muscle in situ (Vicini and Schuetze, 1985) which were also distinguishable on the basis of their kinetics and conductance. Typical values for the E-type channel in embryonic rat muscle are ~5 ms mean open time and 35 pS current amplitude. At birth the second J-type of channel is expressed, with a shorter lifetime (,-~ 1 ms) and a higher conductance (~50 pS). The differences in conductance reflect a different maximum conductance rather than a different affinity for permeant ions. The relative proportion of the channel type correlates with the stage of development of the muscle cell in rat: the E-type of channel (slow and smaller in conductance) exhibits a several-fold reduction in channel mean open time and gradually disappears within the first three weeks of postnatal life, being replaced by the junctional, adult type of channel. In Xenopus myotomal cells in culture the E-type of channel exhibits a sharp decrease in the mean open time (Leonard et al., 1984). After blocking surface AChR in Xenopus muscle cells with ~-bungarotoxin, it was observed that the two types of receptors are synthesized de novo in the absence of innervation (Greenberg et al., 1985). The two types were reported to be immunologically (Hall et al., 1983; Schuetze et al., 1985) and biochemically (Brockes and Hall, 1975) distinct. Myastenic serum, containing antibodies against the AChR, selectively inhibits the activity of E-type of channel at developing rat NMJs (Schuetze et al., 1985). In Xenopus myotonal muscle the changes between the two types of AChR take place within a shorter period. Thus, for about the first 20 hr following the onset of ACh sensitivity, the majority of channels are of the E, slow type (3 ,-~ 3 ms). Shortly before hatching, faster channels with about 1 ms mean open time appear, increasing their number

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with respect to the slower ones during the 2 days following hatching, and by the age ol maturity of the myotomal synapse outnumbering the E-channels (Kullberg and Kasprzak 1985). Schuetze and Vicini (1984) have studied the effect of early denervation on th~ ontogenetic conversion of AChR channels. The system studied was the rat soleus muscle in which mean channel lifetime decreases from about 4.5 ms to about 1.5 ms betweet postnatal days 8 and 18. Early denervation inhibits the normal replacement of the E-typa by the adult type of channel. Although the J- and E-type of channels exhibit a major conductance state which doe not interconvert, a second conductance state which can switch to a state of abou 20% lower conductance has been observed. This phenomenon has received variou interpretations (Hamill and Sakmann, 1981; Auerbach and Sachs, 1983; Trautmann 1982). Developmental changes in AChR channel gating behaviour have also been reporte~ in mouse (Dreyer et al., 1976), frog (Kullberg et al., 1981), human (Adams and Bevan 1985) and bovine (Mishina et al., 1986) muscle, but not in chick muscle (Schuetze, 19801 When the AChR channels in embryonic rat muscle cells are activated by a variety o agonists of different pharmacological efficacy, no preference for opening a particular typ of channel is noticed; the two conductance levels observed in the presence of ACh ar also elicited by nicotine, decamethonium, 3-phenylethyltriethylammonium and six othe agonists (Gardner et al., 1984). In Xenopus myocytes, Auerbach and Lingle (1986) hay observed that the high-conductance AChR channel (65 pS in this species) has a lowe affinity for ACh than the low-conductance (45 pS) channel. They suggested that agoni,, binding (and perhaps gating) rates are different for the two types of channel. Furthe discussion of this subject can be found in Schuetze (1986) and Schuetze and Role (1987 the issue of the molecular changes underlying the developmental changes described her is given in Section 9. 4. MOLECULAR BASIS OF AChR DEVELOPMENTAL CHANGES: PHYLOGENY AND ONTOGENY The changes in AChR gating behaviour reviewed above raise the question of wheth~ embryonic and adult AChR channels are the result of posttranslational modi! cations along development or whether they correspond to two different types of ger product. A definite answer to this question had to await the advent of cDNA recombinal techniques, sequencing of subunits, and the mise au point of ancillary techniques to asse: the effects of chain substitutions, point mutations and so on. This constitutes one of tt most rapidly developing topics in the field and any outline of its contents faces quk obsolescence. 4.1. SEQUENCE HOMOLOGY AND PHYLOGENET1C EVOLUTION OF THE NICOTINIC A C H R

Chemical sequencing methods applied to the first 54 amino acids at the NH-termin end of the four subunits of T. californica AChR indicated extensive homologies amol all chains (Raftery et al., 1980). The suggestion followed that the subunits arose from four-fold duplication of a single ancestral gene and divergence through gene duplicati~ (Raftery et al., 1980). Subsequently, the homology could be assessed throughout the chai: upon application of eDNA recombinant techniques. The partial (Ballivet et al., 198 Sumikawa et al., 1982) and later the full cDNA clones (Noda et al., 1982; 1983a,b. Claudio et al., 1983; Devilliers-Thiery et al., 1983) were sequenced. The evolutionary ori 8 of each subunit could thus be refined: comparison of the amino acid differences per resid between all sets of pairs of chains suggested that a single gene duplication gave rise to tv protogenes, each of which was in turn duplicated, thus originating the ~- and/~-chains the one hand and the 7- and 6-lineages on the other (Noda et al., 1983b). There is great similarity between the sequence of the/~-chain and the sequences of the 7- and 6-subun than there is between the ~-chain and these latter two, suggesting different rates of ehan throughout phylogeny for the ~- and /3-lineages (Noda et al., 1983b).

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4.2. TAXONOMICALCOMPARISONS Once the sequences of the Torpedinidae AChR subunits were known, genomic or cDNA encoding components of AChR from other species were promptly elucidated. Thus, Electrophorus electricus AChR subunits (Conti-Tronconi et al., 1982), calf AChR (Noda et al., 1983a; Takai et al., 1984; Tanabe et al., 1984; Kubo et al., 1985) and the ct- and 7-subunits of human muscle AChR (Noda et al., 1983a; Shibahara et al., 1985) have further extended the list of known sequences. Their comparison (Noda et al., 1983c; Numa et al., 1983) shows that the homology between chains extends through most of the primary structure of the subunits with the exception of small stretches, particularly between residues 342 and 425. This is indicative of the existence of similar structural features in each subunit and hence of similar contributions of each subunit to the overall AChR structure. The most dramatic example of homology found in comparative studies of AChR subunits was the unexpected finding of a fifth subunit in calf AChR (Takai et al., 1985) which shows higher homology with the 7-chain than with the others. This subunit, termed c, is not present in adult muscle, a fact which may help to explain various biologically relevant differences between adult and less than fully developed AChR. It is worth emphasizing that this type of taxonomical comparison was facilitated by the remarkable homology of sequence between species. The availability of sequences from different species in turn permitted the formulation of further hypotheses concerning the evolutionary trends of the AChR molecule. As expected, sequence homology decreases with increasing evolutionary distances between species. The accumulated data suggested that AChR from the sources so far studied descend from the products of an initial four-fold gene duplication, and that the four genes coding for specific functions developed and were subsequently preserved through phylogeny (Conti-Tronconi et al., 1982). Inter-species comparisons reinforce the view that, as long as no gene duplications we are unaware of have occurred, the rate of evolutionary changes in the ct-subunit has been much slower than that in the other subunits. The ~-subunits of human AChR and electric tissue display 80% homology (Noda et al., 1983a). In fact, Torpedo ot is more homologous to calf muscle or human ~ than it is to other Torpedo subunits (Noda et al., 1983a). The high degree of homology indicates that the a-chain is probably essential for AChR function, a good reason for this subunit having withstood chain mutations throughout phylogenetic evolution. The high degree of homology between ~t-chains does not seem to be the case with other subunits. The most homologous chains within a species are the 7 and 6. At the amino acid level, 57% homology is found between these two chains, whereas figures ranging between 36 and 43% are found for the other subunits in T. m a r m o r a t a (Noda et al., 1983b). Sequence homology between 6-chains from mouse and Torpedo is about 60%, i.e. not much greater than the 50% homology between the heterologous Torpedo 7- and mouse 6-chains (La Polla et al., 1984). It has been proposed that the amino acid residues that do encode for characteristic features of the 6-subunit are those involved in intersubunit contacts, occurring on the surface of the chain. These residues, unique to the 6-subunits, appear in a periodic pattern (White et al., 1985).

4.3. LOCALIZATIONOF ACHR GENES

Recently, there have been successful attempts to localize the genes coding for the AChR protein. Thus, Nef et al. (1984) demonstrated that the genes for the chick 7- and 6-subunits are separated by 740 base pairs in the chromosome. Hybridization experiments of genomic clones from human AChR with 7- and 6-chain cDNA probes have indicated clustering of the genes and possibly a common control of their transcription (Shibahara et al., 1985). More recently, Heidmann et al. (1986) have taken advantage of length polymorphism of restriction fragments between two related mouse species to establish the chromosomal location of the genes coding AChR subunits. They found that AChR genes are not clustered within a single region but occur in three different chromosomes. Further, they

342

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co-segregate with genes coding for contractile proteins that are co-expressed with AChR genes at the onset of myotube formation. Thus, the 7- and 6-subunit genes are linked on the same chromosome, i. Let us recall that these two subunits exhibit the highest degree of homology (vide supra). The 0t-subunit gene occurs in chromosome 17 together with the ~-cardiac actin gene; the/3-chain gene is located on chromosome 11. The fact that the 7- and g-chain genes are linked on the same chromosome has led Heidmann et al. (1986) to elaborate on their phylogenetic evolution. Thus, the duplication of the corresponding genes could either have taken place later in evolution as compared to the duplication of the ~/fl lineage or, as is the case with the immunoglobulin genes, the close proximity of the ~/3 genes has caused them to maintain their high sequence homology via gene conversion. 4.4. ONTOGENETIC REGULATION OF A C H R CHAIN GENE EXPRESSION

The evidence reviewed in previous sections supports the hypothesis that genetic switches regulate the expression of different genes coding for at least two AChR subunits, which interchange along muscle development. AChR chain genes co-segregate with genes for contractile proteins; their coordinate expression occurs early on in myotube formation (Heidmann et al., 1986). The levels of g-chain message also appear to be subjected to developmental changes, decreasing at postnatal stages (Sakmann et al., 1985). It has been suggested that such changes are linked to innervation: denervation of rat muscle at birth suppresses the appearance of J-type channels (Schuetze and Vincini, 1984). Schuetze (1986) considered the possibility that innervation could promote the expression of and inhibit that of ),-subunit mRNA. Alternatively, innervation could modulate the stability rather than the level of expression. The finding of partial dispersion of the four AChR genes in three different chromosomes (Heidmann et al., 1986) suggests that their expression is regulated by trans-activating factors rather than by a common cis-regulating mechanism. Transcriptional activation has actually been shown to be responsible for the increase in ~- and g-chain mRNA during differentiation of the mouse C2 cell line (Buonano and Merlie, 1986). Other factors involved in the regulation of AChR biosynthesis are discussed in relation to the phenomenon of denervation supersensitivity in Section 10. 4.5. RAISON D'ETRE FOR EVOLUTIONARY CONSERVED A C H R CHAIN REGIONS Why should different regions of related proteins be more highly conserved than othersl Regions of unusually high homology in a set of related protein sequences are presumabl) essential for coding structurally and functionally important features of the protein Conversely, sequence stretches displaying a low degree of homology can tolerate change, throughout evolution without important impairment of structure or function, probabl3 reflecting their lesser importance in the protein (see e.g. Wilson, 1985). These general trend, appear to be respected in the AChR. As a whole, regions of the AChR molecule whos~ functional importance has been identified by deletion mapping and oligonucleotid~ mutagenesis (Mishina et al., 1985; Sakmann et al., 1985; Mishina et al., 1986; Imoto e al., 1986) show less variation among organisms for all subunit types than do the sequence~ as a whole (Stroud and Finer-Moore, 1985). The exception is the proposed MA amphipathic channel-forming helix (Fairclough et al., 1983). The MA helix is an exceptior in terms of homology, since this region displays lowest sequence homology for the/3-, 7 and 6-subunits. However, if conservative substitutions are computed as matching residue: the degree of structural homology among these putative helices increases (Finer-Moon and Stroud, 1984). These authors emphasize that the proposed role for these helices is t~ provide the hydrophilic wall of a water-filled channel and not to interact directly with ions Hence the essential requirement for this function is amphipathicity, a property which cat be maintained even with substantial changes at the level of individual amino acids Amphipathicity as such is in fact preserved in all sequences.

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5. THE ADULT AChR: A MULTISPANNING MEMBRANE PROTEIN 5.1. STRUCTURE

When the AChR is solubilized in non-denaturing detergents, the monomeric molecule exhibits the characteristics of an asymmetric cylindrical-shaped body, with a Stokes radius of 7 nm, a radius of gyration of about 4.6 nm, a sedimentation coefficient of 9 S, and molecular weight ranging between 290,000 and 303,000 Daltons (see references in Karlin, 1980; Barrantes, 1983). Application of radiation inactivation methods (Lo et al., 1982) and Doppler effect laser light scattering (Doster et at, 1980) have more recently yielded molecular weight estimates in closer agreement with the 267,000 Dalton figure derived from the sequence data obtained in turn by means of cDNA recombinant techniques (see above). In agreement with its ctafly6-subunit stoichiometry (Reynolds and Karlin, 1978; Lindstrom et at, 1979), the AChR monomer appears to lack symmetry when observed in the native membrane (Klymkowsky and Stroud, 1979; Kistler et aL, 1982; Zingsheim et aL, 1980, 1982a,b). Application of low-dose ( < i0 e/A 2) scanning transmission electron microscopy (STEM) and image averaging techniques specially developed to analyze non-crystalline specimens enabled us to produce the first low-resolution maps of the average AChR particle in the native receptor-rich membranes at a resolution of 1.5-2.0 nm (Zingsheim et aL, 1980). In the axis normal to the membrane surface, the AChR molecule is also unevenly distributed with respect to the bilayer, extending 5.5-7.0 nm towards the extracellular space and 1.5~4.0 nm into the cytoplasmic compartment (Ross et al., 1977; Klymkowsky and Stroud, 1979; Zingsheim et al., 1982a; Brisson and Unwin, 1985). When viewed from this axis the two-dimensional projection of the AChR appears as a ring-shaped, rosette-like particle 8-9 nm in diameter with a stain-filled central pit. Three non-symmetrical regions could be identified around the central pit of the AChR in such end-on views. Upon removal of non-receptor proteins from the cytoplasmic face of the membrane, a different profile of the AChR particle becomes visible (Barrantes, 1982b). This is presumably the cytoplasmic-facing end of the molecule. Thus, the AChR monomer displays asymmetry along axes both parallel and perpendicular to the membrane plane. Brisson and Unwin (1985) have subsequently obtained three-dimensional maps of the AChR by Fourier synthesis of images from 'annealed' tubular structures derived from AChR membranes. These crystals were of sufficiently good order to perform reconstructions (l.8-2.0nm resolution) in three dimensions of frozen, unstained and stained specimens. The AChR appears in these reconstructions as a body with a calculated volume of 350,000 .~3, of which only a minor portion is embedded in the membrane. The portion of the AChR protruding about 7 nm into the synaptic cleft displays a fivefold axis of symmetry in a plane perpendicular to the membrane; no symmetric features could be observed in the cytoplasmic portion of the protein, and symmetry could be ascertained in only part of the membrane-spanning domain of the AChR. Given the relative contributions of these three portions, the overall two-dimensional projection of the molecule of the plane of the membrane displays approximately pentagonal symmetry. The AChR exists in the native membrane as closely but not maximally packed particles (Barrantes et aL, 1980; Zingsheim et aL, 1980). Ordered arrays (Brisson, 1980; Klymkowsky and Stroud, 1979) occur exceptionally in the native membranes (another reflection of the lack of symmetry of the individual molecule), and the type of symmetry displayed in such ordered lattices is variable (see review in Barrantes, 1979). As mentioned above, a certain degree of ordering can be induced in membrane-bound AChR by procedures such as annealing (introduced by Brisson, 1980). Rows of AChR particles have been observed by rapid freezing in the cholinergic synapse in situ (Heuser and Salpeter, 1979; see Figs 1 and 2) and in AChR-rich membranes by negative contrast techniques (Barrantes, 1982b).

