A MORPHOLOGICAL STUDY OF THE CHOLINERGIC RECEPTOR … · using a x 10 magnifier lens equipped with...

J. Cell Sci. 29,313-337 (1978) 313Printed in Great Britain © Company of Biologists Limited IQJS

A MORPHOLOGICAL STUDY OF THECHOLINERGIC RECEPTOR PROTEIN FROMTORPEDO MARMORATA IN ITS MEMBRANEENVIRONMENT AND TN ITS DETERGENT-EXTRACTED PURIFIED FORM

JEAN CARTAUD AND E. LUCIO BENEDETTI,Laboratoire de Microscopie Electronique, Institut de Recherdie en Biologie Moliculairedu C.N.R.S., University PARIS VII, 2 place Jussieu, 75005 Paris

ANDANDR£ SOBEL AND JEAN-PIERRE CHANGEUX,Laboratoire de Neurobiologie Moliculaire, Institut Pasteur, 25 rue du Docteur Roux,75015 Paris

SUMMARY

The highly specialized structural features of the postsynaptic membrane of Torpedo mar-morata electric organ have been studied by freeze-fracturing, freeze-etching and negativestaining applied both to intact electroplaques and to isolated purified excitable membranefragments.

The external surface of the postsynaptic membrane, revealed by deep-etching and negativestaining, is characterized by the presence of geometrically packed arrays of repeating particleshaving a subunit structure and a central pit. The isolated receptor protein molecules (molecularweight : 300000) show structural features identical to those characteristic of the repeatingparticles visualized on the external surface of the postsynaptic membrane.

Freeze-fracture cleaves the postsynaptic membrane into 2 asymmetric halves, showing thatnumerous identical paniculate entities are more strongly anchored to the cytoplasmic side ofthe bilayer. On the other hand, geometrically packed arrays of pits or depressions found on theexternal fracture face suggest that the receptor protein extends across the outer leaflet andis exposed to the external surface facing the synaptic cleft.

The homogeneous size-distribution of the particulate entities associated with the post-synaptic membrane element is consistent with the finding that the receptor protein accountsfor at least 40 % of the total protein of purified excitable membrane fractions. These resultsstrongly favour the assumption that the particulate entities represent the receptor protein,which forms oligomeric complexes either of identical polypeptides or quasi-equivalentlyrelated proteins.

Thus, the cholinergic receptor oligomers forming a bidimensional lattice of repeating par-ticles are exposed both to the synaptic cleft and to the cytoplasmic environment. It is proposedthat the regulating properties of the excitable membrane depend upon oligomeric or polymericassociations of receptor proteins spanning the bilayer.

INTRODUCTION

Nerve cells transfer signals between themselves and their sensory or effector part-ners via highly differentiated structures: the synapses. Among these the chemicalsynapse has been most thoroughly investigated: (Couteaux, i960; Gray, 1961;Palay, 1964; Akert, Moor & Pfenninger, 1971). It is well established that transmis-

314 J. Cartaud and others

sion of the nerve impulses across the 20-nm cleft separating the nerve terminal fromthe postsynaptic membrane depends upon a chemical signal. The neurotransmitterdelivered by the nerve ending diffuses across the cleft and hits the postsynapticmembrane, where it causes a selective change of membrane permeability to ions(for references, see Katz, 1962). Important progress has been made during recentyears on the identification of the postsynaptic targets for several neurotransmitters(for references, see Handbook of Psychopharmacology, 1975) but only the receptorfor acetylcholine from fish electric organ or vertebrate neuromuscular junctions hasbeen purified in large enough quantities to enable it to be studied as a protein (seeChangeux et al. 1976; Karlin, Weill, McNamee & Valderrama, 1976; Maelicke &Reich, 1976; Raftery et al. 1974; Raftery, Vandlen, Reed & Lee, 1976).

There are important reasons for investigating the structural organization of thecholinergic receptor within the plane and across the width of the subsynaptic mem-brane. The explanation of some physiological properties that the excitable membraneshares with allosteric enzymes could only come from demonstration of an oligomericor polymeric association of the regulatory protein in the bilayer membrane, as origin-ally postulated by one of us (Changeux, 1969; Changeux, Kasai & Lee, 1970a;Changeux, Podeleski, Kasai & Blumenthal, 19706).

A preliminary attempt to investigate the subunit structure of postsynaptic mem-branes by selective staining of thin sections or by freeze-fracture revealed particulateentities in the plane of the membrane (Sandri, Akert, Livingstone & Moor, 1972;Rash & Ellisman, 1974; Rosenbludt, 1975).

The interpretation of such substructure remained problematic until highly selec-tive markers of the nicotinic receptor site, the a-toxins from snake venom, wereintroduced to label this site at the neuromuscular junction (Lee & Chang, 1966), atthe electric organ synapse (Changeux et al. 1970 a) and recently in the central nervoussystem (Salvaterra, Muhler & Moore, 1975; Polz-Tejera, Schmidt & Karten, 1975).Autoradiographic studies at high resolution have shown that the a-toxin-bindingsites are present at an exceptionally high density in the subsynaptic membrane(Fertuck & Salpeter, 1976), either with a uniform distribution, in the case of the electricorgan synapse (Bourgeois et al. 1972; Bourgeois, Popot, Ryter & Changeux, 1977) orhighly concentrated in the juxtaneural region of the folds, in the case of the neuro-muscular junction (Fertuck & Salpeter, 1974, 1976; Albuquerque, Barnard, Porter& Warnick, 1974).

