Endogenous Chemical Receptors: Some Physical

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ENDOGENOUS CHEMICAL RECEPTORS:

Some Physical Aspects

+9135

Francisco J. Barrantes

Max-Planck-Institut flir Biophysikalische Chemie, 34 Gottingen, West Germany

Chemical receptors are the mediators of specific reactions in a variety of biological processes that range from elementary chemosensory activities in primitive bacteria to the highly sophisticated behavioral mechanisms in man. They have the capacity to specifically recognize a given chemical and to generate the requisite signal for evolving a biological response. Strictly speaking, the word "receptor" was used as such for the first time in the early papers of Ehrlich (see 54, 1 37) (in the discipline now known as immunology), although it was in the context of physiology and pharma cology that it really acquired its "wholistic" connotation. Since then the term has been subjected to considerable use and misuse, often leading to confusion, but above all depriving receptors in many instances of their most superb integrating property. It cannot be over-emphasized that spe cific recognition of a chemical does not suffice to define a receptor [for a clear distinction between receptors and acceptors see (2 1)]. The chemical message encoded in a ligand, say a neurotransmitter or a hormone, is virtually meaningless until it is decoded by its corresponding receptor into purposeful regulatory signals in the target cell. Therefore I adhere throughout this review to the early interpretation of the term, using it to denote both the capacity to specifically recognize a ligand (cognitive or discriminatory property) and the capacity to initiate the chain of events leading to the biological effect (gating property). I have not attempted to cover the whole field of chemical receptors, but have preferred instead to restrict the discussion to some endogenous receptors for hormones and neurotransmitters. This necessarily implies the omission of several important problems in chemoreception. Arbitrary though it may seem, the review progressively concentrates on a single archetypal system, the receptor for the neurotransmitter acetylcholine 287 0084-6589/79106 1 5 -0287$0 1 .00


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(ACh). The methodologies, questions and partial answers, and ambiguities and paradoxes covered in the discussion of this system nevertheless are common to most other chemical receptors currently being investigated. Some concessions to generalization are made in the first part of the paper, which deals with structural and dynamic properties of receptors in situ, in the membrane environment, and in isolation. The second part of the review is devoted to the problem of receptor ligand interactions. The information on this subject has been made available by means of a strategy common to electrophysiology, classical pharmacol ogy, and biochemical endocrinology, and which consists of defining the steps of ligand action in reverse order with the ultimate aim of reaching the molecular basis of ligand potency, efficacy, etc. The opposite perspec tive, namely building up the scale of complexity, starting with rather simple and well-defined systems (this approach being heavily dependent on the availability of such systems), is more akin to the ways of biochemistry and molecular biology. At present we are witnessing a most interesting phase in the study of receptor-ligand interactions: The above two ap proaches are converging with increasing impetus towards the integral char acterization of receptor systems, and cross-fertilization of approaches rather than the offspring of isolated progress in any one discipline is the key to this impetus. I would even venture to say that we are on the threshold of a small-scale revolution in which understanding receptor ligand interactions at the molecular level will call for a drastic redefinition of classical concepts concerning ligand activity. It is hoped that this fragmentary overview on some physical aspects of chemoreceptors will provide an adequate stimulus for experimentation in many needy areas. STRUCTURAL ASPECTS OF RECEPTOR SYSTEMS Localization, Regional Distribution, and Density of Receptors

As early as 1907, Langley (76) conducted a remarkably simple experiment aimed at localizing the "receptive substances" for nicotine. He applied the ligand with a fine brush along the length of muscle fibers and found that the response was maximal at the nerve-ending region. It was only 50 years later, after the introduction of micro-ionophoretic techniques for the application of drugs, that the functional distribution of nicotinic ACh receptors (AChRs) could be electrically traced. This approach has attained high levels of sophistication, along with the development of methods for drug application and recording from voltage-clamped regions of the muscle and techniques for observation of the end-plate with Nomar-