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F.J. BARRANTES 5.2. TOPOGRAPHY OF A C H R SUBUNITS

In addition to the 9 S AChR monomer, a 13 S dimeric form with a Stokes radius of about 8.5 nm and a molecular weight twice that of the monomer is also present in AChR membranes. Interconversion occurs via disulphide reduction/oxidation. The comparison of native membranes with those subjected to cleavage of the disulphide bonds by reducing agents has yielded the putative average position of the 6-subunits (Zingsheim et al., 1982a) in the native membrane-bound AChR. Similar results were obtained by measurement of the angles determined by biotinylated toxin-avidin complexes tagging ct-subunits in AChR trimers (Wise et al., 1981). Analogously, Fairclough et al. (1983) measured the angles subtended by Fab fragments of monoclonal antibodies directed against the ct-subunits on AChR particles. They concluded that the ~--6 bonded dimers are a mixed population of translationally related particles (R--R) and particles related by a C2 type of symmetry (R--'d). The angular distributions of Fab fragments are broadened by the contribution arising from the flexibility about the 6--6 bond, which was calculated to be + 2T. The reason for the different occurrence of symmetrical dimers related by a two-fold axis in native membranes (Zingsheim et al., 1982a) and asymmetrical dimers in lipidenriched or reconstituted vesicles (Fairclough et al., 1983; Bon et al., 1984) is not known. The nature of the interactions operative in the two types of preparation might bear on the discrepancy. Knowledge of the location of the 6 - - 6 bond linking two monomers in a dimer provided a useful landmark for establishing the relative topography of other AChR subunits in the population of dimers displaying C2 symmetry in native AChR membranes (Zingsheim et al., 1982a). Since the ~-subunits carry the recognition site for agonists and antagonists, avidin bound to biotinylated :~-toxin enabled Holtzman et al. (1982) to calculate the angles separating the a-chains. The conclusion was reached that the two a-chains in a monomer cannot be contiguous. The angle between ~-subunits (110° + 30°) was also measured with avidin-biotin in dimers cross-linked via the /3-subunits (Karlin et al., 1983). Native ~-bungarotoxin has also been used for the direct location of its recognition sites on the AChR molecule by low-dose electron microscopy and single particle image averaging techniques (Zingsheim et al., 1982b; Bon et al., 1984). Attachment of a single native toxin to its recognition site on each a-chain produces a significant increase in the mass contributing to the average image of the AChR, roughly one-quarter of the mass per -subunit. A difference map between the toxin-tagged and untreated membranes yields two statistically significant peaks of stain-excluding density. The two ~-chains exhibit contact,~ with a different set of subunits, i.e. they possess different local environments. This provide,, a structural framework within which one can rationalize the functional non-equivalenc~ of the recognition sites (see Section 5.6). Fairclough et al. (1983) have employed immunocytochemistry to measure th~ angle between ~-subunits, which was found to be 144~ + 4 °, i.e. non-adjacent withir the monomer. Small-angle X-ray diffraction of toxin-tagged AChR also suggestec an apical location of the binding sites on the extracellular portion of the molecul~ (Fairclough et al., 1983). Bon et al. (1984) have also carried out an analysis o membrane-bound AChR tagged with native ~-bungarotoxin; their results are in ful agreement with ours (Zingsheim et al., 1982b) and those of Holtzman et al. (1982) Karlin et al. (1983) and Fairclough et al. (1983) on the location of c~-subunits. Th, non-adjacent distribution of ~-subunits is corroborated by recent biochemical studie (Hamilton et al., 1985). Summarizing this rare point of coincidence among all group studying these aspects of AChR structure, (i) the ~-subunits are separated by one othe subunit and (ii) the immediate environment of each ~-subunit is necessarily different. Mos recently, Kubalek et al. (1987) have determined the azymuthal distribution of ACht subunits around the central pit of the molecule: their clockwise arrangement is cq, 13, ~2 7, 6, as determined by image analysis of ~-toxin and Fab-tagged membrane-bound AChR The adjacency of ~2 and ), subunits within the AChR oligomer had been postulate (Barrantes, 1983).

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5.3. COMMONFEATURESOF ACHR CHAINS The distribution of amino acid residues along the AChR polypeptide chains can be analyzed in terms of the occurrence of hydrophobic and hydrophilic stretches, from which the location of segments relative to the membrane lipid bilayer has been postulated. The application of algorithms such as those developed by Kyte and Doolittle (1982) or Hopp and Woods (1981) made apparent the occurrence of four segments of hydrophobic amino acid residues in all receptor subunits (Noda et al., 1983a,b; Fairclough et al., 1983; Finer-Moore and Stroud, 1984). One type of model, the so-called four-helix model, postulated the presence of about 30% of the AChR protein on the cytoplasmic face of the membrane. An alternative type of model (the five-helix model) places a much smaller (ca. 20%) portion of the protein on the intracellular face of the membrane and predicts that in addition to the four transmembrane helices per chain there is a fifth, amphipathic transmembrane helix (Fairclough et al., 1983; Guy, 1984). The basic discrepancy between the two types of model resides in the fact that the five-helix models place the C-terminus on the cytoplasmic face of the membrane. More recently, alternative models have appeared (Criado et al., 1985; Ratnam et al., 1986) which challenge the above two types of hypothesis. A thorough discussion of this issue is given in a recent review (Barrantes, 1988). 5.4. THE SYNAPTIC CLEFT-FACING PORTION OF THE ACHR Lactoperoxidase iodination (St. John et al., 1982), graded proteolysis in sealed, intact vesicles (Strader and Raftery, 1980), chemical analysis of AChR chains and cross-linking with neurotoxins (see references in Conti-Tronconi and Raftery, 1982) indicate exposure of all subunits to the extracellular milieu. The evidence indicates that all subunits are exposed to the extracelluar and cytoplasmic compartments and transverse the membrane at least once. In fact, current models postulate the existence of several transmembrane helices for each chain, bringing the total number of helices within the bilayer to more than 25. The A C h R is an integral, transmembrane glycoprotein. But how are these transmembrane portions arranged? The electron density profile determined by X-ray diffraction techniques indicates that the extracellular mass of the AChR protrudes into the synaptic cleft about 6 nm (Ross et al., 1977; Zingsheim et al., 1982a). The regions between residues 70 and 160 in the four subunits are highly homologous (27 residues are totally conserved). Both the four- and five-helix models postulate that this region is on the synaptic cleft. The 70-160 stretch would carry two important features: sites for N-glycosylation (Asn residues 143 and 145) and the disulphide bond which reacts after reduction with affinity reagents like 4-(N-maleimido)benzyltrimethylammonium (MBTA), as postulated by Numa et al. (1983). Karlin and co-workers have recently established the identity of the cystinyl residues responsible for MBTA binding as Cys-192 and Cys-193 (Kao et al., 1984; Kao and Karlin, 1986; see Karlin et al., 1986). The putative extracellular stretch is also postulated to be rich in amphipathic fl-sheets common to all subunits in the form of a six- or eight-strand fl-barrel domain (Finer-Moore and Stroud, 1984). According to Fairclough et al. (1983) the predominant negative charges in the 'vestibule' of the protein might contribute to the cation specificity of AChR channel. 5.5. THE CHANNEL PORTION OF THE A C H R

Fairclough et al. (1983) and Guy (1984) have put forward specific proposals on the structure of the AChR channel. The fifth, amphipathic helix postulated in their type of model possesses a continuous hydrophobic face on one side and a hydrophilic face on the other, the latter providing a structural substratum for the lining of the internal walls of the ionic channel. Thus the continuous hydrophilic face has the appropriate length to span the lipid bilayer (about 3.8 nm) and a distribution of charges (21 positive, 19 negative,

346

F.J. BARRANTES

10 uncharged) in its residues (Fairclough et al., 1983) to make it ideally suited fo inter-subunit ion-pairing. Other hypotheses on the subunit domains involved in channe formation have also appeared (Kosower 1983a,b,c, 1984). These hypotheses have begul to be tested experimentally, as discussed below (see Section 9), Electrophysiological dat~ have provided some hints on structural aspects of the AChR channel region. For instanc~ the studies of Aguayo et al. (1981) indicate that the AChR channel must be asymmetrica with its selectivity gate located most likely towards its intracellular-facing regior Electrophysiological measurements have also contributed to set limits to the size of the io channel (Adams et al., 1980, 1981; Dwyer et al., 1980), with a pore cross-section of abot 0.65 x 0.65 nm, and to postulate the existence of ion-binding sites (Adams et al., 1981 within the lumen of the AChR channel. Measurements of channel conductance ha~ pointed out discrepancies between the expected ionic selectivity and the reversal potentia leading to the postulation of ion-binding sites within the channel lumen. More accural structural data is needed in order to establish the precise location of these sites. 5.6. AGONIST AND COMPETITIVE ANTAGONIST RECOGNITION SITE(S)

One of the major enigmas still to be resolved is that of the exact location of the ligar recognition site(s) on the receptor molecule. The wealth of information on this topic sten from biochemical studies interpreted in the light of electrophysiological data. Equilibriu and rapid kinetic studies have provided estimates of the number of ligand binding sit and affinities of agonists and antagonists in vitro. Most of the available biochemical da concur with the electrophysiological data in that two ACh binding sites occur per ACh monomer in Torpedo AChR, one in each ~-chain. The quaternary ammonium head grol present in ACh and other small cholinergic ligands most likely binds to an anionic regic on the AChR (Michelson and Zeimal, 1973). Hydrogen-bond formation between the A( molecule and the AChR should contribute additional binding energy (reviewed in Spiw and Albuquerque, 1982). One fortunate feature of the agonist recognition site(s) of the AChR is that an eas reducible disulphide bond lies in its vicinity. Covalent modification of the reduced bon (which lie on the subunits) by affinity reagents was shown to permanently block the acti, of agonists and some antagonists (Damle and Karlin, 1978; Damle et al., 1978). Recent the target cysteine residue(s) have been identified as Cys-192 and Cys-193 (Kao et 1984), two amino acids unique to the c~-subunit (see above) (Noda et al., 1983a). The m~ widely used approach to identify the recognition site(s) consists in their labelling w appropriate covalent ligands and subsequent characterization of the chemically modifi amino acid residues by biochemical techniques. Early experiments paved the way for su recent attempts by making use of two affinity reagents, MBTA and bromoacetylchol: (BAC). These two now classical ligands have been found to label only one ~-subunit 1 AChR monomer in T. californica (Weill et al., 1974; Damle et al., 1978), whereas anot] affinity reagent, p-(trimethylammonium) benzenediazonium fluorobate, labels two sites monomer (Weiland et al., 1979). In fact, BAC labelling is highly dependent on react! conditions, since although only one site is detected in T. m a r m o r a t a monomer at 4°C, bq sites are labelled at 23°C (Wolosin et al., 1980). Since both chains have identical prim structure, one possible interpretation of such a difference (a kind of half-of-the-: reactivity) is based on the different environment of each subunit: the asymmetric azimut orientation of the five subunits in the AChR pentamer determines different contacts the ~ and ~2 chains (Holtzman et al., 1982; Zingsheim et al., 1982b; Bon et al., 191 Posttranslational modifications have also been considered as a source of a-chain mic heterogeneity. The reported difference in the degree of glycosylation of the two ~-ch~ in T. cal(fornica AChR (Conti-Tronconi et al., 1984) was an obvious candidate explaining the non-equivalence of binding sites. It has been suggested that only one of ~-chains is glycosylated and that it is this chain that is labelled with MBTA with affinity (Conti-Tronconi et al., 1984). More recent evidence appears to dispel such propt (Pedersen et al., 1986). There is no evidence pointing to the involvement of other cova

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posttranslational modifications such as methylation (Kloog et al., 1980) or phosphorylation (see Huganir et al., 1984, 1986) in the half-of-the-site reactivity phenomenon, although phosphorylation seems to play an important role in the desensitization process (vide infra). Upon ACh binding 4-6 Ca 2+ ions (Chang and Neumann, 1976) or 6-12 Tb 3+ ions (Rfibsamen et al., 1978) are released from purified Torpedo AChR. Pharmacological experiments have revealed the existence of some ligands that interact differently with each of the two ACh recognition sites. In addition to BAC, curare and related competitive antagonists fall into this category (Neubig and Cohen, 1979; Sine and Taylor, 1981). Dose-response curves normally fall short of providing the actual dissociation constant for each of the sites, since they yield only the geometrical mean of the binding to both sites. On the basis of the above background information, the most recent attempts to identify regions of the or-chains responsible for ligand recognition have made use of reagents like the ones developed by Karlin (1969) and other covalent probes. There are various reasons why this type of reagent is ideal for this purpose: (a) all nicotinic AChRs contain an easily reducible disulphide bond in the vicinity of the ACh subsite; (b) this disulphide bond can be reduced under mild conditions in the resting AChR conformation; (c) agonists trigger a conformational change that stabilizes the disulphide bond; (d) the quaternary ammonium alkylating reagents used as affinity labels have a known pharmacological activity, i.e. they are competitive antagonists of the AChR; (e) the structure of the affinity reagents dictates that the presumptively negatively-charged ACh recognition subsite must be within 1 nm from the alkylated cysteinyl residue (see review in Karlin et al., 1986). Kao et al. (1984) have identified Cys-192 and Cys-193 (unique to the a-chain) as the two sites alkylated by MBTA in T. californica AChR (Fig. 4). Furthermore, they demonstrated that Cys-128 and Cys-142 remain cross-linked under conditions that reduce Cys-192 and Cys- 193. The sulphydryl-directed reagent MPTA has also been found to label a single a-chain cyanogen bromide fragment identified as 179-207 in native T. marmorata AChR (Dennis et al., 1986). Similarly, the photoaffinity reagent p - ( N , N - d i m e t h y l a m i n o ) benzenediazonium fluoroborate (DDF) labels three cyanogen bromide peptide fragments in an agonist-sensitive manner. The major peptide also corresponds to ~ 179-207 (Dennis et al., 1986); this fragment contains the cysteine residues 192 and 193 labelled by MBTA (Kao et al., 1984) and MPTA (Dennis et al., 1986) in the reduced AChR. Synthetic peptides corresponding to this region of the AChR have been found to be capable of binding or-toxins, although such binding does not respond to agonists and displays lower affinities than that of native AChR (Wilson et al., 1985; Neumann et al., 1986). f-~