These observations did not demonstrate unambiguously that the particulateentities revealed by ultrastructural studies corresponded to the acetylcholine receptor.That required a direct comparison (Cartaud et al. 1973) between these structures andthat of the detergent-extracted receptor molecule purified by affinity chromato-graphy (Olsen, Meunier & Changeux, 1972; Meunier et al. 1973; Meunier, Sealock,Olsen & Changeux, 1974).

In the present study, a more complete demonstration of this point is made by theuse of a population of membrane fragments resulting from the subcellular fraction-ation of Torpedo sp. electric organ (Cohen, Weber, Huchet & Changeux, 1972; Nickel& Potter, 1973; Duguid & Raftery, 1973). In these receptor-rich membranes, which

Cholinergic receptor protein from Torpedo 315

most likely derive from the innervated membrane of the electroplaque, the acetyl-choline receptor may constitute up to 40% of the total protein (Sobel & Changeux,1977). The application of freeze-fracturing and freeze-etching, carried out in parallelon these isolated membrane fragments and on the electric tissue in situ, is now des-cribed. In particular, we show that in Torpedo marmorata electric organ the receptormolecule is the major integral protein of the subsynaptic membrane; it is exposedto its outer surface underlying the cleft and penetrates deeply within the membranethickness. Moreover, the receptor molecules form a geometrical lattice of repeatingunits in the plane of the subsynaptic membrane.

MATERIALS AND METHODS

The Torpedo marmorata were obtained from the Marine Station in Arcachon (France) andkept for a few weeks in artificial seawater.

Purification of the Ach-R-rich membranes

All the purification steps were carried out in the presence of io"4 M phenylmethylsulphonylfluoride (PMFS,) as a protease inhibitor and 002 % NaN, to prevent bacterial growth. Onehundred and fifty grammes of freshly dissected electric organ were homogenized in 150 mldistilled water containing NaN3 and PMFS, and then submitted to successive centrifugations.A first low-speed centrifugation (7000 g for 10 min) of the homogenate yielded a supernatantSi and a pellet Px. The latter was homogenized again and centrifuged under the same con-ditions, and supernatant S, was thus obtained. Si and Sa were pooled (S12) and centrifuged at10000 g for 2 h. The new pellet (P3) was resuspended in 32 % w/w sucrose and the resultingsuspension (E,) was layered on top of a discontinuous sucrose gradient (34%; 37-5 %; 43 %)and centrifuged at 100000 g for 6 h. The layer at the 375-43% interface was collected,diluted with 1 vol. of distilled water and centrifuged. The pellet was resuspended in 32%sucrose (E2). E2 was layered on top of a continuous (35-43 %) sucrose gradient and centri-fuged at 100000 g for 6 h. The cholinergic-receptor-rich fractions were pooled and consti-tuted the pure subsynaptic membrane fraction (M). This membrane preparation constitutes asuitable material for the isolation of the receptor protein.

Purification of the Ach-R protein

M was solubilized in 10 mM Tris-HCl, pH 74 , 100 min NaCl, io~s M 2-mercaptoethanol,\o~* M PMSF and 0 0 2 % NaN, containing Triton X-100 (final concentration 4%) . Thesolubilized material was layered on top of a continuous 5-20 % sucrose gradient containing thesame buffer with 0 1 % Triton X-100 and centrifuged for 15 h at 150000 g. A sharp Ach-Rband was then obtained and the Ach-R-rich fractions were pooled to give the purified choli-nergic receptor protein.

A more detailed description of these purifications is published elsewhere (Sobel & Changeux,1977)-

Freeze-fracture and freeze-etching

Columns of stacked electroplaques were carefully dissected from the living fish kept at lowtemperature by immersion in ice-water and rapidly fixed in 2 % glutaraldehyde in 0-2 Mphosphate buffer, pH 72 for 2 h at 4 °C. Small fragments of electric tissue were then preparedand washed in the same buffer. These fragments were impregnated with 20 % glycerol for 2 h,rapidly frozen in Freon R-22 and stored in liquid nitrogen. In other experiments, small frag-ments were frozen immediately after dissection.

Purified subsynaptic membrane fragments (M) were fixed in 2 % glutaraldehyde for 1 h,washed in cacodylate buffer and pelleted. The pellet was then resuspended in a small amount ofdistilled water, small drops were put on to gold disks, frozen in Freon R-22 and quicklytransferred in liquid nitrogen.


Carbon-platinum replicas were made in a Balzers BAF 300 and 360 apparatus. All thespecimens were fractured at — 140 °C and etching was performed at — 100 °C for 30 s-2 min.Micrographs were always printed as positive images (shadows are white) and were mountedwith the direction of shadowing from bottom to top. The recently proposed nomenclature forfreeze-etching is used throughout (Branton et al. 1975).