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ski optics (57, 73, 1 08, 1 3 7). Langley's basic observation, confining ACh chemosensitivity to a discrete area of the target cell (the end-plate region), has been found to hold for all fast-contracting muscles that contain AChRs. A few microns away from this zone the sensitivity falls markedly [spatial decrement (73, 107)], being about WOO-fold lower 500 /Lm away from the high-sensitivity region. Serotonin and dopamine receptors (58) also appear to be discretely located. AChRs in slow-contracting muscles (4) and monoamino monocarboxylic acid receptors in molluscan neurones (72), on the other hand, have been observed to occur diffusely over the cell surface. Diffuse disposition of otherwise discretely located AChRs also occurs, however, in two special circumstances: before innervation (49) and after denervation ( 1 0). It is interesting to note that in spite of the existence of two types of arrangement at the cell surface, the receptors in both clustered and diffuse dispositions have similar average lifetimes, in the order of 20 hr (55). The receptors for analgetic-like substances have been mapped electrically in brain neurones (20). Their location con forms to that outlined by autoradiographic techniques. After the introduction and radiolabeling of the quasi-irreversible choli nergic blocking agent a-bungarotoxin (a-toxin), a series of studies appeared on the light and electron microscope localization of AChRs by autoradio graphic and immunohistochemical techniques. Uniform distribution of re ceptors in adult muscle was claimed at first, though later studies have shown that AChRs are concentrated at the crest of the junctional folds adjacent to the nerve terminal, in a specialized region of thickened mem brane constituting 50% of the end-plate (56). Immunoperoxidase staining at the electron microscope level confirmed this topography (44). Indirect immunofluorescence (25) and direct fluorescence techniques (6, 7) have also been used to localize AChRs in fixed cells. On living muscle cells a reversible antagonist, cholinergic fluorescent probe has been employed ( 1 4). Synaptic location of the fluorescent probes was observed in adult muscle, as were discrete patches (hot spots) and diffuse distribution in developing muscle (6, 7, 9). The spatial occurrence of various other receptors has also been studied. Randomly dispersed insulin receptors and small clusters of the same have been mapped on liver plasma membranes using ferritin-labeled insulin and freeze-etching electron microscopy (103) or 125I-labeled insulin autora diography (17). Melanocyte-stimulating hormone receptors on melanoma cells also occur in discrete patches, as revealed by autoradiographic ( 1 4 1 ) and fluorescence ( 140) techniques. Immunofluorescence localization o f the luteinizing hormone receptors on testis cells (67) adds to the list of surface receptors that occur in clustered arrays. The resolution of the fluorescence microscope localization of /3-adrenergic receptors on cerebellum neurones