MPTA

site

OOF sit~ I~.~MBT^ site KOYROWKHWVYYTCCPOTPYLO

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,,,

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FIG. 4. Schematic diagram showing the location of the ligand recognition site on the ~t-subunit of the AChR. The ACh subsite is tentatively identified by the position of the covalent affinity reagents (competitive antagonists) MBTA (Kao and Karlin, 1986) and MPTA (Dennis et al., 1986). Cysteines 192 and 193, unique to the ~t-chain, are the target of these two reagents. Pedersen et al. (1986) have also identified the curare binding site in a tryptic fragment of the ~t-chain starting at Ser-173. Dennis et al. used a photoaffinity reagent, DDF, to identify the ligand recognition site; it overlaps the MBTA/MPTA site. J P T 38 3

F

348

F . J . BARRANTES

Binding studies on whole cells (BC3H-1) have shown that competitive antagonist such as cobrotoxin and ~-bungarotoxin bind non-cooperatively and with very higl affinity (Ko ~ 0.1 nM) to the ligand recognition site but with Hill coefficients les than unity, implying non-equivalence of the sites (Sine and Taylor, 1981); but in th Torpedo AChR the two sites appeared equivalent in membrane-bound and detergent solubilized AChR (Ellena and McNamee, 1980). Their affinity does not seem to underg changes like those induced by agonists (Weiland and Taylor, 1979; but see Qua,~ et al., 1978). Agonist binding has two effects: channel opening and desensitization. At the molecula level these processes involve conformational transitions of the AChR. The reporte quenching of AChR intrinsic fluorescence involved processes occurring at rates compatibl with the desensitization phenomenon (Bonner et al., 1976; Barrantes, 1978; Kaneda et al 1982, and see Section 7). But in addition to these rather slow transitions, fluorescent techniques using extrinsic probes, e.g. non-covalently bound ethidium (Schimerlik et al 1979a,b) or the covalently bound fluorophore 5-(iodoacetamide) salicyclic acid (Dunn al., 1980), have shown changes other than desensitization. The two latter reagents increa,~ their fluorescence intensity upon agonist binding. The former extrinsic fluorescent prot probably acts as a non-competitive antagonist of the AChR, and thus the fluorescem changes probably reveal different eonformational transitions of the receptor protei~ Pyrene cholinergic derivatives have also been used as extrinsic probes of the ACh (Barrantes et al., 1975; Tan et al.. 1980). When tested for their pharmacological activit most pyrene-alkyl-trimethylammonium compounds behaved as competitive antagonis (Tan et al., 1980). Rapid kinetic studies made apparent conformational transitions of tt membrane-bound AChR following binding of the pyrene derivatives (Tan and Barrante 1980). Another pyrene compound bound covalently to the fl- and 7-chains of T. californi~ was used more recently (Gonzalez-Ros et al., 1983). Quenching of the pyrene fluorescen, by nitromethane decreased with desensitizing concentrations of Carb, whereas ~-bungar, toxin had the opposite effect (Gonzalez-Ros et al., 1983). These results were interpret~ with a simple model in which the agonist-binding site becomes less exposed to solvent up~ agonist binding, while the bulk of the AChR molecule becomes more accessible except fi its membrane-embedded domains. Similar conclusions were reached in a study of ACh intrinsic fluorescence quenching by nitroxide spin labels in the absence or presence of fi cholinergic ligands (Barrantes, 1978). By definition the competitive antagonist binding site overlaps the agonist bindil site. In the case of snake neurotoxins (Mr 7000-8000) this recognition site is predicted cover 2 x 3 nm of the AChR surface (Low, 1979; Kistler et al., 1982; Stroud, 198 Fairclough et al., 1983). Binding of these toxins to the surface has been indirectly visualiz, in electron micrographs by using gold-labelled antitoxin antibodies (Klymkowsky al Stroud, 1979) and avidin binding to biotinylated toxin (Holtzman et al., 1982), in t dimension perpendicular to the membrane by X-ray diffraction (Fairclough et al., 198: and more directly using difference image autocorrelation methods (Zingsheim et c 1982b). When two ~-toxins are bound they lie about 5-7 nm apart from one another, the surface of the AChR molecule. This was also established by fluorescence ener transfer measurements in which one toxin was labelled with fluorescein and the ott with tetramethylrhodamine--two adequate donor-acceptor fluorophores--at Lys(Johnson and Taylor, 1982; Cheung et al., 1984; Johnson et al., 1984). Bungaroto~ derivatives have been shown to label both the ~- and 6-subunits in T. californ, membranes (Witzemann et al., 1979) but in in situ studies they cross-link all four subun (Nathanson and Hall, 1980). Given the size of the snake toxins, their binding site is lik, to overlap more than one AChR subunit. Interaction of spin-labelled neurotoxins with 1 AChR suggested that Lys-27 in the former was in the vicinity of a disulphide on the AC1 (Tsetlin et al., 1982). Lys-27 is presumed to be about 1.6 nm from the high-affinity ago~ binding site (Fairclough et al., 1983). Using radiolabelled tubocurarine, Pedersen et (1986) have recently been able to localize the binding site for this competitive antagor on a proteolytic fragment which starts at Ser-173 of the ~-subunit (see Fig. 4), terrr

Muscle endplate cholinoreceptors

349

V8-20. This fragment also carries the binding site for the non-competitive antagonist meproadifen (see below). The curare recognition site also overlaps that for ~t-bungarotoxin and MPTA. 5.7. NON-COMPETITIVE ANTAGONIST RECOGNITION SITE(S)

A wide variety of chemically heterogeneous compounds which block or modulate the agonist-stimulated increase in cation permeability fall into the category for noncompetitive blockers (NCBs). Within their pharmacologically active doses they do not bind at the agonist-recognition site itself but at secondary or 'regulatory' sites. This category of compounds comprises aminated local anaesthetics like lidocaine (Steinbach, 1968), procaine (Katz and Miledi, 1975), tetracaine (Cohen et al., 1974), the alkaloid histrionicotoxin (HTX) extracted from the South American poison-dart frog, the hallucinogenic psychoactive tranquilizer phencyclidine (angel's dust; Albuquerque et al., 1973), fatty acids (Brisson et al., 1975; Andreasen and McNamee, 1980), the antipsychotic neuroleptic chlorpromazine (Anwyl and Narahashi, 1980), aliphatic alcohols (Spivak and Albuquerque, 1982), and triphenylmethylphosphonium (TPMP ÷, Lauffer and Hucho, 1982). In neurons, even drugs known to activate cAMP-dependent phosphorylation of the nAChR, albeit in a different concentration range, like forskolin, exert local anaesthetic-like effects (Akagi and Kudo, 1985; McHugh and McGee, 1986). In t~itro observations have led to the operational classification of NCBs into HTXsensitive and HTX-insensitive (see e.g. Heidmann and Changeux, 1981). In vit, o electrophysiological studies have put more emphasis on whether their action is dependent on membrane potential or not. Thus, charged quaternary or tertiary aminated local anaesthetics like lidocaine and its derivatives QX-222 or QX-314 (Neher and Steinbach, 1978; Neher, 1983), procaine, tetracaine are more effective blockers at the depolarized NMJ, whereas chlorpromazine, HTX, phencyclidine, or dibucaine are not affected by membrane potential. It is perhaps somehow unexpected that such a wide range of chemically different compounds have several properties in common in terms of their action mechanism. One such common property is their ability to stabilize the AChR in the desensitized state. AChR affinity for some non-competitive antagonists increases upon binding of agonists and some competitive antagonists; binding of [3H]phencyclidine to Torpedo AChR-rich membranes is 103-104-fold faster when Carb is simultaneously applied (Oswald, 1983a; Oswald et al., 1984). This enhanced rate is not totally unambiguous, however. Heidmann and Changeux (1986) interpret such phenomenon as a reflection of NCB binding to the active AChR conformation. Binding of local anaesthetics to saturable site(s) on the AChR stabilizes the AChR in a high affinity state for agonist (Heidmann and Changeux, 1979; Boyd and Cohen, 1984). Their binding occurs on multiple sites on the membrane-bound AChR. A saturable, high-affinity, allosteric binding site for non-competitive blockers has been proposed (Heidmann et al., 1983; Oswald et al., 1984; Haring and Kloog, 1984). Two classes of sites for [3H]phencyclidine have been reported for T. ocellata AChR membranes (Haring and Kloog, 1984). One class exhibits high-affinity (Kd = 6-9 #M) and one site per AChR, and the other two low-affinity (K0 = 85/~ M) with two sites per AChR monomer. It has been postulated that calcium channel antagonists may interact with the AChR channel as well, since they interfere with phencyclidine binding (Epstein and Lambert, 1984). It has been reported that in the millimolar concentration range non-competitive antagonists can bind to the agonist recognition site. Heidmann et al. (1983) reported a single high-affinity site per AChR monomer for phencyclidine, meproadifen, and Triton X-100 (K0 5/~M). This site could be competitively blocked by perhydroHTX (K0 0.15/~ M). Chlorpromazine and trimethisoquin binding is not HTX-sensitive, and exhibits 10 30 low-affinity sites per AChR monomer. Since the stoichiometry of low-affinity sites depended linearly on the lipid-protein ratio in reconstituted AChR membranes, it was suggested that such sites occur at the lipid-protein interface, distant from the channel itself (Heidmann et al., 1983). Non-competitive blockers of the low-affinity type exhibit affinities

350

F. J. BARRANTES

related to thier partition coefficient in lipids and their charge. Two NCB sites have been detected on the AChR with ESR techniques (Earnest et al., 1984). A third 'non-saturable' binding site in the bulk lipid bilayer has also been proposed (Heidmann et al., 1983). The high lipid solubility of several of the non-competitive blockers suggests that even if there is one saturable high-affinity binding site per AChR monomer responsible for blocking agonist-stimulated ion flux, the lipid membrane provides the receptor with a locally high concentration of drug which may have its own effects on AChR function through specific effects on the lipid-protein interface, or by affecting the local charge environment, or by a combination of both (Chan and Wang, 1984). This appears to be the case with spin-labelled local anaesthetics which sense strongly immobilized spectra in the presence of agonist, suggesting agonist-induced conformational changes of the AChR protein (Palma et al., 1986). The possibility that the probe's signal arises partly from protein-lipid interactions has been considered by these authors. Photoaffinity labelling has been the choice technique in attempts to identify non-competitive binding sites on the AChR (Lester et al., 1980; Oswald and Changeux, 1981a,b; Kaldany and Karlin, 1983; Heidemann and Changeux, 1984; 1986; Miihn et al., KotzybaHibert et al., 1985; Oswald et al., 1985). Thus a radioactive photoaffinity derivative of the local anaesthetic trimethisoquin (5-azido [3H]trimethisoquin) has been used by Oswald and Changeux (1981a), who showed labelling of the 6-chain; this could be inhibited competitively by unlabelled trimethisoquin and phencyclidine; labelling of the or-chain could be prevented by agonists and competitive antagonists. Karlin and co-workers have made use of alkylating derivatives of local anaesthetic-like substances like quinacrine mustard to localize NCB sites. Its parent compound, quinacrine, had been previously used by Grfinhagen and Changeux (1976a,b) as an extrinsic fluorescent probe to monitor the desensitization process, and by Adams and Feltz (1977, 1980a,b) and Tsai et al. (1979) to define its local anaesthetic-like activity at the NMJ. Kaldany and Karlin (1983) found that quinacrine mustard labelled the ~- and fl-chain,, of the AChR, at variance with the results of Oswald and Changeux (1981a,b), whc localized the NCS on the 6-chain. It is therefore possible that there are distinct binding sites for different NCBs. A closer approximation to the localization of the NCB site(s) has been recentl~ attempted by using affinity labelling and proteolytic mapping of the tagged polypeptidc in the AChR protein (Pedersen et al., 1986). The ligand chosen was [3H]meproadifer mustard, a reactive analogue of proadifen and meproadifen (Dreyer et al., 1986). Thi, ligand specifically tags the ~ and to a lesser extent the fl AChR subunits in the presenc~ of high agonist concentrations. In earlier studies Cohen's group had used the parenl compound, meproadifen, to characterize its equilibrium binding properties with th~ membrane-bound AChR (Krodel et al., 1979). Upon proteolytic digestion of the meprod. ifen mustard-tagged ~-subunit, the site could be identified within a 20 kDa peptide, namec V8-20, which also carried the competitive antagonist recognition site (as measured b3 tubocurare and ~-bungarotoxin binding) and the reactive disulphide bond in its vicinit~ (see Fig. 5). N-linked carbohydrate was also present in the proteolytic peptide, whicl" begins at ~ Ser-173 (Pedersen et al., 1986). Miihn et al. (1984) employed triphenylmethylphosphonium (TPMP +) in stopped-flo~ experiments; the probe was mainly incorporated into the or-chain in the absence o cholinergic ligands but, in addition to this, extra sites on the 6- and fl-chains was observec in the presence of cholinergic drugs. More recent results from Hucho's laborator: (OberthiJr et al., 1986) localized TPMP ÷ on position 262 of the M2 helix of the 6-chain A note added to their paper comments on their finding of additional tag on residues 241 and 254 of the 2- and fl-chains respectively. The nature of the amino acid tagged wa,. presumed to be serine. These findings bear relationship to the recent point mutagene sis/patch-clamp studies of Imoto et al. (1986), which have led to the contention that th~ putative transmembrane M2 segment and the adjacent M2/M3 bend region of tht 6-subunit might be involved in determining the rate of ion transport through the AChl; channel (see Section 9).

Muscle endplate cholinoreceptors

351

M2-MS bend region

lUC----7 r---ll

Y a

0

II I0 I0

I I I

II rl II

MII

//

_

MI

~

-

-

j II II II

M2/

] I I

I I

M3

M2--~___.._

Ii

["""71 I | Ii I II

MA

M4

M3

A~" PTnS-GEK [MTLSISVLLSLTVFLLVIV ELIPSTSSAVPLIGK

| |

ppnA-GEKIMSLSISALLAVTVFLLLLA DKVPETSLSVPIIIR PAgAGGQK]CTLSISVLLAQTAFLF'LIA BKVPETSLNVPLIGK 6 PAES-GEK[MSTAISVLLAQAVFLLLTS 13RLPETALAVPLIGK

'OUINACRINE MUSTARO s i t e

LCHLORPROMAZINE sitQ TPMP+ s i t e

FIG. 5. Tentative location of non-competitive blocker (NCB) site(s). Use was made of quinacrine mustard (Cox et al., 1985, and see Karlin et al., 1986) to identify the NCB site of the MI segment of the ~t-chain. The NCB chlorpromazine reacts covalently with Ser-262 in the 6-chain and Ser-254 in the fl-subunit (Giraudat et al., 1986). These two serine residues appear to be homologous. Additional labelling is observed in Leu-257 of the fl-chain. Hucho and co-workers (1986) labelled Ser-262 in the f-chain with TPMP ÷. Thus, two reagents identify the NCB site on the M2 hydrophobic segment, mainly in the f-chain, whereas the quinacrine mustard site and meproadifen site (Dreyer et al., 1986) appear to be located closer to the M1-M2 region in the ct-subunit. The M2-M3 bend region in the 6-chain, which point mutations suggest involved in channel gating properties (lmoto et al., 1986), is also indicated in the scheme.