Negative staining

Both membrane fragments (M) and purified cholinergic receptor protein were studied bynegative staining. The membrane suspension was usually layered on the carbon-coated grid,washed with o-i % aqueous ammonium formate and stained with 1 % sodium phosphotung-state (pH 7-2) or with 1 % aqueous uranyl formate according to Leberman (1965). Negativestaining of the purified receptor protein was carried out using a buffered solution with o-i %Triton X-100 containing about 10-100/tg of receptor protein per ml. To that solution, baci-tracine was added to a final concentration of 10 fig per ml in order to improve the spreading ofsamples on a carbon-coated grid. A drop of this solution was put on to a carbon-coated gridand the excess of the solution was then withdrawn with filter paper. The grid was then washedwith o-i % ammonium formate, negatively stained with uranyl formate and rapidly dried.

All the specimens were studied in Philips EM 300 and EM 400 Electron Microscopesoperating at 80 kV and fitted with a jo-fim objective aperture. Photomicrographs usuallytaken between x 10- and 50000 direct magnification were subsequently enlarged using apointed illumination enlarger device (Durst Variput).

Measurements of particle sizes and particle densities

Particle sizes were determined from positively printed micrographs enlarged to x 200000using a x 10 magnifier lens equipped with a micrometer grating (Polaron Equipment Limited).The diameter of the particles was measured taking the width of their shadows as parameter.Large and elongated structures - probably resulting from the apposition of few unresolvedelementary particles - were discarded. Approximately 200 particles were measured for eachhistogram.

Estimates of particle density were made on the larger areas of membrane obtained, enlargedto x 100000, by direct counting over transparent graph paper. A few /tm* of membranesurface were analysed.

Since measurement of particles on replicas is fraught with uncertainty due to the fact thatthe angle of shadowing on the fractured surfaces may vary considerably, we have analysed alarge population of particles in different areas of the membrane displaying similar shadowlengths.

RESULTS

General organization of T. marmorata electroplaque

The electric organ of T. marmorata is made up of an ensemble of prisms which areoriented perpendicularly to the surface of the body and span its entire thickness.Each prism consists of stacks of electroplaques (Fig. 1) which receive numerousnerve endings on the ventral face. The dorsal face - the non-innervated one - ischaracterized by the absence of nerve terminals and by a dense network of micro-invaginations (0-1-0-2 jum in diameter and a few /«m in length) which extend intothe cytoplasm (Fig. 3).

Freeze-fractures of the ventral face of the electroplaque reveal numerous nerveendings distributed along the excitable membrane (Figs, i, 2). From newborn toadult, the size (and possibly the density) of the nerve terminals increases: they cover

Cholinergic receptor protein from Torpedo

Fig. i. Freeze-fracture view of the electric organ of a small Torpedo. Stacked electro-plaques are seen showing fracture planes of innervated (ventral) and non-innervated(dorsal) faces. From this picture it can be estimated that large areas ( ~ 50 %) of theinnervated face are associated with nerve endings, if, innervated face; nif, non-inner-vated face, x 6000.

31 CEL 29


approximately 50% of the exposed electroplaque surface in new-born animals (Fig. 1)but form an almost continuous layer in older specimens (Fig. 2). These nerve endingscontain large (80 nm) synaptic vesicles in varying amounts and some mitochondria.The process of Schwann cells investing the nerve endings are also frequently observedon the replica.

The presynaptic membrane in situ

At high magnification the internal organization of the presynaptic membrane ischaracterized by randomly distributed particles which remain mainly associated withthe P face after cleavage. In contrast with what seems to occur at the myoneuraljunction (Rash & Ellisman, 1974; Heuser, Reese & Landis, 1976), no double rows ofparticles, presumed to be the site of vesicle release, have been observed in the pre-synaptic element.

The postsynaptic membrane in situ

In a fixed fragment of electric tissue, the postsynaptic membrane is usually frac-tured across the membrane width. Therefore only a few surface views of fracturedareas are found (Fig. 2). More frequently the fracture splits the inner core of in-foldings of the innervated membrane (Fig. 4). At high magnification the postsynapticmembrane is characterized by the presence of numerous particles mainly associatedwith fracture face P (Fig. 4A). Their size-distribution is given in Fig. 5. Only oneclass of particles is found and the average diameter measured in different replicasranges between 6 and 8 nm. The density of the particles on fracture face P is approx-imately 8000 particles//tm2 of membrane surface. In some areas the particles appearlocally arranged in linear rows. Only a few particles are present on the correspondingfracture face E. Replicas of this inner membrane surface are characterized by arrays ofdepressions or pits displaying the same geometrical arrangement and spacing as theparticles associated with the P face (Fig. 4B). It is remarkable that the size distri-bution of the particles present on the P face is identical to that of the few particulateentities found on face E (Fig. 5).