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on the whole has not sufficed to ascertain a detailed topography (90), since studies in this direction for the most part have not gone beyond a general mapping within regions of an organ [see, for instance, the autoradio graphic localization of analgesic receptors (74) or of brain receptors for the better characterized inhibitory neurotransmitter, 'Y-aminobutyric acid (35)]. Though not explicitly stated, practically all the above-mentioned studies describe the localization of receptors on the cell surface membrane. This fact carries more weight than at first is apparent, since it not only reflects the current state of localization studies, but also points to a characteristic common to most chemical receptors, i.e. their location at the interface between the intracellular space and the external milieu. This is probably the earliest acquired characteristic of eukaryotic cell receptor systems, and dates back to the origin of chemosensory activity in primitive protozoa. Intracellularly located receptors are a case apart; steroid receptors, for instance, occur mainly in the cytoplasmic and nuclear compartments (e.g. 1 02). Internalization of receptors normally located at the plasmalemma is also observed (e.g. insulin-receptor complexes), a phenomenon with po tentially important functional implications (see 130, 136). The intracellular disposition of receptors in relation to their life cycles has been studied in more detail in the case of AChRs (55). The same methodology described above for localization studies has been applied for quantitative determination of AChR sites. A correlation be tween the density of a-toxin sites determined by autoradiography and the electrical response to ionophoretically applied ACh has been observed. There is general agreement on the quantitative relationship between the two parameters: The a-toxin site density is roughly proportional to the square of the ACh dose, D, which in turn is related to the conductance elicited, g, by the relation-sites = glDn, where n is the Hill coefficient of about 1 . 7 (75). Nevertheless, the interpretation put on this relationship varies; the wealth of evidence would seem to favor the possibility that the quadratic ratio can be accounted for on the same basis as the dose response curve for agonists: Two molecules of ACh would be needed to activate the receptor. Of course this explanation is provisional, in that the stoichiometry of ACh to toxin sites and of toxin sites to AChR-associ ated channels has not been firmly established. In absolute figures, the number of toxin sites per end-plate [2-6 X 107 (75)] and their high density have clearly facilitated the quantitation of this particular system. Earlier studies reported densities of 7000-8000 a-toxin sites per ,..,m 2 in mouse motor end-plate. When these values are corrected for the observed juxta neural distribution of sites, an even larger value (20,000-30,OOOI,..,m2, ±30%) can be calculated for the receptor-rich portions of the folds, falling



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sharply to 50 sites/f-tm2 only 1 5 f-tm away. Only 10-20 toxin sites/f-tm2 occur in extrasynaptic regions. Initially, densities of about 30,000 toxin sites/f-tm2 were reported for the electroplax (23, 24), but technical improve ments have led to revised figures of 50,000 ± 30% and 370 ± 250 sites/ f.Lm2 in synaptic and extrasynaptic regions, respectively (see 30, and refer ences therein). A most complete review on this subject has been given (55). That the AChR is clearly a unique case is made apparent by comparing the above figures with those of other receptor systems where available. The number of insulin receptors per square micrometer, as determined by ferritin-labeled antibodies in the adipocyte (fat cell) ghost membrane, is only about 200 ( 1 33). 125I-Iabeled insulin autoradiographic studies re vealed about 1 05 total sites on the hepatocyte plasmalemma ( 1 7). The number of l3-adrenergic receptors per cell varies considerably from about 200 in the S49 lymphoma cell line to about 80,000 in skeletal muscle in tissue culture (see 8). When these figures are normalized according to cell size, the average numbers are 8 receptors/f-tm2 in the 849 cells and in turkey erythrocytes, and about 100-200/f-tm2 in muscle or HeLa cells (8). The reason this point is considered at all here is that the figures given above do not merely represent a statistical curiosity, but in many instances constitute a requisite for the selection of specimens for the applica tion of biophysical techniques, some of which are illustrated in this review. For instance, the density of adrenergic receptors in 849 and red blood cells does not suffice for their observation with the fluorescent l3-adrenergic blocker 9-aminoacridino-propanolol (8). Aside from this consideration, the absolute number of receptors in a given cell type has a more fundamen tal significance: It reflects the subtle equilibrium between synthesis, inter nalization, and degradation of such receptors, which fact the cell may well use as a means of regulating its sensitivity towards the ligands. The ligands in turn exert a regulatory activity on the number of receptors present, this being of considerable physiological and medical importance (e.g. 53, 55). Most Receptors Are Deeply Integrated Within the Membrane Framework

A considerable gap exists between the number of receptor-mediated func tions identified by pharmacologists and the number of receptors now being characterized in biochemical terms as distinct molecular entities. In addi tion to the paucity of some species (e.g. insulin receptors constitute only 0.000 1 % of the protein content in liver), the great majority of receptors are intimately associated with the plasma membrane. This latter fact has posed more difficulties than originally expected in the isolation and charac-