Changeux's group has also tackled the issue of the exact location of the NCS. The high-affinity NCBs chlorpromazine and phencyclidine, whose slow blocking action of AChR channels appears to be supported by recent patch-clamp studies (see Section 8.6), have been used in photolabelling experiments. Before UV irradiation chlorpromazine exhibits a single high-affinity binding site; after photoirradiation the bound probe tags all four AChR subunits (Heidmann et al., 1983). The suggestion has been made that NCB sites within the ion channel are made accessible when the channel opens (Oswald and Changeux, 1981b; Heidmann and Changeux, 1984), since the rate of labelling of all AChR subunits with chlorpromazine increases in the simultaneous presence of cholinergic agonists; addition of agonist prior to the non-competitive ligand abolished the effect. Cox et al. (1985) rebutted this interpretation; in their experiments with quinacrine azide (see above) the conclusion was reached that the rapidly desensitized form of the AChR was labelled. More recently, Heidmann and Changeux (1986) have produced evidence which they interpret as more firm support of their former hypothesis. In the presence of agonist, chlorpromazine would bind to the active conformation of the AChR 100-1000 times faster, in an almost diffusion-controlled manner. Giraudat et al. (1986) have identified the Ser-262 in the 6-chain as the amino acid residue tagged by chlorpromazine on the hydrophobic M2 segment (Fig. 5). Ser-254 and Leu-257 were correspondingly identified on the fl-subunit (Giraudat et al., 1987), also on the M2 segment. In Torpedo m a r m o r a t a AChR, Ser-254 in the fl-chain appears to be homologous to Ser-262 in the 6-subunit. Oberthuer et al. (1986) identified position 262 on the g-chain as the site of TPMP ÷ covalent attachment, and more recently Hucho et al. (1986) identified a homologous position on the 6- and fl-chains. Another approach recently used to tag the non-competitive ligand recognition site is the use of extrinsic fluorescent probes. Using fluorescence lifetime measurements and also the same strategy successfully employed in the determination of distances between the two competitive antagonist recognition sites, Taylor and co-workers (Taylor et al., 1986) have shed some light on the environment of the non-competitive recognition site. Non-covalently bound ethidium has been used as a probe; its lifetime can be substantially modified by PCM and is sensitive to the binding of agonists at a distant site. Ethidium binds at a hydrophobic site and not within a water-filled region as one would expect for the AChR channel. The distance between the non-competitive antagonist site (one per AChR monomer) and the agonist recognition site is larger than 5 nm.

352

F.J. BARRANTES

6. FUNCTIONAL CORRELATES OF AChR STRUCTURE 6.1. CORRELATIONBETWEEN LIGAND BINDING AND A C H R CHANNEL ACTIVITY

Although as reviewed above both biochemical and pharmacological studies indicate that the two agonist binding sites differ in structure, independent evidence points to the involvement of both sites in the agonist-dependent channel activity of the AChR. For example, when one agonist site is blocked by MBTA, ion flux follows on from agonist binding to the second high-affinity site (Delegeane and McNamee, 1980). Agonist (ACh) binding to these sites does not follow a simple isotherm, however, but appears to be weakly cooperative under desensitizing conditions (Fels et al., 1982; Prinz and Maelicke, 1983). The Hill coefficient for channel opening is 1.97 _ 0.06 in ritro (Neubig and Cohen, 1980), suggesting that the binding of two (or more) agonists is required for channel opening. Karlin (1967) and Colquhoun (1973) have discussed the meaning of the Hill coefficient for a variety of kinetic schemes and shown that the Hill plot is not expected to be linear for many such models. A further complication is that the Hill slope varies with voltage (Chabala et al., 1986), suggesting that the overall agonist affinity is voltage-dependent. The variations of the Hill coefficient with voltage at high agonist concentration were interpreted by Chabala et al. (1986) as a manifestation of strong negative cooperativity. Both electrophysiological and flux measurements concur in that at least two ACh molecules are required for efficient channel opening and that the dissociation constant for agonists lies in the 10-400/~M concentration range. Were it not for the desensitization phenomenon, ACh could effectively open most of the available channels. The actual number of agonist sites involved in channel gating is rather difficult to determine in t,itro. The greatest handicap in this type of study is the poor time resolution of ion-flux measurements. Active A C h R species mediating channel activity are in fact driven into the thermodynamically favoured desensitized state in the course of the ion-flux assay. Obviously, the resulting data is biased to different extents by the proportion of desenitized/active AChR, which is of course agonist concentration-dependent. In addition, the ratio of resting to desensitized AChR in the absence of agonist varies for different AChR membrane preparations (Griinhagen and Changeux, 1976a,b; Weiland et al., 1977; Barrantes, 1978; Neubig and Cohen, 1980). Equilibrium dissociation constants for Carb binding to the resting (Ko = 30 pM) and desensitized (Ko = 10 100 nM) states of the AChR (Weiland et al., 1977; Quast et al., 1978) differ substantially from those determined to induce half-maximal activity in electrophysiological studies of frog muscle (0.5-1 mM) (Dreyer et al., 1978; Dionne et al., 1978). However, improved ion-flux measurements of purified AChR, utilizing 22Na+ (Neubig and Cohen, 1980) and 86Rb+ (Hess et al., 1983) flux or T1+ quenching (Moore and Raftery, 1980) yielded dose-response curves similar to those determined by electrophysiological analysis (see below). The possibility of correlating electrophysiological, binding and flux studies using the very same cell is not an easy task: the recent success in obtaining single-channel recordings from Electrophorus electrocytes (Pasquale et al., 1986) opens an interesting avenue in this respect. Complex kinetic schemes have been postulated to account for the differences observed in equilibrium and kinetic measurements (Neubig and Cohen, 1980; Hess et al., 1983), and the possibility that the high-affinity binding sites identified by MBTA are involved with desensitization while low-affinity sites located on other subunits are responsible for channel opening has been suggested (Dunn and Raftery, 1982; Dunn et al., 1983). These researchers observed changes in the fluorescence of a bound, extrinsic probe indicative of a conformational change; this occurred within the time scale of channel opening even when the high-affinity sites were blocked by BAC and the receptor was in the desensitized state. Additional regulatory binding sites for agonists such as suberyldicholine on Electrophoru,s A C h R (Pasquale et al., 1983) and a voltage-dependent inhibitory ACh binding site in Electrophorus (Takeyasu et al., 1983) and T. californica (Shiono et al., 1984) have been described. The location of these sites is not identified, but direct channel blockage, which is consistent with some electrophysiological measurements (Sine and Steinbach, 1984a,b), was assumed not to occur in these cases.

Muscle endplate cholinoreceptors

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6.2. MACROSCOPIC AGONIST RESPONSE AT THE MOTOR ENDPLATE The relation between the agonist concentration and the postsynaptic current was one of the questions addressed earlier by means of classical electrophysiological techniques. It was observed that upon application of ACh to muscle cells by ionophoresis the evoked current did not increase linearly (it should depend linearly on the number of channels open) but supralinearly with dose (i.e. yielding Hill coefficients ranging between 1.3 and 3), suggesting that more than one agonist molecule is needed to open a channel (Katz and Thesleff, 1957; Hartzell et al., 1975). The range of concentrations which could be explored with these techniques was, however, limited to the low-concentration end (Adams, 1975b) because of the desensitization phenomenon (Katz and Thesleff, 1957). In single-channel measurements, on the other hand, measurements of the dose-response curve were restricted to the high-concentration end, where desensitization intervened (Sakmann e t al., 1980; Siegelbaum et al., 1984) such that AChR channel activities, leaving the desensitized state, could be observed one at a time. Attempts to provide a more quantitative description of the dose response curve pointed to the need to know more accurately the applied agonist concentration, either by resorting to diffusion calculations (Dreyer et al., 1978; Dionne et al., 1978) or by perfusion of known agonist concentrations onto the whole NMJ. The latter approach is hampered by the limitations on the rate of agonist application (Adams, 1981). Furthermore, since the response fades during prolonged contact due to receptor desensitization the rate at which agonist is applied and reaches its target should be rapid compared to the fastest likely rate of desensitization (See Section 7). When the latter phenomenon is taken into account, a Hill coefficient of 2 is obtained for the dose-response curve (Pennefather and Quastel, 1982; Trautmann, 1983). The interpretation of ionophoresis experiments on the basis of diffusion calculations has also led to the conclusion that at least two agonist molecules are needed to activate AChR channels, although (a) small deviations from a perfect square law are observed at the foot of the Carb response curve (Dionne et al., 1978) and (b) in the case of ACh itself the exponent in the power law may be greater than two (Dreyer et al., 1978). The first type of deviation may be related to the difficulty in obtaining responses with low enough Carb concentrations; alternatively, perhaps rare or brief channel openings triggered by single agonist molecules could be the cause of the observed deviations. The second anomaly may be related to the factors involved in the determination of cholinesterase activity in the diffusion calculations (Salpeter, 1987). The rapid ionophoresis method provides a means of applying high agonist concentrations without the distorting effects of desensitization. Values of half-maximum ACh concentration (K0.5) obtained by ionophoresis methods are usually in the range of 400/~M and 40 pM for Carb and ACh respectively, in general agreement with those obtained in recent experiments produced by bath application of agonist yielding dose-response curves corrected for desensitization (Trautmann, 1983). Another parameter which can be derived from compounding the half-maximum agonist concentration with estimates of channel density per unit area is the maximum fraction of AChR channels able to operate (Ymax). For a linear reaction scheme involving binding of two agonist molecules and subsequent isomerization of the liganded complex to the open-channel conformation: 2k +

R , 2k

k+

" AR

- k-

fl

" A2R -

" A2R*

(1)

the fraction of activatable receptors will be: [ A 2 R * ] / ( [ R ] + [AR]) and the ratio of activated receptors to activatable receptors is given by: Y = C ( [ A ] Z / 2 [ A ] + Kd)

(2)

where [A ] is the agonist concentration, Kd is the dissociation constant of the agonist, and C is a constant. Values of Ym,x are usually in the range of 0.5~).9. The estimates are obviously of considerable physiological relevance, since they place upper limits on the

354

F.J. BARRANTES

ability of a quantum of ACh to open AChR channels. These estimates are difficult tc confirm by more direct rapid kinetic studies in vitro, although the series of studies or BC3H-I cells provide convincing evidence of good correlation between binding ant ion-flux parameters (Sine and Taylor, 1980, 1981). Uncertainties still remain concernin~ the number of functional AChR molecules. Furthermore, fast desensitization may obscure the results. The wide range of Ym,x values obtained reflect these uncertainties, h electrophysiological experiments, however, it is possible to measure indirectly the fractim of the channels that are open. One way of doing this is to analyze fluctuations in the mea~ number of open channels (channel 'noise' analysis).

6.3. CHANNEL CLOSED--OPEN TRANSITIONSAND FLUCTUATION (NoisE) ANALYSIS

On thermodynamic grounds, the time the channel remains open is the time needed fo the open channel to accumulate sufficient energy, and subsequently overcome the energ,. barrier for the channel closing step. Individual AChR channels have different initial energ,. profiles, i.e. they follow a Boltzman's distribution. Their capacity to accumulate energy. depends on interactions with their environment. Hence, open-times reflect these variation: in energy levels and they can be considered random variables which follow a Poissol (exponential) distribution (Colquhoun and Hawkes, 1977, 1981, 1982). The mean value o this distribution is the average open-time, ~, which for the simplest open-close transitim model is the reciprocal of the rate constant, ~, for the closing transition: closed - /~ - open. :¢

(3

In the presence of a constant concentration of an agonist, each channel may be activate~ several times. In analogy with the closing step, the activation step is a random proces which depends upon random collisions of agonist molecules with AChR molecules. Whel these collisions generate sufficient energy for the agonist-AChR channel to exceed a give~ energy barrier, channels open. The independent, random activation of a large number o channels will sum up to produce an overall membrane current response, I(t), whicl fluctuates about a mean level, [. Fluctuation analysis is based on the fact that eacl successful activation of an ion channel produces a brief pulse of current of fixed amplitude i, and of duration t. The amplitude of the current pulse is the product of the channc conductance, 7, and the driving force on ion movements, V,,-V,,, where Vmis the membran potential of the cell and Vo is the null potential, at which no current flows when the io: channel opens (also called the reversal potential). The fluctuations of the current respons can be analysed to yield estimates of 7 and r. Briefly, the analysis is based on two majo assumptions: (1) channels have only two observable states, open and closed, and (2 agonist-induced transitions between closed and open states (and subsequent open-close. transitions) can be described as Poisson processes.

a2/l = i(1 - - p )

(4

where a 2 is the variance of the current fluctuations about the mean level, [, and p is th probability of a channel being open at a given agonist concentration. The so-called powe spectrum of the current fluctuations, S ( f ) , is given by:

S ( f ) = S(0)/[1 + (2~tzf)z].

(~'

S ( f ) measures the contribution to the variance, ~ 2, of membrane current fluctuations fror individual frequencies, f, of oscillation. S (0) is the zero frequency asymptote of S ( f ) , an z is the average open-time of the underlying single channel events. If a low probabilit (p << l) of channels being open is assumed, i can be calculated from Eqn 4, and y can b calculated if the driving force is known. A graph of the power spectrum can be fitted wit the curve representing Eqn 5, and the frequency F, ('corner frequency'), at whic S(f)/S(O) = 0.5, determined. At this value, z = 1/2~f [see Eqn (5)]. At this stage c

Muscle endplate cholinoreceptors

355

analysis the mean current response, I, to the agonist can be represented in terms of three parameters, 7, z and n, the average frequency of channel openings for a particular agonist concentration: [ = nzT(Vm-Vo).