The membrane organization described so far is observed not only in infoldingslocalized beneath the nerve terminals but also in membrane areas present betweensynapses. Fig. 6 shows the P fracture face of a plasma membrane segment far awayfrom the nerve ending. It is noteworthy that in the extrasynaptic areas the structuralfeatures - including the size and density of the particles - are identical to those found

Fig. 2. Freeze-fracture replica of the innervated face of an electroplaque. The post-synaptic membrane is mainly fractured across its width and only the infoldings areseen in front view (arrows). Two nerve endings are shown containing numeroussynaptic vesicles (sv). The presynaptic fracture face P shows randomly distributedparticles. No double rows of particles are detectable, pre, presynaptic membrane frac-ture face P. x 20000.Fig. 3. Freeze-fracture replica of the non-innervated face of an electroplaque, showingnarrow imaginations extending a few microns into the cytoplasm, x 20 000. Inset:The fracture face P is characterized by many 8'5-nm particles and by large depres-sions which represent the fractured contour of invaginations (arrows), x 120000.


in the subsynaptic region of the excitable membrane. These findings are in goodagreement with the observations of Rosenbludt (1975), that the subunit patternvisualized on thin sections of Torpedo electric organ covers the entire innervated faceincluding the extrasynaptic areas.

The structural homogeneity of the ventral face of T. marmorata electroplaquecontrasts with that of the neuromuscular junction from high vertebrates, where onlya small fraction of the plasmalemma - the juxtaneural area of the folds - contains ahigh density of cholinergic receptor sites revealed by a-bungarotoxin binding (Fertuck& Salpeter, 1976) and shows structural differentiations believed to represent recep-tive areas (Rash & Ellisman, 1974). This homogeneity is also of special interest forthe preparation of receptor-rich membrane fragments from homogenates of electrictissue (Cohen et al. 1972; Sobel et al. in preparation).

Attempts to visualize, by deep-etching, the external surface (ES) of the postsynapticmembrane which is directly in contact with the synaptic cleft were unsuccessful evenon unfixed and unglycerinated specimens. Only with isolated membrane fragmentsin vitro was it possible to obtain images of the etched external surface (see below).

All the results presented till now concern observations carried out on replicas offreeze-fractured electric tissue which has been previously fixed in glutaraldehyde andimpregnated with glycerol. Replicas of unfixed specimens have also been obtained bythe conventional freezing technique. Fig. 7 shows the structural features of the frac-ture faces produced in an unfixed specimen. In contrast with the aspect revealed infixed material, the P fracture face of a postsynaptic membrane invagination showsdouble rows of irregularly shaped particles of variable size. Also visible are some 7-8nm rounded particles identical to those already described in fixed specimens. Thecomplementary face E is characterized by arrays of small particles (6 nm in diameter).Larger particles of irregular shape are also found in close apposition to these arrays.Glycerol treatment of the unfixed specimens does not strikingly modify the topographicdistribution of the inner membrane particles as in other tissues (see Mclntyre, Gilula,& Karnovsky, 1974). The only difference noticed is a better preservation of the tissueduring the freezing process. The interpretation of these results is not immediatelyapparent (see Heuser et al. 1976). Experiments using rapid freezing devices are inprogress in our laboratory.

Ultrastructure in vitro of isolated membrane fragments rich in acetylcholine receptor

Additional information regarding the structural organization of the subsynapticmembrane was obtained either by negative staining or by freeze-etching of isolatedmembrane vesicles. These membrane fragments were purified by ultracentrifugation

Fig. 4. A, freeze-fractured face P of a subsynaptic membrane invagination. Note thepresence of closely packed identical 7-nm particles. Some of them are locally arrangedin linear rows or in small clusters. The inner aspect of PF close to thesynaptic cleft is identical to that found deep in the imaginations, x 120000. Arraysof geometrically packed particles are shown in the inset; x 250000. B, freeze-fractured face E of a subsynaptic membrane invagination characterized by the presenceof few 7-nm particles and arrays of geometrically arranged pits with a repeatingdistance of approximately 10 nm. x 120000; inset, x 250000.


21-3


from crude homogenates of electric organ using a recently improved method (Sobel& Changeux, 1977, manuscript in preparation) derived from that of Cohen et al.(1972). About 40% of their protein consists of the acetylcholine receptor and, on thisbasis, they were assumed to derive from the innervated face of the electroplaque.These membrane fragments make closed vesicles with a diameter ranging from o-ito 5 /tm (Fig. 8 A) and respond to cholinergic antagonists by a change of permeabilityto 22Na+ ions (Ha2elbauer & Changeux, 1974; Popot, Sugiyama & Changeux, 1974,1976).

4 0 r

20

PF

4 5 6 7 8 9Particle sizes, nm

60 r

40

20

EF

4 5 6 7 8 9Particle sizes, nm

Fig. 5. Histograms of particle sizes in the postsynaptic fracture faces P and E.

In negatively stained preparations, these 'excitable microsacs' collapse and theobserved membrane sheets are characterized by a particulate pattern. In the bestpreparations studied (specific activity 4000 nmol 3H-a-toxin sites per g of protein)up to 99 % of the membrane fragments exhibit closely packed 7-nm particles (Fig. 8 B).Each individual particle has the appearance of a rosette with a central pit or holewhich retains the stain and often shows a subunit pattern. In some of these particles5-6 subunits can be identified (Fig. 8 c). In some areas the particles appear regularlypacked, with a centre-to-centre distance of 8-9 nm. In some membrane fragmentsthese arrangements present a well defined geometry (Fig. 9 A) such as linear rows ordistorted hexagonal patterns. In the latter case, the photographic rotational techniqueof Markham, Frey & Hills (1963) (Fig. 9 c) yields a marked enhancement of the


at.