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terization of these entities, a subject outside the scope of this review.l Concerning the nature of this association, a variety of physicochemical criteria support the view that most endogenous receptors belong to the category of "integral" membrane-bound glycoproteins. In the case of the AChR, statistical techniques based on amino acid composition and elemen tary thermodynamic parameters--discriminant analysis (lI)-have been applied in the absence of other rigorous quantitative criteria for establishing their integral nature. This method of analysis has since been applied in combination with additional physicochemical parameters to substantiate the conceptualization of the isolated AChR as an integral membrane pro tein (30, 32, 54, 1 14). One of the distinctive features of integral membrane proteins is their binding capacity towards detergents, which constitutes one of the bases for their solubilization (e.g. 42, 53). The average mass of non-ionic detergent Brij 58 bound per mass of protein was calculated to be 0. 1 8 and 0.04 for the monomeric and dimeric forms of the AChR ( 1 1 9). Direct estimates of the amount of detergent bound using radiolabeled Triton X-lOO and cholate range from 1 1 to 50% of the apparent molecular weight of the protein-detergent complex (see 54). This has obvious implications for the determination of the size and molecular shape of the purified AChR (see below). A further line of evidence bearing on the integral nature of the AChR in the plasma membrane stems from ultrastructural studies. Freeze-fracture replicas of the neuromuscular junction from various animal species show intramembranous particles, often in paired rows with a typical "herring bone" disposition, in areas of the postsynaptic membrane where AChRs are expected to have maximal density ( 1 1 6). The particles range between 80 and 1 40 A in diameter, with a packing density of 3000 to 6ooolp,m2 . A smaller type of intramembranous, closely packed, and often regularly arrayed 60-A particle is observed in freeze-fractured specimens in associa tion with the extemal leaflet of the fractured bilayer (E face), more apparent in unfixed excitable membranes from electric tissue (29, 30) A third type of particle was reported to occur in the inner, cytoplasmic leaflet (P face) in 'chemically fixed specimens, differing from the aforementioned ones in size (60-90 A) and packing densities [8000- 1 O,OOO/p.m2 (29, 30)]. Finally, projections of 80- 1 20 A in width were observed in thin sections of choli nergic synapses with a density of l O,OOOIp.m2 ( 1 23). Electron microscopy of the purified AChR-rich membranes reveals parti cles of 80-90 A in diameter, with a central pit when uranyl acetate is .

1 The reader may find it helpful in following the historical evolution of the subject to compare several of the reviews (42, 54, 55).



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used [Figure 1 (30, 125)] or without clear substructure when phosphotung stic acid is employed. The difference here is probably due to the varying degree of penetration of the two stains in the direction perpendicular to the membrane. These particles have often been reported to occur in ordered arrays and have been interpreted as constituting the morphological counter part of the AChR. The observation of particles with similar charcteristics in negatively stained preparations of purified AChR-detergent micelles was used to support this view (30, 9 1 , 1 25). Cartaud et al (30) have claimed that more than 99% of the purified membranes exhibit closely packed, often hexagonally ordered 70-A particles; however a previous report indi cated that up to 20% of these membranes had no discernible structure and that more than 99% of them did not contain ordered arrays ( 1 25). Furthermore, the densely packed particles revealed by negative staining reach densities of 1O,000/l-I-m2 , a figure that exceeds that of the intramem branous particles of similar size apparent in freeze-fractured specimens from the same AChR-rich membranes (30). Three types of inconsistencies therefore appear to obscure interpretation of the above structural studies: (a) the disparities in particle size; (b) the variance in particle distribution; and (c) the numerical discrepancies between the average density of a-toxin sites [5 X 105/l-I-m2 (30)] and that of particles in purified membranes from the same tissue, several-fold lower. This latter incongruency cannot be straightened out until the more basic disagreements on the ratios of ACh to a-toxin sites [ 1 : 2, 1 : 1, 2 : 1 (see 43, 54)], the number of toxin sites per receptor monomer, and the subunit composition of the AChR have been resolved. Points (a) and (b) may be dealt with together; aside from measurement uncertainties, these inconsistencies can be accounted for partly in terms of methodological differences (e.g. use of or absence of fixation, shadowing or staining proce dure, freezing artifacts, etc), but even then an underlying pleomorphism may become more evident. In my opinion such pleomorphism is a reflection of the dynamic balance statically captured by these structural techniques between different states of aggregation of the AChR. One aspect of this is discussed below (the clustered-diffuse receptor equilibrium that takes place both in development and in the adult synapse). The aforementioned balance probably reflects the inherent property of the AChR as a biopo lymer to selfaggregate as a function (among other things) of the redox state of the membrane, this being intimately connected with the breakdown and formation of S-S bonds between oligomers. The existence of higher order polymers is of interest at this point. Such structures, apparent in sedimentation equilibrium measurements (53), could account for example for the larger particles observed in freeze-fractured specimens, for which Rash et al (1 1 7) calculated molecular weights in the order of 106 daltons.