(6)

In summary, the amplitude of the variance of the noise, ~2, is related to the fraction of channels that are open and the ratio of the current variance to the mean current will equal i (the single-channel current) for low enough agonist concentrations, falling to exactly half i when S ( f ) / S ( O ) = K0.5. Measurements indicated that for ACh K05 = 20/~M (Adams, 1982). One drawback of macroscopic noise measurements is that they cannot determine the limiting value at high agonist concentrations because the variance becomes very small. Probably the strongest assumption of noise analysis is the independence of channel opening (Katz and Miledi, 1972; Anderson and Stevens, 1973). Testing of this hypothesis (Neher et al., 1978) at the single-channel level led to the conclusion that the probability of channel opening follows a Poisson distribution, implying that there were no interactions between neighbouring AChRs, i.e. validating the independence assumption. Although the stationary probability of multiple channel openings does follow a Poisson distribution, Yeramian et al. (1986) have recently challenged this dogma, showing that interactions between receptors do occur, leading to an increased probability of other channel openings when one channel is already open. There is agreement between data from noise analysis of current fluctuations and that from application of other techniques. Using the property of certain channel blockers (see Sections 5.7 and 8.6) which exhibit non-competitive antagonism at rates proportional to the fraction of unblocked channels that are open by simultaneous application of agonist, dissociation constants for ACh (about 20vM) and Carb (200 ktM) could be calculated (Adams and Feltz, 1980b). Finally, the application of rapid kinetic techniques has enabled the measurement of equilibrium and kinetic flux parameters in vitro using AChR-enriched membrane fragments from Torpedo electric organ. Use is made in these cases of stopped-flow fluorescence techniques (Moore and Raftery, 1980) and rapid mixing and quenching techniques (Hess et al., 1983; Neubig and Cohen, 1980; Walker et al., 1982). The data obtained in vitro shows concurrence with values observed in vivo with electrophysiological methods, if allowance is made for membrane potential differences. In principle, membrane potential can be established in vitro by resealing vesicles in appropriate ionic media, but this parameter is seldom found in the literature. Membrane hyperpolarization in vivo shifts the dose-response curves to lower agonist concentrations (Sheridan and Lester, 1977). Conversely, the voltage sensitivity of the fraction of open channels is very much reduced when most of the channels are open (Adams and Sakmann, 1978a). 6.4. MACROSCOPICKINETIC MEASUREMENTS Though rapid enough for some purposes, bulk application of agonist is too slow to reveal delays in the opening of channels even in the case of a simple linear monoligandedactivation scheme (a simplified version of scheme l) of the type: A +R.

k~ , A R ~ k2

~ ,AR*. ot

(7)

Fortunately it is possible to apply various rapid perturbation methods to study the electrophysiological epiphenomena of AChR-ligand interactions. These methods consist of altering a pre-existing agonist-AChR complex at equilibrium, and following the relaxation back to an equilibrium state (which may or may not be the same as the original state). We have already discussed the contribution of a spontaneous perturbation method (noise analysis) although without specifically mentioning the nature of the perturbation, i.e. the spontaneous thermal perturbations or fluctuations from an equilibrium condition

356

F.J. BARRANTES

induced by the presence of the neurotransmitter (Katz and Miledi, 1972, 1973a,b; Anderson and Stevens, 1973). Quite recently this type of analysis was taken to an unprecedented level of resolution when Sigworth (1985, 1986) analyzed the 'microscopic' noise in single-channel measurements. Fluctuations on a time scale of ~ 1 ms were reported to be associated with the open-channel current levels (Sigworth, 1985). When Sigworth (1986) tested the possibility that such fluctuations were associated with the gating transitions that open and close the AChR channel, he found no evidence of such a coupling, but was able to place values to the energy barrier for the open~zlosed conformational transition, i.e. much smaller than 0.3 k T rms. This boundary implies that the fluctuations in the AChR channel transition rates are either too small or they are weakly correlated with the open-channel current fluctuations. That is, molecular motions underlying the open-channel current fluctuations are not strongly coupled to open-close conformational changes of the AChR. Of course, the interpretation of the noise in the latter case is different from that causing macroscopic noise. More typically, perturbation methods externally impose a change from the equilibrium condition. The voltage-jump method, for instance, consists of imposing step changes in membrane potential (Adams, 1975a; Neher and Sakmann, 1976b; Sheridan and Lester, 1973). Another perturbation method is the agonist-jump which exploits the displacement from equilibrium either by very brief, spontaneous ACh pulses as in the m.e.p.c.s. (Land et al., 1980, 1981, 1984) or induced by photoisomerization of light-sensitive compounds which acquire agonist activity upon irradiation (Lester et al., 1980; Lester and Nerbonne, 1982; Chabala et al., 1985, 1986). All these studies have provided evidence for a single exponential relaxation process with a single time constant. This has been widely interpreted as showing that, at least as far as AChR-channel closed-open transitions are concerned, only two kinetically distinct states are involved, as in scheme (3) above. When two agonists inducing separately short- and long-channel mean open times are applied together, the channels opened by combined activation have a short mean open time, as if they were controlled by the 'faster' original decay time (Trautmann and Feltz, 1980). Relatively much slower processes (such as desensitization, see Section 7) and very fast processes (see Section 8.2) whose existence is now clear were not apparent in the observed relaxations [data is usually filtered (0.5 1 KHz) before analysis]. In spite of these limitations, the oversimplified 'single-relaxation type' experiments have been fruitful and have provided plenty of information on the physiology and pharmacology of the cholinergic synapse. One of the earliest observations is that l/z increases with agonist concentration (Sheridan and Lester, 1977), a result which has been subsequently verified (Sakmann and Adams, 1979). The original voltage-jump studies of Sheridan and Lester (1975, 1977), which were performed on Electrophorus electroplaques, reported a roughly linear increase on agonist concentration, with a slope (for ACh or Carb) of about 107M ~ s " . It was subsequently established that the relation is no longer linear at low agonist concentrations (Sakmann and Adams, 1979) and with high concentrations of Carb the reciprocal relaxation time versus agonist concentration flattens off. The low concentration limiting time constant, often interpreted as equivalent to the mean channel open time, varies for different agonists, being typically about 2 ms for ACh (Katz and Miledi, 1972; 1973a; Auerbach et al., 1983). The slope of the linear portion also varies for different agonists: it is about 10 times steeper for ACh than for Carb. The relationship between reciprocal relaxation time and agonist concentration also shows dependence on voltage in the low agonist concentration limit. Chabala et al. (1986) have also found a strong voltagedependence of the Hill coefficient. Because of bandwidth limitations, macroscopic kinetic information has not been adequate to unambiguously assign rate constants to individual steps in a plausible kinetic scheme, still less to deduce such a scheme ab initio (Adams, 1987). Nevertheless it does enable a few predictions to be made on possible rate constants. Early work suggested that agonist binding was very rapid and that the subsequent conformational transition(s) isomerization step(s) were rate-limiting (Anderson and Stevens, 1973), such that 1/~' = 1/~t, the true lifetime of the open-channel state. At present there is growing consensus on the

Muscle endplate cholinoreceptors

357

notion that binding of ACh must occur at least at a rate of 2 x 107M -~ s -j, and that channel opening (fl) is much faster than channel closing (ct). Recent work also suggests that AChR isomerization is faster than agonist dissociation and that there is no overall rate-limiting step in AChR receptor activation (Colquhoun and Sakmann, 1981, 1983; Odgen and Colquhoun, 1983). Work on the voltage sensitivity of the macroscopic transition rates suggests that most of the voltage effects on channel mean open time are exerted via ~, the channel closing rate, but that fl is also voltage-dependent, decreasing with membrane depolarization (Chabala and Lester, 1984; 1985). 7. DESENSITIZATION 7.1. THE SIMPLEST KINETIC SCHEMES ACCOUNTING FOR THE PHENOMENON

In vivo the conductance increase induced by continuous application of agonist gradually wanes rather than remaining constant; if the agonist is washed out and then reapplied, the conductance response does not return to control unless the recovery interval is quite long. The process responsible for this reversible loss of response resulting from prior exposure to agonist is termed receptor desensitization. There is now agreement that the relatively slow transformation from low to high affinity of the AChR molecule observed in vitro represents one of the forms of desensitization revealed by electrophysiological experiments. Since the pioneer work of Katz and Thesleff (1957) it has been thought that desensitization represents a gradual conversion of receptors to a refractory state(s), whose onset kinetics depend on agonist concentration and whose recovery kinetics on the contrary are a slow unimolecular process independent of the nature and concentration of the agonist eliciting the process. Thus desensitization rates determined by changes in intrinsic (protein) fluorescence of the AChR itself differ for the agonists suberyldicholine, Carb and ACh in T. marmorata, but the final fluorescent state of the AChR is in each case the same, implying that the ultimate form is independent of the ligand (Barrantes, 1978). Desensitization may proceed from monoliganded receptors, as seen in stopped-flow fluorescence studies of Torpedo and Electrophorus (Bonnet et al., 1976) and by rapid ion-flux measurements (Dunn et al., 1980; Hess et al., 1983). Katz and Thesleff (1957) also suggested that the refractory state was one of high agonist affinity, and it has now been directly shown that affinity does indeed increase in parallel with loss of sensitivity (e.g. Heidmann et al., 1983). The simplest kinetic scheme to account for these properties is given by: A +R.

KR kl k2

.AR*.

KD = k3/k 4 k3 k4

~AD

(8)

where KR is the equilibrium dissociation constant for agonist binding to resting receptors and Ko the isomerization constant for desensitization k3/k 4. In this linear reaction scheme the resting receptor (R) first binds ACh and opens ( A R * ) (ligand binding and channel opening are compounded in a single step in this simplified scheme) and then the bound complex undergoes an isomerization step to an inactive form (AD). It follows that the ACh concentration producing half maximal binding would initially be KR, whereas after desensitization had reached equilibrium it would be K R K o / ( K o + 1). Katz and Thesleff (1957) were able to show that desensitization onset kinetics could be slower than the corresponding offset kinetics, which is incompatible with the simple linear scheme. This observation led to the postulation of the simplest two-state cyclic model: A +R. M=D/R

Kg kl

k, k7 A +D.

"AR k, k,

xo

.AD

(9)

358

F.J. BARRANTES

One essential feature of this model is the postulation that desensitized receptor (D) can exist even in the absence of agonist. This hypothesis has been verified by a variety of rapid kinetic techniques in vitro (see below). Before agonist addition the AChR is predominantly in the resting state R. The wealth of information on this subject has been obtained in vitro using Torpedo AChR membranes (Neubig and Cohen, 1979; Heidmann and Changeux 1979) and the BC3H-1 muscle clonal cell line (Sine and Taylor, 1979, 1980, 1981, 1982) In the presence of agonist the AChR is finally driven into the desensitized state D, a thermo. dynamically favoured conformation. It is important to know whether the D populatiot is large at normal endplates, because that would affect the functioning of the synapse. Bu desensitization has also been important on a practical issue: since it is slow enough an~ involves relatively high agonist affinities, desensitization was the first AChR-agonis reaction pathway to be studied by rapid kinetics in vitro (see Table 1 below). It also le~ to the postulation (Weber et al., 1975) and verification of the occurrence of agonist-induce~ conformational changes (Grfinhagen and Changeux, 1976a,b; Bonner et al., 1976 Barrantes, 1978; Heidmann and Changeux, 1979) in the AChR protein. In the next sectiol we shall review the developments which led to some correlations between ligand bindin~ and agonist-mediated state transitions of the AChR, including desensitization. 7.2. AGONIST AFFINITIESAND ACHR STATE TRANSITIONS

Agonist and (reversible) antagonist binding is competitive with the binding of e-toxim, quasi-irreversible competitive antagonists of the AChR. Given the slow kinetics of toxi~ binding, the association of these curare-mimetic toxins is in fact practically irreversibl when examined over short time intervals. Advantage has been taken of these intrinsi properties of ~-toxin and AChR ligand kinetics for the measurement of apparent kineti and equilibrium constants in competition experiments. The inhibition of the initial rate c e-toxin binding by the competing ligand has thus been extensively used as a convenier measure of occupation of the AChR binding site (Weber and Changeux, 1974). Earl binding studies failed to show, however, the expected correlations with the potencies c agonists deduced from electrophysiological studies. Equilibrium dissociation constanl determined for agonists with detergent-solubilized AChR, or after prolonged exposure c membrane-bound receptor to the ligand, indicated affinities far too high to satisfactoril account for the functioning of the cholinergic synapse. Only later did it become clear th~ solubilization of the AChR resulted in alterations in agonist affinities. The case of th anomalous agonist affinities with the AChR in a native membrane milieu was tool puzzling. Membrane-bound preparations of the AChR displayed agonist affinities suc that the calculated rate of ACh dissociation was far slower than the decay rate of endplal currents in vivo. Other physiological phenomena such as repetitive activation of individu~ channels were also incompatible with such high affinities. Weber et al. (1975) found a explanation for this puzzle: after prolonged exposure to the agonist, the experimental] determined affinities did not reflect binding to the active state but to an agonist-induce higher affinity state of the AChR. It was the presence of the agonist itself which slow increased the apparent affinity of the complex, affecting not the e-toxin binding kineti~ but the capacity of the agonist to compete with c~-toxin association (Weber et al., 197 Weiland et al., 1977). Reversal to the original state and binding affinity was also four to be a slow process. Direct measurements of [3H]acetylcholine binding by fast filtration methods ha, provided an additional means of monitoring AChR state transitions in vivo (Boyd ar Cohen, 1980a,b). The capacity to induce such AChR transitions is shared by all agonis and to a much smaller extent by some antagonists, according to some authors (Quast al., 1978; Neubig and Cohen, 1979) but not others (Weiland et al., 1977; Weiland ar Taylor, 1979) (cf. Table 1). The so-called metaphilic antagonists (Rang and Ritter, 197, are a case apart. These ligands bind with higher affinity to receptors in the desensitiz~ state and themselves are capable of inducing the low-to-high affinity state transitk (Weiland and Taylor, 1979; Weiland et al., 1977).

Muscle endplate cholinoreceptors

359

Various groups have analyzed the kinetics of the state transitions in the AChR from

Torpedo membranes (Bonner et al., 1976; Weiland et al., 1976, 1977; Quast et al., 1978; Barrantes, 1978; Heidmann and Changeux, 1979; Boyd and Cohen, 1980a,b, 1984) and in clonal muscle cells (Sine and Taylor, 1979). For the two-state cyclic scheme (9) above, the kinetic constants obtained by various procedures for Torpedo AChR are in notably close accord (Table 1). In the absence of agonist the low-affinity (or activatable) state of the receptor is favoured, i.e., M = 0.1-0.2. Thus, the D state of the receptor possesses an affinity that is about three orders of magnitude greater than the R state. Binding at equilibrium should be reflected by a smooth hyperbolic function; the overall equilibrium constant Keq reflects the dissociation constants for both states, KR and K o, and the equilibrium distribution between states, M: Keq = (l + M)/(1/KR + M/KD).