Fig. 6. Freeze-fracture view of the innervated electroplaque surface showing subsynap-tic and extrasynaptic membrane areas. The structural features of the subsynaptic(arrow) and extrasynaptic (double arrow) domains are essentially identical. Comparealso the latter area to that shown in Fig. 4A. X 60000.

324 J. Cartatid and others

hexagonal organization for n = 3. Other rotation photographs with different n(n = 5 and n = 7) produced nonsense images.

The particulate pattern visualized by negative staining can be consistently revealedby deep-etching of the isolated fragments. The fracture produces again 2 asymmetricinner membrane fracture faces which are dotted by particulate entities either ran-

Fig. 7. A, in unfixed specimens the fracture face P of the subsynaptic membrane ischaracterized by linear arrays or double rows of irregularly shaped particles, x 120000.B, replica of unfixed postsynaptic membrane showing that the fracture face E ischaracterized by a geometrical lattice of 6-nm particulate entities. Some large andirregularly shaped particles are also seen in close association with the lattice, x120000.

domly distributed or closely packed in clusters of various dimensions. By deep-etching, patches of particulate entities are detected on the external surface of 'right-side-out' microsacs, interspaced with smooth-etched surfaces (Fig. 10). Thesepatches occasionally show geometrical arrays of packed subunits or more irregularly

Fig. 8. Negatively stained purified excitable membrane fraction. At low magnifi-cation, it is clearly demonstrated that the purified fraction consists almost exclusivelyof membrane sheets displaying a subunit pattern (A). The surface feature of a mem-brane sheet is characterized by the presence of packed arrays of pitted particles (B).High-magnification micrographs (c) and (n) illustrate the ultrastructure of theparticles revealed by negative staining. Each particle consists of a ring-shaped moleculeformed by five or six subunits. Edge view (D) of the membrane sheets show thatthe lateral projection of the particle is consistent with it having a cylindrical shapeand forming regular rows. A, x 50000; B, x 120000; c, D, x 500000.

Cholinergk receptor protein from Torpedo


clustered paniculate entities (Fig. 10). In the most favourable preservation of thestructure, the particles exposed by deep-etching have a subunit structure and acentral pit and resemble the rosettes revealed by negative staining. The centre-to-centre distance is of about 9-10 nm and this value is comparable to that measured inthe negatively stained preparations. Some microsacs show both etched (ES) and

Fig. 9. A lattice organization of the excitable membrane can be revealed both innegatively stained (A) and deep-etched (B) membrane fragments. The hexagonalpattern of the lattice is enhanced (c) by rotation (n = j) according to Markham(1963). A, x 250000; B x 350000; c, x i 000000.


fractured (PF) faces. In these fragments the etched surface is usually characterizedby particulate entities and the fractured face (PF) by numerous particles. Very oftenthe particles are aggregated and the etched surface usually shows clustering of thesuperficial structures in the corresponding areas (Fig. 11 A). A few vesicles, probably'inside-out' or inverted ones, display a completely smooth-etched surface and afractured face characterized by few particulate entities and few holes. The structuralfeatures of this fractured face (Fig. 11 B) closely resemble the fracture face E of thepostsynaptic membrane in situ.

Ultrastructure of the purified acetylcholine receptor protein from. T. marmorata

The detergent-extracted acetylcholine receptor protein was purified from T.marmorata receptor-rich membrane fragments by a recently described method whichavoids affinity chromatography (Sobel & Changeux, 1977, and manuscript in prep-aration). The specific activity of the preparations was 7000 nmol 3H-a-toxinsites/g protein, i.e. close to that found by other authors with different techniques(Raftery et al. 1976). Negatively stained preparations usually display a rosette-likestructure with an average diameter of 8 nm. Each molecule shows again a subunitpattern and a densely stained central pit is often visible (Fig. 12). Although some pic-tures suggest a pentameric or hexameric organization of the receptor molecule, itis not possible yet to ascertain the exact number of subunits from these micrographs.Better resolution appears difficult to achieve with purified proteins by conventionaltransmission electron microscopy, mainly because of the considerable irradiation ofthe specimen (for references, see Engel, Dubochet & Kellenberger, 1976). Anotherdifficulty may come from an eventual deformation of the hydrophobic receptor mole-cule as a result of its interaction with the carbon supporting film.

Similar images have already been presented in the case of the purified acetylcholinereceptor from Electrophorus electricus (Cartaud et al. 1973; Meunier et al. 1973,1974; Nickel & Potter, 1973) and from Torpedo californica (Eldefrawi & Eldefrawi,1975). The structural similarity between the isolated acetylcholine receptor moleculesand the particles covering the receptor-rich membrane fragments is evident.