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Figure I (Top left panel) Minimal electron-dose « 10 e-IA2) micrograph of a negatively stained (1% uranyl formate) AChR-rich membrane fragment from Torpedo marmorata, taken with a scanning-transmission electron microscope in the dark field mode. The image was photographically reversed in contrast to show stained regions in dark. Particles of about 70 A in diameter are observed throughout. End-on views are characterized by a central pit ,of stain. Magnification: x 500,000. (Top right panel) Purified AChR from Torpedo in 0.1% sodium cholate buffered medium processed as above for electron microscopy. The particles can be observed in end-on, lateral, and intermediate projections. The most striking feature revealed by comparison of the solubilized AChR and the membrane-bound particles is the larger size of the former. More detailed observation of the isolated particles shows two characteristic regions of the molecule; in one of them two protruding "fingers" can be observed (double arrows), whereas the other, larger region has a globular shape. The projected diameter of the smaller region agrees with that of the end-on views in the membrane-bound particles, whereas neither end-on projections nor lateral views of the larger region in the purified AChR do. Furthermore, the central pit of stain in the membrane-bound particles shows correspondence with the stain-filled partition in lateral views. Therefore it appears likely that the smaller portion of the purified AChR is the one revealed by the surface images of thc membrane fragments, and that the larger globular part is anchored in the bilayer (P. Zingsheim and F. J. Barrantes, unpublished observations). (Bottom) Negatively stained (1% uranyl acetate) tubular structure obtained by the fusion of AChR-rich membrane fragments from Torpedo. Rows of particles with a remarkable degree of order can be observed. Magnification: X142,OOO . (Inset) Optical diffraction pattern obtained from a selected region of the electron micrograph, including about 200 ordered particles. The two sets of spots are probably due to the superimposition in the image plane of two lattice arrays from the upper and lower surfaces of the flattened tubular structure. The resolution of the higher order spots is 30 A . (Courtesy of A. Brisson, Centre d'Etudes Nucleaires de Grenoble.)



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Symmetrical structures can be generated-as attested by electron micros copy (Figure 1) and X-ray diffraction (see below)-if particles are allowed to close-pack in two dimensions. Two types of packing symmetries have been reported to occur in membrane preparations: one, with a PI plane lattice group ( 1 25) and the other with a P2 lattice (29, 30). Regardless of the natural occurrence of such ordered arrays, A. D. Brisson (unpub lished data) has managed to produce tubular structures derived from AChR-rich membranes that exhibit a remarkable degree of order (Figure 1) with the pz-Iattice type of symmetry. This development might obviously enable the application of electron diffraction techniques on unstained speci mens in the manner successfully attempted by Unwin & Henderson ( 1 39) on the purple membrane. X-ray diffraction studies also support the existence of ordered structures in the AChR-rich membranes (50, 1 25). Assuming a hexagonal plane sym metry similar to that occasionally observed in electron micrographs, Du pont et al (50) calculated a center-to-center distance of about 90 A from the equatorial reflections, which they attributed to the organization of particles in the plane of the membrane. Raftery et al (1 14) have estimated that the ordered domains extend for about 500 A, and that the distance between particles is 1 73 A. The unit cell parameters obtained in the Ross et al study ( 1 25) were a = c 9 1 A, f3 = 1180, with a crystallographic plane lattice group PI; the optical transform of their electron micrographs and reconstructions there-of were not compatible with the hexagonal sym metry. As far as the detailed structure of the membrane-bound AChR is concerned, published images do not possess sufficient resolution to elucidate this point. Recent unpublished results from our laboratory (P. Zingsheim and F. J. Barrantes) on the purified AChR from Torpedo (Figure 1) give some indication of the overall structure of the oligomer and its possible mode of insertion in the membrane. =