(10)

Taylor and his group (Weiland et al., 1977; Weiland and Taylor, 1979) made extensive use of a dilution method to study the kinetics of recovery from the D state. After driving the AChR into the D state by exposure to a high concentration of agonist, subsequent dilution allows the AChR to revert to the original low-affinity state. Measurement of ligand binding before the occurrence of complete reversion to the R state enabled the obtention of both KR and KD for the two detectable AChR states (Weiland et al., 1979). Early attempts to establish that AChR activation proceeds from the R state and that conversion to the D state is accompanied by impairment of AChR channel gating properties were only partly successful because of technical limitations. The rapid rate of ion influx in AChR-containing vesicles precluded quantitative correlations between the kinetics of state transitions and ion permeability due to rapid equilibration of the tracer ion. This limitation could be overcome by using clonal muscle cells, given the larger ratio of internal volume to membrane surface area of the system. This permits initial rates of Na + influx to be measured more accurately than in submicron size vesicles at low agonist concentrations (Sine and Taylor, 1979, 1980, 1982). At high agonist concentrations the rates of desensitization become limiting for they exceed the temporal resolution of the measurements. Within a wide agonist concentration range, however, correlations between ligand occupation and the kinetics of desensitization could be established. The agonist binding function at equilibrium (Y) and a state function for desensitization (D) are both expressions of the same three constants: KR, KD and M. In the absence of cooperativity the expression for both functions are given by:

AR+AD Y=AR+AD+R+D=A D:

I+M

[

I+M

]-',

(1/KR+M/Ko) ~-A

(11)

~ + A ~K~RJ_j .

Membrane-bound Torpedo AChR and BC3H-1 cells have similar ratios of KR/Ko. M is 0.1-0.2 and 1 × 10 4 in Torpedo and BC3H-I cells respectively (Sine and Taylor, 1982). K~qin BC3H-1 cells differs from KR only by a factor of 2-3 while this factor is 30 in Torpedo AChR. When M becomes very small, Keq approaches KR. As M becomes large, Keq-.-,K o. At high ligand concentrations the extent of desensitization will be inversely proportional to M. The extent of desensitization also differs between fish and BC3H-I cell AChR receptors: it appears larger in the Torpedo AChR where essentially complete antagonism of the ion flux can be achieved (Sugiyama et al., 1976). 7.3. THE NEED TO INVOKEADDITIONALREACTIONPATHWAYS Electrophysiological experiments aimed at testing the cyclic model require rapid application and removal of defined agonist concentrations at the NMJ. Early voltage-

360

F.J. BARRANTES

clamp experiments along these lines made apparent a single slow exponential onset o ' desensitization, with a rate constant that increased linearly with agonist concentratior (Adams, 1975c). Following agonist removal it was observed that recovery proceeded ii two phases, in the second and minute time ranges respectively. More recent wort (Trautmann, 1983; Feltz and Trautman, 1982; Chestnut, 1983) has shown that two phase are also present in the onset, the fast phase having been overlooked in earlier worl (Scubon-Mulieri and Parsons, 1978). The slow rate constant shows an approximatel,. linear concentration dependence. However, this relation breaks down at very lov concentrations, since the recovery rate (i.e. zero agonist concentration) is comparable t~ the onset rate. This has also been found to be the case for rapid kinetic studies in vitr. (Neubig et al., 1982; Sine and Taylor, 1979, 1980). In the cyclic kinetic scheme [Eqn (91 there are two recovery pathways: D --*R and A D ~ A R , so that the observed overall rat, constant is a weighted sum of the individual rates, the weights depending on the agonis concentration. Similarly, the onset rate for desensitization does not uniquely determine th extent of desensitization achieved at equilibrium. This prediction is also fulfilled by th available experimental data. Though the simplest cyclic model (scheme 9) can account for most of the electrophys iological and biochemical observations, there exist additional rapid components of onse and recovery of desensitization which have remained largely unexplained. Early obserw tions of Magleby and Pallota (1981) reported the development of refractoriness to AC at the NMJ in the time-course of a few milliseconds. But the fast time constant fc desensitization found in most electrophysiological experiments falls typically in the rang of 2 to 20 s and it decreases as the agonist concentration is raised. Similarly, the tim constant for dissociation of ACh from the slowly desensitized AChR state measured i v i t r o (Table 1) is also in the range of several seconds. However, rapid dissociation ( agonist from a still desensitized receptor would not be expected to lead to appreciab] recovery of electrophysiological responsiveness. Two-step desensitization kinetics chara~ terized by rates in the orders of milliseconds and seconds respectively have been measure in reconstituted T. c a l i f o r n i c a AChR (Walker et al., 1982) an observation which has le to consideration of the existence of an additional desensitization process. In addition, other experiments have indicated that the relative weight of the fast recovm phase of desensitization depends on the length of time for which the agonist has bee acting. If the agonist was present for a long time, the fast component of recovery is vel small, most likely because a large proportion of the AChRs are driven into the slow desensitizing form D. Unlike the slow process, fast desensitization may proceed via bo, mono- and biliganded AChRs (Pennefather and Quastel, 1982; Hess et al., 1982). As tt agonist concentration is further increased more receptors are driven into the rapid desensitized state, suggesting that the slowly desensitizing state is favoured by monolig~ tion, whereas rapid desensitization is favoured by biligation. Calcium appears to increa: the amplitude of the fast phase of AChR desensitization (Oswald, 1983b). Consideratic of these features leads to a more complicated kinetic scheme: Rapid desensitization pathway: A + D ' , - -

....

AD'-

.. A2 D '

I

L

Activation pathway:

A +R

-

~R.2 " A R

-

2KR

" A vR

Slow desensitization pathway:

A +D

-

~D.2 " A D

-

2~0

.. A2 D

(1

D " is the rapidly desensitizing state of the receptor. The number of open channels assumed proportional to A 2 R , which may be a compound state including the t r open-channel state A 2 R * . In this scheme the slow recovery rate is independent of the natu

k~ ks M = k~/k7 = D / R KR Ko k3 k4 K~q

0.37s i 0.01 s - '

6 . 6 x 10 6M

Carbamoylcholine (Bonner et al., 1976)

O.O004s i 0.004s-' 0.I 2 × 1 0 5M 5× 108M 0.012s-I 0.0003 s i 5 × 10 v M

O.O08s 0.017s 0.47 1.0× 10 6 × 10 1.5s -I 0.006 s ~

6M 9M

~ '

Suberyldicholine (Barrantes, 1978) O.O019s -l 0.004s t 0.48 4 × 1 0 SM 1.2× I0 7M 0.14s i 0.006 s - i 1.7 × 10 -6 M

Carbamoylcholine (Quast et al., 1978)

0.0005s I 0.0023s ~ 0.22 3×10-5M 2 . 5 × I0 - s M 0.15s-1 0.00054 s - I 1.2 × 10 -7 M

Carbamoylcholine (Boyd and Cohen, 1980b)

TABLE I. Equilibrium and Rate Constants Fitted According to Cyclic Scheme (Eqn 9)

Carbamoylcholine (Weiland et al., 1977)

O'O005s I 0.0023s i 0.22 5× 10-7M 1 . 4 x 10 - g M 0.06s i 0.0008 s i 8 × 10 9 M

Acetylcholine (Boyd and Cohen, 1980b)

0.16s t 0.027 s - J

4.1 x 1 0 - 6 M

Acetylcholine (Bonner et al., 1976)

O

O

.q-

362

F.J. BARRANTES

of the agonist whereas the fast rate depends on agonist concentration. Apparently, the two agonist molecules must dissociate from the AChR in order for recovery from desensitization to occur. Differences were apparent when analyzing the proportions of resting and desensitized AChR (M) in the absence of agonist that are characteristically found in Torpedo and BC3H-i cell preparations respectively. Another difference between the two is the extent of cooperativity found in the concentration-dependence for agonist occupation. Both the muscle cells and the isolated Torpedo vesicles show Hill coefficients for activation between 1.5 and 2.0 (Cash et al., 1980; Neubig and Cohen, 1979, 1980; Sine and Taylor, 1979, 1980). However, the Hill coefficients for Torpedo AChR occupation are close to 1.0 (Weiland et al., 1977; Quast et al., 1978; Boyd and Cohen, 1980a,b). In the BC3H-1 cells the Hill coefficient varies between 1.3 and 1.7, reflecting positive cooperativity for AChR occupation (Sine and Taylor, 1979, 1980). However, reports on negative cooperativity have also appeared (Chagala et al., 1986). The positive cooperativity model involving ligand biligation yields an agonist binding function F'~oop and the state function for desensitization Dcoop: F~oop- (A/KR)(I + A/KR) + M ( A / K o ) ( 1 + A/Ko) (1 + A/KR)Z + M(1 + A/Ko) '

(14)

O oo _-[l

05/

-I

'

Differences in the extent of cooperativity for occupation and activation require consideration of at least three states of the AChR: open channel, closed channel (activatable) and desensitized. The cooperatively for AChR occupation at equilibrium depends on the value of M; the Hill coefficient will approach the theoretical limit of the number ofsubunits wher M is of similar magnitude to Ko/KR. As M approaches unity, as it does with the Torpe& receptor, the apparent cooperativity for occupation decreases. In early patch-clamp work Sakmann et al. (1980) found that using high enoug~ desensitizing ACh concentrations, AChR channel opening became non-random; opening: occurred in bursts of activity, separated by silent periods lasting hundreds of milliseconds In an even slower time scale, bursts themselves were seen to be superarranged in clusters separated by silent periods lasting many seconds. This behaviour resembled at leas qualitatively the pattern one would expect if channels were returning from long (clusters or short (bursts) desensitized states. Long interburst and intercluster intervals point to th existence of additional closed states, although not necessarily corresponding to desensitize~ states. The lifetimes of these closed states differ so radically from the two recover processes unveiled by macroscopic recordings that it is likely that bursting does not aris from conventional desensitization. The apparent closed times in patch-clamp recording correspond to the true closed times divided by the number of active channels in the patc~ a fact which precludes direct comparison. But the actual observation of a cluster of bursl signifies that even though many channels may be present in the patch only one chanm is active, and therefore the observed interburst interval of about 0.2 s can be ascribed t the true channel closed time. The intercluster interval can be interpreted either as th long-lived desensitized state (several minutes) divided by the number of channels in tl~ patch or as the shortqived desensitized state (Adams, 1987). The reader is referred t Section 8 for discussion of other channel silent events which need to be distinguished fro1 desensitization in patch-clamp experiments. 7.4. MODULATIONOF ACHR DESENSITIZATION A further look at the cyclic schemes (9) and (13) above raises another physiological relevant question: if the R and D states pre-exist in equilibrium before receptors a exposed to agonist, which factors govern the amount of AChR in each state? The rap kinetic techniques in vitro and other biochemical studies cited above have shed light c

Muscle endplate cholinoreceptors

363

this matter. In general, AChR-rich vesicles from Torpedo, which undergo a rather lengthy isolation procedure, are found to contain an overwhelming population of their AChRs in the R state, that is, M <1 in scheme (9) (Neubig and Cohen, 1979; Heidmann and Changeux, 1979). AChR phosphorylation, previously suggested to account for the differences between synaptic and extrasynaptic AChRs (Saitoh and Changeux, 1981), has now been found to affect the rate of desensitization (Eusebi et al., 1985; Huganir et al., 1986; Albuquerque et al., 1986; Middleton et al., 1986). Phosphorylation of the AChR is catalyzed by at least three different protein kinases: a cAMP-dependent kinase, protein kinase C and a tyrosine-specific protein kinase (Huganir and Greengard, 1983; Huganir et al., 1984; Zavoico et al., 1984; Eriksonn et al., 1986). Greengard's group has characterized some of the sites on which these kinases act. The cAMP-dependent kinase catalyzes the phosphorylation of the serine 354 residue of the y-subunit and the serine 361 on the a-chain. These covalent modifications are without effect on the ion fluxes induced by ACh, but they increase 7-8-fold the rate of the rapid desensitization process (Huganir et al., 1986). In order to demonstrate this, phosphorylation of the membranebound AChR by an exogenous cAMP-dependent kinase was performed in vitro, the AChR purified, and its effect on desensitization assessed upon reconstitution in lipid vesicles. When either of (or both) the a- or 7-subunits were phosphorylated, the rate of the rapid phase of desensitization (see schemes 14 and 15 above) augmented 7-8-fold (Huganir et al., 1986). The in vitro studies have been substantiated by the simultaneous demonstration in vivo that forskolin, a drug which activates the endogenous cAMP-dependent protein kinase, also increases the rate of desensitization in developing rat myotubes, presumably also affecting the rapid desensitization process (Middleton et al., 1986). Albuquerque et al. (1986) also observed that the degree of desensitization at the frog or rat NMJ increased upon forskolin treatment, using either electrical stimulation or rapid ionophoretic application of ACh in situ. Forskolin does not appear to increase the rate of desensitization of neuronal AChR (Akagi and Kudo, 1985; McHugh and McGee, 1986). Given its lipophilic nature, and the fact that the effect of forskolin does not appear to be mediated by activation of adenylate cyclase, it is probable that forskolin acts like a general anaesthetic (McHugh and McGee, 1986), in a manner reminiscent of the action of volatile anaesthetics (Young et al., 1978). Drugs which activate protein kinase C have also been reported to increase the rate of desensitization in developing myotubes in culture (Eusebi et al., 1985). In the case of this kinase, it has been proposed that phosphorylation occurs on the a- and s-chains within a region of 16 amino acids common to that where the cAMP-dependent kinase acts (Huganir et al., 1984), but biochemical evidence for the existence of an endogenous C-type kinase, as demonstrated for the cAMP-dependent kinase (Eriksonn et al., 1986), is so far lacking. A tetradecapeptide comprising this region was recently synthesized (Souroujon et al., 1986). Antibodies raised against the peptide bound to the a-subunit and inhibited its phosphorylation by the cAMP-dependent kinase. Finally, a tyrosine-specific kinase phosphorylates tyrosines on the fl-, 7- and a-chains (Huganir et al., 1984). Most local (Bonner et al., 1976; Weiland et al., 1976, 1977) and volatile (Young et al., 1978; Young and Sigman, 1981; Young et al., 1981) anaesthetics and some competitive antagonists (Quast et al., 1978) have been shown to enhance agonist-induced rates of desensitization in Torpedo AChR. However, competitive antagonists are not expected to produce such an effect (Rang and Ritter, 1970) and in fact Covarrubias et al. (1984) did not observe acceleration of desensitization rates by competitive antagonists. The cholinergic antagonists affecting desensitization were termed metaphilic by Rang and Ritter (1970). Similarly, tetracaine was found not to accelerate but to decrease agonist-induced rates of desensitization in vitro (Blanchard et al., 1979). Desensitization onset kinetics become faster upon increasing membrane potential (Magazanik and Vyskocil, 1976) and Ca 2+ concentration (Fieckers et al., 1980). In the presence of acetylcholinesterase inhibitors (i.e. longer residence time of ACh in the synaptic cleft) desensitization is established upon repetitive nerve stimulation (Magleby and Pallota, 1981). J P T 38 3--G

364

F.J. BARRANTES 8. T H E F I N E S T R U C T U R E

OF AChR CHANNEL

FUNCTION

8.1. GENERAL PROPERTIES OF THE A C H R CHANNEL Since its application to the first recordings of ionic currents t h r o u g h individual biological ( A C h R ) channels (Neher a n d S a k m a n n , 1976a) the so-called p a t c h - c l a m p technique has revolutionized electrophysiology. Single-channel recordings from a small (a few square microns) patch of cell m e m b r a n e c o n t a i n i n g A C h R s permit the o b t e n t i o n of basic knowledge which was hitherto inferred or in some instances assumed. The technique allows direct o b s e r v a t i o n of the r e c t a n g u l a r - s h a p e d single-channel c u r r e n t pulses a n d the different kinetics of c h a n n e l open-state i n d u c e d by different agonists, as well as the testing ot predictions c o n c e r n i n g specific reaction schemes of c h a n n e l f u n c t i o n (Fig. 6). The A C h R ion c h a n n e l is b r o a d l y selective for cations a n d virtually i m p e r m e a b l e tc anions. The selectivity for cations at the N M J is rather weak a n d follows the order Cs > R b > K > N a > Li for the m o n o v a l e n t s a n d M g > Ca > Ba > Sr for the divalent

FIG. 6. An unstained skeletal muscle fibre enzymatically isolated from the interosseal muscle of Rana pipiens. The fibre was photographed with Nomarski interference contrast optics and is approximately 50 pm wide and 1,I cm long. The darker area in the lower middle portion of the muscle fibre is the apparent endplate region. The waveforms on either side of the figure represent unitary currents flowing through ion channels recorded using the patch-clamp technique: using ACh (right-hand side; the first current trace in the upper right has an amplitude of 2.4 pA and a mean open time of 10.2 ms at a transmembrane potential of -80 mV) and batrachotoxin (left side). The latter are inwardly flowingcurrents through a sodium channel (the current in the bottom trace has an amplitude of 1.9 pA and a mean open time of 30 ms at a transmembrane potential of -90mV). Courtesy of Drs. C. N. Allen, A. Akaike and E. X. Albuquerque, Department of Pharmacology and Experimental Therapeutics, University of Maryland.