DISCUSSION

The neuromuscular junction and the electroplaque synapse have been the subjectof a considerable number of structural studies (for reviews, see Couteaux, 1958,i960; Bloom & Barrnett, 1966). The postsynaptic membrane of such synapses meritsparticular attention because of its physiological significance as the target of the neuro-transmitter, and attempts were made to reveal the structure of its receptor molecule.The first significant results came from conventional electron microscopy. 'Densifi-cations' of the outer membrane layer were observed on the postsynaptic membraneand viewed either as a thickening localized at the top of junctional folds in neuro-muscular junctions (Birks, Huxley & Katz, i960; Couteaux, 1958; Fertuck &Salpeter, 1974), or as projections extending into the synaptic cleft. This latter aspecthas been found in many neuromuscular junctions (Rosenbludt, 1972, 1974, 1975)


and interpreted as related to, or even representing, the receptor for acetylcholine.A non-uniform distribution of these projections was reported at the amphibianmotor endplate (Rosenbludt, 1974) but in Torpedo electric organ such projections havebeen found regularly spaced (7 nm) and distributed all over the innervated face ofthe electroplaque, without preferential localization under the nerve terminals(Rosenbludt, 1975).

Freeze-fracture experiments performed on neuromuscular junctions brought ad-ditional evidence supporting the non-uniform nature of the junctional fold membranesand led to the demonstration of a morphological specialization of the juxtaneuralregion of the folds (Rash & Ellisman, 1974; Heuser, Reese & Landis, 1974; Peperet al. 1974; Heuser et al. 1976). These specializations either consisted of aggregates ofparticles with a density of 8700 particles//an2 (Peper et al. 1974) or of irregular rowsof large 11-14-nm particles on the P fracture face (Rash & Ellisman, 1974).

In contrast with the structural inhomogeneity of the postsynaptic membrane inthe neuromuscular junction, we show here by freeze-fracture that the ventral inner-vated surface of Torpedo electroplaque is structurally substantially homogeneous.In fact, the same morphological profile is found in both extrajunctional and post-junctional areas of the innervated face. This unique feature, which is consistent withthe findings of Rosenbludt obtained on thin sections (Rosenbludt, 1975) is of par-ticular interest for biochemical experiments and may explain why subcellular frac-tionation of Torpedo electric organ yields large quantities of excitable membranefragments (Cohen et al. 1972; Duguid & Raftery, 1973; Sobel & Changeux, 1977).

The substructure of the postsynaptic membrane in Torpedo electric organ has beenstudied by freeze-fracture, which shows that numerous particulate entities areassociated with the P fracture face (Orci, Perrelet & Dunant, 1974; Cartaud, 1975;Clementi, Conti-Troncori, Peluchetti & Morgutti, 1975; Rosenbludt, 1975).

Different results have been reported in the literature regarding the size and shapeof the particles localized on the P face. Rosenbludt reported that the particle diameterranged between 10 and 15 nm (Rosenbludt, 1975), while Orci et al. (1974), in agree-ment with our observations, have found a diameter of 8-5 nm. Regular arrays of theseparticles were not reported but only linear arrangements (Rosenbludt, 1975). On thecomplementary fracture face E, more or less regular hexagonal (Orci et al. 1974) orsquare (Rosenbludt, 1975) lattices of particles having a repetitive distance of 6 nm

Fig. 10. Deep-etched membrane fragments. The external surface (ES) is visualizedin this micrograph. Arrays of closely packed pitted particles are found, interspersedwith smooth areas (A), X 120000. At high magnification the depression in the par-ticles is more easily visible (arrows) (B). X 250000.

Fig. 11. Freeze-etched purified membrane fraction showing 'right-side-out' (A) and'inside-out' (B) vesicles. The 'right-side-out' vesicle is characterized by a fractureface dotted with numerous particles and by an etched surface (es) showing thecharacteristic particulate pattern as shown in Fig. 10. Note that smooth areas can beseen either on PF or on adjacent ES face, indicating some topographic relation betweenthe superficial and the internal particulate entities. The 'inside-out' vesicle showsa completely smooth-etched surface and a fractured area characterized by the pres-ence of some particles and also some depressions. These features resemble those ofpostsynaptic membrane face EF as shown in Fig. 4B. x 120000.


were reported. In the present study, we have never been able to find clear evidencefor such a structure, even in our best replicas. Instead, a smooth surface containingarrays of pits and a few scattered particles was observed. Only in unfixed specimenswere lattices of 6-nm particles consistently observed. The differences between themorphological aspect obtained either on fixed or on unfixed specimens are not easilyunderstood. Recent observations from several laboratories reinforce the evidence thatextremely rapid freezing of the sample is essential for stabilization of the moleculararchitecture of biological membranes, in particular when these structures have notbeen previously fixed in aldehydes (Heuser et al. 1976; Gulik-Krzywicki & Costello,1976). Thus, a definite interpretation may only come from studies with a rapidfreezing device.