Dynamics of Receptors in the Membrane

The existence of clustered as well as diffusely located receptors in the plasmalemma raises queries as to the nature of the physical factors involved in the formation, stabilization, and disassembly of the patch structures, as well as the dynamics of these processes. This is a central consideration in current theories on the coupling of some hormonal receptors and the enzyme adenylate cyclase (see below). The functional implications of the dynamic balance in the case of neurotransmitter receptors has also received attention (55). Along these lines, one obvious and interesting possibility is the involvement of clustered arrays in oligomer-oligomer cooperative phenomena (34, 69, 80). A specific relationship has been postulated to exist between AChR clusters and the stabilization of the synapse (33).


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Other specific cases where the diffuse-clustered equilibrium of hormonal receptors have been invoked to play a decisive role are discussed in a recent review ( 1 36). A variety of systems and techniques have been explored with the aim of indentifying the above classes of receptors. Two populations of AChR have been found in developing muscle using photobleaching techniques and fluorescent-labeled a-toxin (9). Ninety percent of the labeled AChR occurs in areas with an average density of lOOOIp.m2• This type of AChR is relatively mobile, with an average two-dimensional diffusion constant of 5 X 1 0-1 1 cm2 sec-I at 22°C. Since the life-time of this type of AChR is in the order of 20 hr (55), the receptor would experience Brownian motion displacements in the order of 20 p.m during the time it spends in the membrane. The AChR clustered into small, high-density patches of 1 0-60 p.m2 has little if any lateral diffusion in vivo ( 1 0-1 2 cmZ sec- I). Rotational immobilization of the AChR from Torpedo in its membrane bound state has been detected using saturation transfer electron spin reso nance ( 1 26). An active site-directed, covalent affinity label was used; from the lack of motion a lower limit of about 1 msec was calculated for the rotational relaxation time of the AChR. Biologically active fluorescent derivatives of insulin and epidermal growth factor hormone have been used also for measuring translational motion of their corresponding recep tors ( 130). The receptors originally diffusely distributed in the plasma lemma were found to be very mobile; average diffusion coefficients of 34 X 1 0-10 cmz sec-I were calculated. The receptor-hormone complexes aggregated into patches as the temperature was raised from 23° to 37°C. This thermal dependence resembles the observed behavior of other nonre ceptor integral membrane proteins, in terms of their ability to aggregate into patches as the lipid phase transition is traversed (144). A possible cause of receptor cluster formation has been considered re cently in physical terms (59), using basic thermodynamic concepts intro duced in 1876 by Gibbs (60) in relation to the problem of homogeneous nucleation. Only a reduction of dimensionality distinguishes the treatment from that used in the theory of the growth of a clump in a matrix, as currently dealt with in metallurgy. In brief, the nucleation process (cluster ing) depends on the balance between a favorable contribution, the change in free energy upon transferring receptors from the bulk membrane phase (the matrix) to a patch (the nucleus), and an offsetting free energy contri bution due to the boundary tension generated between the two phases. As is also common in materials science, the pressure terms are neglected (see below). The variation of a G as a function of patch radius (one could also consider it as a function of the number of receptors) resembles an activation energy profile; a maximum in free energy is reached at r rc, =