Muscle endplate cholinoreceptors

365

cations (Adams et al., 1980; Dwyer et al., 1980); K? permeates only slightly better than Na ÷. H ÷ can block the AChR-operated channel (Huang et al., 1978; Goldberg and Lass, 1983). Other cations can also block the AChR channel (Watanabe and Narahashi, 1979; Farley et al., 1981). It is generally accepted that agonists open AChR channels in an all-or-none fashion, developing 'standard', fixed conductances (at a given membrane potential) of about 15-30pS. Spivak et al. (1983) found that conductances may vary among different compounds when semi-rigid agonists are used. Such conductances reflect the passage of about 104 ions/ms, although high-resolution single-channel recordings allow the observation of very brief ACh-induced openings whose corresponding movement of charges is of the order of only 300 (Sigworth, 1985). It is also generally accepted that more than one ACh molecule is needed to accomplish channel opening at the NMJ (see above). Once opened, the channel remains in this state for a few milliseconds, the length of the burst depending on the nature of the agonist (Neher and Sakmann, 1976a), pH (Landau et al., 1981), temperature (Katz and Miledi, 1972; Hoffmann and Dionne, 1983) and membrane potential (Magleby and Stevens, 1972). Voltage alters the shape of the energy barriers in the AChR channel, thus affecting the mean dwell-time of permeant ions within its lumen. This in turn affects mean channel lifetimes, which are approximately 0.8 ms for 3-phenylpropyltrimethylammonium, 3.2 for ACh and 5.6 ms for suberyldicholine (Colquhoun et al., 1975). These average values probably reflect differences in channel closing kinetics (Jackson et al., 1982), though many other thermodynamic reasons could be invoked. Seasonal changes can affect single-channel conductances by 35% without affecting channel lifetime (Lewis, 1984). Simultaneous application of two different agonists that would separately produce distinct short and longish mean open times results in cross-potentiation: when a 'faster' agonist (i.e. one inducing short open time, like Carb) and a 'slower' agonist (inducing long open times, like suberyldicholine) are applied together, the channels opened by the combined activation have a short mean open time (Trautmann and Feltz, 1980). As we have seen in previous sections, junctional channels have a characteristically shorter mean open time and larger conductance than extra-junctional AChR channels (Katz and Miledi, 1972; Neher and Sakmann, 1976a,b), and these characteristics vary along ontogeny. Although curare is classically considered a competitive inhibitor of cholinergic transmission, causing a parallel shift of the ACh dose-response curve (Jenkinson, 1960) and a block of open channels in neurons (Marty et al., 1976) and NMJs (Colquhoun et al., 1979; Colquhoun and Sheridan, 1982) (see also Section 8.6), there is now compelling evidence that tubocurarine also exhibits agonist-like activity. Thus, the early report that curare depolarized embryonic rat muscle (Ziskind and Dennis, 1978) has been confirmed at the patch-clamp level (Trautmann, 1982; Morris et al., 1982, 19832; Jackson et al., 1982; Takeda and Trautmann, 1984). The latter authors observed two conductance levels with curare: a full level of ~40 pS (the same as that elicited by ACh) and a partial conductance level of about ! 3 pS. Two hypotheses were entertained to explain the partial conductance: (a) partial channel blockage and (b) a partially open AChR channel conformation. 8.2. BRIEF OPENINGS

When agonists are compared, the potencies and mean open times are ranked in the same order, but the range of potencies is much larger (1-200) than that of the mean open time (1-4) (Trautmann, 1983). If the definition of a normal burst given above is accepted (one apparent opening of the channel being a burst of oscillations between the open and closed states), then the differences in mean open times among agonists could be due to the differences in the dissociation of the agonists. Brief openings of AChR channels were first reported by Colquhoun and Sakmann (1981) and have since been described by several others (Dionne and Leibowitz, 1982; Sine and Steinbach, 1984b; Jackson et al., 1983; Takeda and Trautmann, 1984; Labarca et al., 1984). Colquhoun and Sakmann (1981) suggested that they might reflect opening of

366

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monoliganded receptors, but attempts to test this idea by examining the agonist concentration-dependence of the proportion of brief and 'normal' openings has provided contradictory data (Sine and Steinbach, 1984a,b; Takeda and Trautmann, 1984; Labarca et al., 1985). It has not yet been elucidated whether the brief opening constitutes ar abnormal situation, present only in cultured cells or after profound desensitization. 8.3. SPONTANEOUSOPENINGS It has been suggested that spontaneous openings occur in the absence of agonist (Brehn et al., 1984; Jackson, 1984, 1985, 1986) with frequencies of about 2 x 10-3 s -~ per AChF

(Jackson, 1986). Spontaneous openings also show brief and normal open times, suggestin~ that the state of ligation of the receptor may not be the only controlling factor. The rat~ of spontaneous openings increase as the membrane is hyperpolarized. The presence of thq agonist increases the rate of opening by a factor of about 10 7 and the mean open time b: a factor of 5 (Jackson, 1986). Singly-bound tethered agonists open both long and shor duration channels (Chabala et al., 1985). 8.4. FLICKERINGOR MULTIPLEGATINGPHENOMENON Before the seal between a patch pipette and the plasmalemma could be made to reacl resistance in the order of gigaohms (i.e. the 'pre-gigaohm era'), 'flickering' of the channe passed inadverted. Nelson and Sachs (1979) observed short-duration closed states whic'~ broke a long-duration event into a burst of repeated openings in embryonic muscle cell in culture. These short interruptions within a burst of openings are now described as th multiple gating phenomenon. With the advent of gigaohm seals, the improved resolutio enabled the characterization of the short closures (Colquhoun and Sakmann, 1981). Thes interruptions or gaps (flickers) within the apparent open state, in fact separate openin events, and hence the openings immediately following a gap were qualified as 'Nachschla~ [backlash, see Barrantes (1983) for a discussion of the coining of these words]. In recer years the Nachschlag or mutliple gating phenomenon has been assimilated to the flickerin itself. As stressed in a previous review of the subject (Barrantes, 1983) the observation c the gaps forced a redefinition of a channel opening event: what was earlier called a channq opening is now more correctly described as a normal ('N') burst o f openings. Burst is a ten coined by Nelson and Sachs (1979); Colquhoun and Hawkes (1977) had predicted, o theoretical grounds, the occurrence of such events. In the pioneer work of Nelson an Sachs (1979) suberyldicholine was used as agonist, and the possibility was raised that tt agonist itself could block the channel, a phenomenon previously observed with loc~ anaesthetics (see below). Some local anaesthetics induce short-duration closed tim~ interpreted as intervals during which the channel is occluded. Colquhoun and Sakman (1981) subsequently showed the agonist concentration-independence of these closure thereby ruling out channel blockage by the agonist itself; in their interpretation, tt gaps result from multiple closed-open-closed transitions during single-receptor si occupancy. The average number of gaps is about 3/burst in frog muscle (Colquhoun and Sakman 1981) and 0.3/burst in snake muscle (Dionne and Leibowitz, 1982). The number of gal per burst is apparently not affected by membrane potential, but gap duration is decreas~ by hyperpolarization (Leibowitz and Dionne, 1984). Given the possibility that the ga] represent brief returns to the closed A R state, simple schemes like (7) above predict th the number of gaps per opening will be fl/k2 and the mean flicker duration will be k2. Sin both those quantities can be measured, the relevant rate constants k2 and fl can 1 obtained. Short gaps are both much faster (40-70 ps) (Colquhoun and Sakmann, 198 and more numerous than the long gaps of about 200/as (Dionne and Leibowitz, 198] Assuming the former represent returns to A R will give much larger estimates of fl and than assuming the latter. Colquhoun and Sakmann (1981), analyzing short gap duratio obtained values of fl larger than 20 ms. Sine and Steinbach (1984b) have reported t

Muscle endplate cholinoreceptors

367

occurrence of long gaps with durations closer to l ms. Auerbach and Sachs (1984) described three types of gaps: (a) those with zero current, (b) those with non-zero current (a substate) and (c) those too short to have amplitudes that could be determined. 8.5. MULTIPLE CONDUCTANCE STATES(THE EXISTENCEOF SUBSTATES)

The transitions to a lower conductance ( ~ 10 pS) state from the main conductance level which the open channel displays in developing muscle cells (Hamill and Sakmann, 1981) have been discussed in Section 3.5 in relation to the ontogenesis of AChRs. Within the framework of this section, it should only be mentioned that one possible explanation for these transitions is that they arise from unbinding of an agonist molecule from the AChR. The other hypotheses have been formulated in connection with the partial conductance level (~ 13 pS) observed with curare (Takeda and Trautmann, 1984). Subconductance levels elicited with ACh exhibit reversal potential and selectivity indistinguishable from those of the main conductance level. Hamill and Sakmann (1981) pointed out that transitions between the two levels were not in equilibrium; subconductance episodes occurred after but not before normal bursts. Trautmann (1982), on the other hand, found the two levels to be in equilibrium. For Auerbach and Sachs (1984) the subconductance state is an ensemble of different states, with a conductance ~ 10% that of the main state and whose lifetime is dependent on the nature of the agonist, and the main state and subconductance state are close to equilibrium. 8.6. CHANNELBLOCKING

In addition to the desensitization phenomenon, other mechanisms can lead to decreased AChR channel conductance without affecting agonist binding. A particularly important one is channel blockage. A great variety of molecules reduce ACh response by such mechanisms, including local anaesthetics, full and partial agonists. Sine and Steinbach (1984a), for instance, have found that high concentrations of ACh (>0.3 mM) or Curb (> 1 mM) produce a voltage- and concentration-dependent reduction in the mean singlechannel current in clonal BC3H-1 cells. Suberyldicholine (above 3/a M) produces a voltageand concentration-dependent increase in the number of brief duration low-conductance interruptions (substates, see previous section). These observations were interpreted as arsing from channel occlusion by the three full agonists used. In vitro 86Rb+ flux measurements in Torpedo vesicles have established the agonist concentrations at which the maximal ligand-induced fluxes are self-inhibited: they are 110, 211, 3.0, 39 and 8.9 mM for ACh, Curb, suberyldicholine, phenyltrimethylammonium and (-)nicotine respectively (Forman et al., 1987). A simple kinetic scheme has been postulated to explain the action of channel blockers since Adams (1976a, 1977) developed an explanation for barbiturate and procaine channel blockage at the NMJ: A +R.

~ "AR*+B.

r

a

b

"AR*B.

(16)

In this model blockers seem to bind selectively to the open form of the AChR channel at a ratef. The blocked channel can then only unblock (at a rate b) by dissociation of the blocker, being unable to close until it has lost the blocking molecule. The simple model provides an explanation for the biexponential kinetics displayed in the presence of blocker either in macroscopic (Adams and Feltz, 1977; Colquhoun and Sheridan, 1981) or single-channel recording (Neher and Steinbach, 1978). When the two relaxations are well separated in time, they become: 1/zf= ~ + fl + f [ B ] + b, 1/z s =

[B]flf + ~b + fib

+ fl + f [ B ] + b"

(17) (18)

368

F.J. BARRANTES

The expected dependence of time constants on blocker concentration is predicted and usually found. The model also specifically postulates that the block occurs within the channel, either physically or electrostatically occluding it. The gate which controls the channel opening could also control access to the blocker binding site, which may not be necessarily on the conduction pathway. One possibility is that such site resides in the channel vestibule, its occupancy lowering the probability of ions entering the channel Alternatively, in a view akin to allosteric interactions, the blocker binding (heterotopicl site could be located anywhere on the AChR protein, even distant form the channel lumen and exert nonetheless its effect by stabilizing a non-conducting form of the AChR (see Section 5.5). There are, however, major pieces of evidence favouring the simplest versior of the model, i.e. physical occlusion of the channel lumen itself, for most of thc channel-blocking agents. The voltage-dependence of equilibrium binding of many blockers as is observed wit~ some local anaesthetics has led to the suggestion that the binding site(s) are located at leas~ halfway through the membrane. Both the forward ( f ) and backward (b) blocking rate~ show voltage-dependence (Neher and Steinbach, 1978). Of course it could be argued, a~ it often is, that the voltage-dependence does not reflect movement of the blocker througt the imposed field, but instead a voltage-sensitive conformational change of the AChl~ protein. Barbiturates block AChR channels as uncharged species and show no marke~ voltage dependence (Adams, 1976a). Tertiary and quaternary amine non-competitive blockers appear to interact with thq AChR from the extracellular side of the membrane (Aracava et al., 1984; Aracava an~ Albuquerque, 1984). It is the positively charged form of a tertiary amine local anesthetic the one which binds with high affinity to reconstituted AChR (Earnest et al., 1984 Blickenstaff and Wang, 1985). Patch-clamp studies have also shown that non-competitiw blockers do not affect single-channel conductance [but see the results of Ruff (1982) oJ QX-222 blockage] but decrease channel lifetime; this effect is generally voltage-dependen (Koblin and Lester, 1979; Albuquerque et al., 1980; Lambert et al., 1983), althougl voltage-independent blockage has also been observed (Ribera et al., 1985). The observa tion that the binding of some NCBs to site(s) on the AChR is dependent upon the applie~ voltage has led to their further classification as 'open channel blockers'. Procain, (Adams, 1976a), quaternary local anaesthetics (Neher and Steinbach, 1978) and gallamin (Colquhoun and Sheridan, 1981) among others fall into this category. In addition to th more 'classical' local anaesthetic substances like lidocaine (Neher and Steinbach, 19781 other chemicals which promise to be useful probes to identify the NCB site(s), lik chlorpromazine and phencyclidine (see Section 5.7), have been tested in patch-clam] experiments (Changeux et al., 1986). Depending on the affinity of the blocking agent for its site, two possible mechanism of action differing in their kinetics have been described. Fast channel blocking is exhibite, by low-affinity compounds at high concentrations. This is reflected by the flickerin phenomenon in single-channel recordings. The interpretation is that flickering results i this case from binding/dissociation of the blocker at rates which are fast relative to th channel closing rate. S l o w channel blocking is observed on the other hand wit high-affinity blockers which effectively reduce the mean duration of the bursts: dissociatio of the blocker relative to channel closing kinetics is too slow to manifest itself in flickering This is the case, for instance, with chlorpromazine and phencyclidine (Changeux et al 1986). These two positively charged compounds, in the concentration range of 10-200 mlV led to shortening of mean burst times in C2 myotubes. Many blockers exhibit similarity to permeant ions, and in the case of decamethoniun which acts as an AChR channel blocker, its permeation has been confirmed (Creese an England, 1970). Within a series of homologous bisquaternary ammonium drugs dec~ methonium behaves as an open channel blocker in the presence of ACh. Shorter chai compounds like hexamethonium produce a long-lasting blockage that is in fact relieve~ and not enhanced, by applying ACh to open channels at slightly positive membrar potentials (which pushes the molecule out of the channel). This has been interpreted