An important advance in the interpretation of the structural features described inthe subsynaptic membrane came from a correlation of morphological studies withbiochemical investigations, developed in parallel on the purified cholinergic receptorprotein. The use of snake venom a-toxins as specific labels of the acetylcholine recep-tor sites made possible the study of the distribution and density of these sites at theultrastructural level by high-resolution autoradiography in both the electroplaquesynapse (Bourgeois et al. 1972, 1977) and the neuromuscular junction (Fertuck &Salpeter, 1974, 1976; Porter, Barnard & Chiu, 1973; Porter & Barnard, 1975). Aswill be discussed later, a good agreement exists between the results produced bydifferent technical approaches. It has been shown that the particulate entities observedin vitro on receptor-rich membrane fragments from Torpedo exhibit a striking struc-tural similarity to those found in highly purified preparations of detergent-extractedacetylcholine receptor from E. electricus electric organ (Cartaud et al. 1973). Thedata reported here on the receptor protein purified from T. marmorata electric organconfirm this interpretation.

These results on the ultrastructure of the receptor protein obtained by negativestaining of the isolated preparation are consistent with the biochemical data support-ing the concept that the receptor molecule is an oligomer of approximately 300000Daltons made up of smaller subunits (major component 40000 Daltons) (for reviews,see Sobel & Changeux, 1977; Changeux et al. 1976; Karlin et al. 1976; Raftery et al.1976). We have demonstrated by negative 9taining that the molecule exhibits a ringshape, with an apparent diameter of 8—9 nm. The number of subunits in each oligomeris not readily apparent. Either five or six subunits are found in different molecules.Although the Stokes radius determined in hydrodynamic experiments cannot bereadily interpreted in terms of dimensions of the receptor molecules, the value of theStokes radius of 7-3 nm found with both Torpedo and Electrophorus purified receptor(Meunier et al. 1971; Raftery, Schmidt, Clark & Wolcott, 1971) fits rather well withthe 8 nm diameter measured directly on electron micrographs.

Fig. 12. A, purified cholinergic receptor molecules negatively stained with uranylformate. This low-magnification micrograph shows the presence of homogeneous8-9 -nm pitted structures. Some aggregates are also visible, x 250000. B-D, highmagnification of some selected areas in which the detail of molecules is clearly visible.The molecules are homogeneous in size and are characterized by an electron-densecentre and a subunit pattern, x 500000.


Acetylcholine binding to its specific receptor site at the postsynaptic membranetriggers a rapid change of membrane permeability. The receptor molecule (or itscomplex with the ionophore, if the ionophore is different from the receptor proteinsensu stricto), must be both accessible to diffusible ligands from the external surface ofthe excitable membrane and span the hydrophobic core of the membrane in order toassure ionic translocation across the membrane. Moreover, since antibodies againstthe purified receptor protein block the response of the electroplaque membrane tocholinergic antagonists (Sugiyama, Benda, Meunier & Changeux, 1973), a signi-ficant surface of the receptor molecule has to be exposed on the outside of the mem-brane surface. The assumption that the receptor complex is an 'integral' membraneprotein, in terms of the definition of Singer & Nicholson (1972) is also supported by thehydrophobic character of the detergent-extracted protein (for reviews, see Changeuxet al. 1976 and Raftery et al. 1976). By negative staining and deep-etching applied toisolated membrane fragments, in which up to 40 % of the proteins is the acetylcholinereceptor, we confirm that the external surface of this acetylcholine-sensitive membraneis characterized by exposed particles having a shape and subunit organization identicalto that of the isolated receptor protein. From this comparison, it can be assumed thatpart of the cholinergic receptor protein is associated with the postsynaptic membraneand is exposed to the synaptic gap. On the other hand, the fracture surfaces of thepostsynaptic membrane, in particular the P face, are characterized by particulateentities which correspond to elements spanning the width of the membrane lipidcore. In contrast to unspecialized areas of the plasma membrane, the inner membraneparticles visualized by the fracture of the postsynaptic membrane have homogeneoussize of 6-8 nm. The latter particles remain mainly associated after cleavage with thecytoplasmic half of the membrane. However, in our best replicas we have also foundthat on the corresponding fracture face E pits are present, the distribution of whichis identical to that of the particles remaining associated with the P fracture face. Thesimplest interpretation of these images is that the inner membrane particles extendthroughout the lipid bilayer, as in other specialized areas of the plasma membranesuch as gap junctions (Benedetti & Dunia, 1975; Goodenough & Gilula, 1974).

From these observations it is, however, difficult to demonstrate, in a definitivemanner, the existence of a structural continuity between the inner membrane par-ticles associated with the fracture face and the receptor molecules identified on theexternal etched surface of the excitable membrane fragments. Similarly in othermembrane systems, the evidence of such an association is essentially indirect and basedon the comparison of topographic distribution and the size of the particles visualizedeither by the fracture or by deep-etching. In the case of the postsynaptic membrane,the size of the inner membrane particles is rather homogeneous and correlates wellwith the dimensions of the receptor oligomers determined both by deep-etching andby negative staining. The inner membrane particles observed on the fracture faces,even in the best replicas, do not reveal the subunit pattern characteristic of the re-ceptor protein visualized on the etched surface. The current interpretation of thisdiscrepancy is that a deformation of the hydrophobic protein moiety occurs duringthe fracture process, giving a round particle on the fracture face whatever the original