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a critical radius. If r becomes larger than rc, then the patch will tend to continue to grow, eventually forming a "cap." A patch with r < rc is unstable and "dissolves"; rc was calculated to be in the order of 8 X 1 0-5 cm for a cell 10 J.Lm in diameter with 1 04 receptors of about 1 05 mol wt (59). Among the factors not specifically considered by the model, "intrinsic" aspects such as chemical differences between clustered and diffusely located AChR have been documented (55). It has been proposed that these differences originate as post-translational modifications of dupli cate gene products, and phosphorylation, glycosylation, or even proteolytic cleavage have been invoked to account for such differences (see 55). A variation of 1 0-30 charges per AChR oligomer can be calculated from the differences observed in the isoelectric points of synaptic and extrasynap tic receptors (see also 55). Among "extrinsic" factors, the boundary effects (59) could also be affected by specific interactions with neighboring lipids, like the ones that have been observed recently in AChR-rich membranes (85). No relationship between endogenous receptor patches and cytoskele ton components has been documented as yet. Most recently a neuronal substance, possibly a protein, has been found to induce the clustering of the AChR in developing cloned muscle cells ( 109). A sixfold increase in receptor density was observed upon addition of this 100,OOO-mol-wt pro tein. The AChR in spherical, 2-day-old embryonic muscle cells are uni formly distributed around the cell perimeter. Orida & Poo ( 1 04) found that application of a steady electric field of 10 Vfcm for 1 . 5 hr induces the clustering of the receptors on the pole of the cell facing the cathode. Concanavalin A, which is known to bind to the AChR (9 1 ), inhibits the electrically induced phenomenon.2 This interesting study should be consid ered in conjunction with that of Mustacich & Weber (94), which deals with the electrically aided transfer of proteins between two phases. The latter authors consider the "mixed" case of ligand-induced transfer of proteins between the two phases and corresponding alteration of their rotational freedom upon binding (94). These phenomena are particularly relevant in this context, since the partitioning of receptors between two phases also plays a role in the nucleation process, and alterations in the distribution of the AChR both in diffuse and patch areas by ligand binding have been reported (3 1). Also, the neglected pressure effects mentioned above might be considered in view of the relationship between the chemical 2

Concanavalin A, a lectin extracted from the Jack bean Canavalia ensi/ormis, is probably

an oligosaccharide-receptor in the vegetable. It is unlikely that evolution has preserved a specific receptor for this receptor in highly specialized eukaryotic cells of higher vertebrates, or that the AChR or the insulin receptor are also receptors for Concanavalin A, for instance. This is a classical case where the term "acceptor" (21) is more appropriate for describing the surface glycoproteins in animal cells that react with an exogenous ligand of this type.


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potential and pressure in each phase; changes in chemical potential elicited by ligand binding on patch receptors might alter the state of diffuse recep tors and their partition in the membrane and vice versa. The physical state of receptors in the membrane, in terms of the dynamic balance discussed above, will certainly be found to play a major functiona� role in various systems. It is predictable that significantly different forms of this balance operate in neurotransmitter and hormonal receptors, prima rily because of the marked disparity in the time these two types of receptors spend in their occupied (liganded) state. Physical Properties of Isolated Receptors