Muscle endplate cholinoreceptors

369

a closing of the channel with the small blocker still inside (trapping) by shutting off the gate. The studies of Neely and Lingle (1986) on amphibian NMJ current block by chlorisondamine (a ganglionic blocker) suggest that channel unblocking can only be produced in these cases by subsequent agonist addition. Chlorisondamine would act not only as an open-channel blocker but would also drive to a stable blocked state. One still unexplained aspect is that if the gate was located on the extracellular portion of the channel (the vestibule, for instance) the blocker would be prevented from leaving the channel for the extracellular space but not from entering into the cell. That is, relief from block should eventually occur through this route. A one-gate model does not rationalize this apparent discrepancy (Adams, 1987). In the case of compounds with fast unblocking rates, the simple channel block model anticipates a linear increase in the duration of the single-channel bursts as the concentration of the blocker is raised. Patch-clamp measurements of AChR channels in the presence of QX-222 (Neher and Steinbach, 1978; Neher, 1983), pempidine (Lingle, 1983) or mecamylamine (Lingle, 1983; Varanda et al., 1985) showed that the bursts of openings were shorter than would be predicted by the simple channel block model. Trapping is one possible reading of this deviation. Macroscopic electrophysiological measurements have also made apparent another type of anomaly. By definition an open channel blocker should produce little reduction in the peak conductance of the e.p.c, or m.e.p.c, because very little block can occur during the rising phase. However, in several cases where the forward blocking rate is known, calculations show that the m.e.p.c, peak is much more reduced than anticipated. Furthermore, anomalies have been observed in early relaxation experiments (Adams, 1977) which can also be accounted for in terms of blocking of the closed channel, presumably at a site identical with or close to the open channel site (Adams, 1977; Tiedt et al., 1979; Masukawa and Albuquerque, 1978). This type of blocking differs from the (previous) closed block mechanism by trapping: it occurs before channel opening. The two types of block can be described by addition of an arm to the simpler linear scheme (16) of channel blockage (Adams, 1981): - 0

C b

f'[B]

fl. BC-

(19)

b' I f'[B]. Ct"

,

"-

OB

In the case of the simple (closed) channel-blocking mechanism fl and ~t are negligible but f i s appreciable. In the trapping mechanism ~ is relatively large but b and f a r e insignificant. Thus, two closed-blocked states could exist: one in which the blocker enters the channel but cannot get beyond the closed gate, and one in which the blocker is trapped behind the closed gate (Adams, 1987). This further expands to cyclic scheme (19) above to include an additional state BC': BC'.

"

C -

BC.

-

0

l

(20)

. OB

9. THE ROLE OF INDIVIDUAL AChR SUBUNITS ASSIGNED THROUGH GENE MUTATION HYBRIDIZATION AND SINGLE-CHANNEL RECORDING A few years ago cloning of an integral membrane protein with an apparent molecular weight of more than a quarter of a million Dalton presented a formidable goal. As Anderson (1987) points out, this was due to the multi-subunit nature of the AChR, each

370

F.J. BARRANTES

subunit being encoded in by separate mRNAs (Mendez et al., 1980; Anderson and Blobel, 1981), a fact which precluded the possibility of employing DNA-mediated gene transfer for cloning (Pellicer et al., 1980), as had been successfully applied to other proteins. Cloning of the AChR thus relied on the abundance of the mRNAs coding for the four chains (~0.5% each), the limited NH-terminus sequence data available at that time (Raftery et al., 1980) and the existence of subunit-specific antibodies. The ability to unravel the specific contribution of AChR subunits and regions thereof to receptor gating properties is now at hand. The availability of cloned DNA has prompted the adoption of molecular genetic techniques, expression of the messages in heterologous cellular systems and assay of the AChR functionality upon insertion of the protein in the plasmalemma of the cell. The amphibian (Xenopus) oocyte has been the heterologous system of choice, given its enormous biosynthetic capacity, and most recently the availability of a technique for stripping away the vitelline membrane and thus enabling the application of singlechannel recording to the oocyte (Methfessel et al., 1986). The recently introduced yeast system (Fujita et al., 1986) is a promising avenue, given the wealth of information available on the molecular genetics of this organism. So far the combination of the above techniques has rapidly produced significant advances, either by using hybrid AChR mRNAs from different species or by the use of mutants generated by site-directed, point mutagenesis. Mishina et al. (1984, 1985) and White et al. (1985) have undertaken site-directed mutagenesis experiments and tested different combinations of normal and modified cloned AChR subunit DNAs to elicit permeability responses. Deletions and replacements ot critical amino acids were performed by Mishina et al. (1985) and assayed with conventional electrophysiological techniques. Altered ~-subunit and normal/~-, 7- and 6-chains elicited abnormal responses to ACh, a result which stresses the importance of the ~-chains in channel gating phenomena. White et al. (1985), using the hybridization approach. synthesized mRNAs coding for Torpedo ~-,/~- and 7-subunits, combined these with mouse 6 subunit and injected them into Xenopus oocytes. The hybrid produced larger response~ than the normal message, i.e. higher ACh binding, larger single-channel conductance. smaller mean channel closed times and slower rates of desensitization were observed witl~ the hybrid than with the native species. Sakmann et al. (1985) have further characterized the nature of the supra-normal responses by using single-channel recording of normal and site-mutated cDNA and hybrids of calf and Torpedo subunits. When injected in Xenopus oocytes normal Torpe& AChR exhibited average currents of about 23 nA whereas calf AChR had currents oJ about 1600 nA. Such a huge difference in macroscopic currents could have been due tc the ability of calf AChR to let more ions pass through its channel, to a higher probabilit3 of maintaining the channel open for a given agonist concentration or to the ability ol Xenopus oocytes to express more calf than Torpedo AChR. The latter possibility was rulec out since ~-bungarotoxin binding capacity was the same in both cases. Single-channe measurements showed that the basic difference between AChR channels of the two specie,, resided in the average duration of the elementary currents. That flowing through Torped~ receptors was much shorter (~0.6ms) than that of calf AChR channels ( ~ 8 m s ) conductance of both channels being about equal (~ 40 pS). In spite of these differences th~ two native receptors were similar in ion selectivity and transport properties. Substitutior of the calf 6-subunit in the Torpedo AChR altered its gating behaviour, making it simila~ to the calf AChR, whereas substitution of the calf ~-subunit was less effective in alterin~ the gating of the Torpedo channel. Thus, the mean current duration of the ~-hybrk (~-chain substitution) is between those of the Torpedo and calf native forms, while th~ 6-substituted hybrids are more similar in duration to the calf wild form. Tentatively, th~ 6-subunit has been postulated to govern the rate of channel closing. The time for whic[ an AChR channel remains open is apparently determined by inherent characteristics of th~ c~- and ~-subunits. Calf muscle DNA codes for an AChR subunit, ~ (Takai et al., 1985), which is hardl,. detected at early fetal stages and progressively increases towards adulthood (Mishina e

Muscle endplate cholinoreceptors

371

al., 1986). Calf E-subunit shows higher sequence homology with the 7-subunit than with any other AChR chain and, as discussed above, can replace the Torpedo ,/-subunit to express functional AChR channels when combined with the a, fl and ~ Torpedo subunits (Mishina et al., 1985). Most recently, Mishina et al. (1986) have found that Xenopus oocytes injected with mRNAs of a-, fl-, 7-, 6- and E-subunits of calf AChR display two different types of channel behaviour. If either the 7 or the E mRNAs is omitted, only one type of channel results; single-channel properties of fetal calf muscle AChR are observed with the a-fl-V-6 combination (AChR.¢), whereas the a-fl-6-E (AChR~) messages apparently code for adult-type channel. Thus the developmental variations in the relative proportions of the 7- and E-chains (Mishina et al., 1986) support the contention that the molecular basis for the changes involved in the replacement of embryonic type for adult-type of AChRs consists of the progressive diminution of the ~,-subunit type of mRNA expression (which occurs prenatally) by postnatal c-chain mRNA expression. In other words, the embryonic AChR initially has the 'classical' a2f176 composition, the 7-subunit message decreases as c-subunit message increases, and finally adult bovine muscle exhibits the ctzflE6-chain composition (Mishina et al., 1986). This correlates with the patognomonic long open time/small current amplitude characteristics of embryonictype AChR and brief open time/large amplitude adult-type of response which the corresponding mRNAs elicit when injected into Xenopus oocytes. Previous studies (Sakmann et al., 1985) had pointed to the importance of the 6-chain in determining conductance properties of AChR channels. Most recently, Yoshii et al. (1987) studied 16 hybrid AChRs synthesized from different combinations of all subunits from Torpedo and mouse. The authors were able to dissect the contribution of the different chains to three properties of the AChR: (a) the assembly at the Xenopus oocyte plasmalemma, which was maximal for hybrids containing 7r (Torpedo) and 7M (mouse); (b) an operationally-defined concentration of ACh eliciting an arbitrary conductance at - 6 0 mV, which was lowest for aM and //r and (c) control of voltage sensitivity, a property which appears to be governed largely by /3. Hybrids containing//M showed the greatest voltage sensitivity. The possibility was discussed that differences in charge distribution at the amino acid level are responsible for awarding greater sensibility to the//-containing hybrids. The region containing amino acids 210-220 is particularly prodigal in charge differences between the AChR chains, and it lies between the highly conserved M2-M3 region thought to be involved in ion permeation (Giraudat et al., 1986; Hucho et al., 1986; Imoto et al., 1986). The combined use of cDNA recombinant techniques and single-channel current recordings has recently produced further insight into the contribution of individual AChR chains to channel function. Normal bovine AChR channel displays a smaller conductance than bovine AChR channel at low extracellular cation concentration. When the 6-subunit of Torpedo AChR is replaced by the bovine 6-chain, channel conductance becomes similar to that of the native bovine AChR (Imoto et al., 1986). In an attempt to locate more precisely the region of the 6-chain responsible for the functional difference, Imoto et al. (1986) constructed chimaeric 6-subunit cDNAs with different combinations of Torpedo and bovine genes. By testing the conductances elicited by hybrid and chimaeric AChRs expressed on the surface of Xenopus oocytes, the authors were able to pin down the region of the S-chain comprising segments M2 and the adjacent bend between segments M2 and M3 as the one responsible for the difference in K + conductance between the Torpedo and bovine AChR channels. This may bear a relationship to the recent finding (Giraudat et al., 1986; Oberthfir et al., 1986; Hucho et al., 1986, and see Fig. 5) that M2 contributes to the recognition site for non-competitive antagonists (see Section 4.6). The bend portion between the putative transmembrane segments M2 and M3 in Torpedo 5-chain possesses one negative and one positive charge, whereas its bovine counterpart has two positive charges. All other AChR subunits contain negatively charged residues in this bend portion (Noda et al., 1983a; Kubo et al., 1985). Imoto et al. (1986) propose that the negatively charged residues might contribute to or form part of the mouth of the AChR channel, attracting permeant cations towards its lumen. The AChR version obtained by substitution

372

F.J. BARRANTES

of the so-called D-12 chimaeric f - c h a i n for the T o r p e d o 6-subunit would thus precisely reduce the n u m b e r o f negative charges in the channel mouth.

10. D E N E R V A T I O N

SUPERSENSITIVITY

The pathological condition which arises when the innervation o f adult skeletal muscle is suppressed is complex in nature, involving a variety o f changes in the metabolism anc physiology o f the muscle cell. A p a t o g n o m o n i c sign upon denervation is the appearanc~ o f an increased sensitivity o f the sarcolemma to the natural neurotransmitter ACh, termec 'denervation supersensitivity'. F a m b r o u g h and co-workers were the first to show th~ inhibition o f supersensitivity by actinomycin D (an R N A synthesis blocker), thus pointin~ to the involvement o f transcriptional control o f this p h e n o m e n o n (see most comprehensiw review in F a m b r o u g h , 1979). The effects o f denervation on channel type conversion durin~ development (Schuetze and Vicini, 1984) were discussed in a previous section. We nov k n o w that augmented A C h R synthesis is characteristically present in denervation super sensitivity (see reviews in Pumplin and F a m b r o u g h , 1982; Salpeter and Loring, 1985), an~ that such change is preceded by a corresponding increase in the a m o u n t s o f m R N A codinl for A C h R subunits (Merlie et al., 1984; Klarsfeld and Changeux, 1985; G o l d m a n et al. 1985). Shieh et al. (1987a) have contemplated two possibilities to a c c o u n t for thes, changes: (i) denervation triggers an u n k n o w n signal that activates the genes coding fo A C h R subunits or (ii) the change in m R N A levels could be posttranscriptional in nature e.g. due to a longer m R N A half-life. In order to test these possibilities, Shieh et al. (1987a used a c R N A probe specific for exon 7, a 224 nucleotide-long probe for the ~-subunit c the chicken A C h R gene, to quantitate the concentration o f mature ~-subunit m R N A an. its precursor after denervation. They found that the rate o f t r a n s c r i p t i o n o f the R N ! message for the ct-subunit is increased, this in turn causing increased message content an, stimulation o f A C h R synthesis in denervated muscle. In a related paper, Shieh e t a (1987b) have also found an augmented synthesis in the message coding for the 7 and A C h R subunits two days after denervation.

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