structure of the protein (Miller, 1976). Moreover, the density of the particles associ-ated with the fracture faces amounts to 8000-10000 particles/^m2 and is close to thedensity of the receptor rosettes on the etched surface (10000-12000 particles//tm2).In that respect, it is noteworthy that the latter density can be compared to thedensity of the acetylcholine receptor sites measured by quantitative autoradiographyin situ using tritiated a-toxin from Naja nigricollis, a specific label for the cholinergicreceptor. This density was 50000 ±16000 toxin-binding sites//tm2 of postsynapticmembrane surface (Bourgeois, Popot, Ryter & Changeux, 1973) in E. electricuselectroplaque. This value is significantly higher than the number of particlesdirectly counted by electron microscopy but the receptor oligomer probably carriesmore than one toxin-binding site (Meunier et al. 1973). Another indication that theinner membrane particles could be associated with the exposed receptor moleculesis the fact that both form a geometrical assembly of repeating particles in theplane of the etched surface and the fractured surface.

X-ray diffraction of receptor-rich membrane fractions reveals a lattice organiza-tion of particles in the plane of the membrane (Dupont, Cohen & Changeux, 1973;Raftery et al. 1975), with a centre-to-centre distance of diffracting units of 9-1 nm,which is in complete agreement with our morphological results. Despite the fact thatthe contribution of protein elements within the lipid bilayer cannot be easily inferredfrom the electron-density profile derived from the Patterson function, the X-raydata tentatively indicate that the remarkable thickness of the membrane element(9-10 nm) probably results from a dense sheet of proteins which distorts the usualbilayer profile on one of its surfaces.

The electron-microscopic observations presented in this paper provide some evi-dence supporting an asymmetric distribution of the receptor proteins within thethickness of the membrane. Although spanning a considerable width of the lipidbilayer, the receptor molecule would be exposed mostly on the outer surface of themembrane. This model is different from that proposed for Halobacterium halobiumpurple membrane, where the entire protein is almost completely buried within thelipid bilayer and forms there an hexagonal lattice within the plane of the membrane(Unwin & Henderson, 1975; Henderson & Unwin, 1975). In the case of the purplemembrane and in contrast with the subsynaptic membrane, negative staining anddeep-etching apparently do not reveal surface features as should be the case if thephotoreceptor protein were largely exposed at the membrane surface (Blaurock &Stoeckenius, 1971).

In the subsynaptic membrane, we have shown that both in situ and in the isolatedfragments the receptor molecules may appear regularly packed into a 2-dimensionallattice. It is not yet clear whether or not some specific functions of the receptormolecules, such as the cooperative binding of cholinergic ligands, is a consequence ofits lattice organization (for discussion, see Changeux et al. 1976).

The density of acetylcholine receptor sites in the neuromuscular junction has beenrecently reviewed (Porter et al. 1973; Fertuck & Salpeter, 1974). Mainly from a-toxin labelling experiments, the density of acetylcholine receptor site in the subsyn-aptic membrane was reported to range between 5000 and 30000 sites//tm2. More

22 CEL 29

334 J- Cartaud and others

recent studies using quantitative high-resolution autoradiography indicate that thedistribution of the a-bungarotoxin sites is not uniform throughout the subsynapticmembrane but is concentrated in the juxtaneural part of the folds (Fertuck & Salpeter,1974; Albuquerque et al. 1974; Fertuck & Salpeter, 1976; Porter & Barnard, 1975).The density in these 'patches' may reach 30000 toxin-binding sites//tm2, a valuesimilar to that found earlier in the synapse of E. electricus electric organ (Bourgeoiset al. 1973, 1977). Such high receptor concentration may 'saturate' the membranesurface leaving little place for other protein, in agreement with our 'quasi-crystalline'pictures of negatively stained excitable membrane fragments.

On the other hand, the hypothesis that some of the internal particles seen in thefractured postsynaptic membrane may represent acetylcholinesterase molecules isunexpected, since the enzyme can be selectively extracted from electric organ hom-ogenates without the use of detergents (Massoulie1 & Rieger, 1969) and thus, belongsto the category of the 'extrinsic' membrane proteins (Singer & Nicholson, 1972).In addition, the purified receptor-rich membrane fragments contain little ( < i % )acetylcholinesterase. Furthermore, ultrastructural studies of the purified enzyme(Rieger, Bon, Massoulie' & Cartaud, 1973; Cartaud, Rieger, Bon & Massoulie", 1975;Dudal, Herzberg & Silman, 1973) reveal that the acetylcholinesterase is a rathercomplex molecule and does not resemble the acetylcholine receptor molecule.Future work should lead to the visualization of acetylcholinesterase molecules in thesynaptic membrane and to the understanding of its relationship with the acetyl-choline receptor protein.

The authors wish to thank Mrs Louise Labaronne for the photographic work. This work wassupported by the Centre National de la Recherche Scientifique and the Delegation G6n6ralea la Recherche Scientifique et Technique, Convention n° 74-7-01-73.

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(Received 28 February 1977)

A MORPHOLOGICAL STUDY OF THE CHOLINERGIC RECEPTOR … · using a x 10 magnifier lens equipped with...

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