As already stated, most receptor proteins are thought to be integral mem brane constituents, needing detergents for their extraction, purification, and maintenance in solution. Although in some cases the physical proper ties of detergent micelles have been the object of detailed investigation (e.g. 1 8, 1 1 8), extrapolation of these properties to those of the comicelle after incorporation of receptor proteins is not always straightforward. In general this point is ignored, and one reads about the properties of deter gent-solubilized receptors as if the detergent were simply the solvent phase; in fact it is the properties of the modified detergent micelle that are being studied in many cases. Detergents may alter both the physicochemical and functional properties of receptors. Perhaps the most important physical property lost upon solu bilization in detergent systems is that of the vectorial sidedness of the receptor, which Nature awards at the moment of insertion of these special proteins in the plasmalemma. This is clearly the case with the most thor oughly studied system, the AChR. Among the modifications of functional character, at concentrations well below those used for solubilizing the receptor, detergents act as local anaesthetic-like substances in the living electroplax (27). Once the receptor is solubilized, and depending on the nature of the detergent used, the binding properties of cholinergic ligands are modified with respect to those found in the membrane-bound state (e.g. 32, 1 1 5). Transitions between affinity and physical states observed in vivo and in the membrane-bound AChR are also lost upon detergent solubilization ( l 3, 32, 54). Finally, physical (i.e. not including restorage of function) reconstitution of the receptor into lipid vesicles is severely hampered once the AChR has been in contact with certain detergents (see 26). Drastic alterations in the functioning of some hormone receptors caused by detergent treatment have been documented (88), in particular the coupling of hormonal receptors with adenylate cyclase. In those cases where a particular effort has been made to account for the contribution of the detergent, some physical properties of isolated



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receptors have been determined. Sedimentation studies of AChR in deter gent micelles, for instance, have provided information on the hydrodynamic characteristics of this receptor under nondenaturing conditions; average sedimentation coefficients of 9 and l 3S have been identified with the mono meric and dimeric forms of the AChR oligomer, respectively ( 1 1 9) . The ratio of these two major species [higher order polymers are also observed (53)] can be manipulated by chemical modification of the -SH groups. The dimeric, heavy form has tentatively been attributed to the native species in situ. The Stokes radii of the AChR have been reported to be about 70 and 80-95 A for the light and heavy forms, respectively (54, 1 19). Analysis of monomeric AChR-Triton X- lOO complexes with neutron scattering showed a radius of gyration of 44 ± 7 A for the micelle ( 1 48). A prolate (2. 5 : I axial ratio) or oblate (4 : 1) ellipsoidal shape was inferred; if the AChR were a comp�ct sphere, a Rg of 32 A could be expected. The partial specific volume of the AChR from Electrophorus was calculated to be 0.74 cm3/g (9 1 ). Minimal electron dose dark-field electron microscopy of the purified AChR form Torpedo reveals asymmetric particles (Figure 1) and suggests a possible mode of insertion of the AChR in the lipid bilayer (P. Zingsheim and F. J. Barrantes, unpublished observations). The insulin receptor is one of the few hormonal receptors for which physical properties are known (e.g. 4 1 , 42). The detergent-solubilized recep tor has a Stokes radius of 70 A and a sedimentation coefficient of 1 1 S. A molecular weight of 300,000 and a frictional coefficient of 1 . 5 have also been estimated (41). None of these values are corrected for bound detergent, which could amount to 20-30% of the protein weight. Physical State of Lipids in Receptor Systems

Many of the functional and physical properties of surface receptors in the plasmalemma are necessarily influenced by the state of neighboring lipids, and vice versa. In trying to explain the relative rotational and transla tional immobility of some receptors, their coupling with other membrane constituents, and the formation and stability of the patch structures, consid eration of the physical state of the lipids becomes indispensable. The in formation available to date is fragmentary; aside from the experimental data summarized below, this important issue is often treated in terms of speculative "fluid models." Using acylcholine, active site-directed spin labels (28), the conclusion was reached that fluid lipids surround the AChR in the membrane ( 1 9). More recently, in addition to the fluid bilayer component a highly immobi lized lipid moiety has been observed in the same membranes with electron spin resooa.:nce techniques (85). This immobilized component certainly extends beyond a single layer of lipid around the AChR oligomer and


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can be sensed equally well with stearic acid, steroid, or phospholipid spin probes, suggesting that the lipid "annulus" has no specific lipid require ments, The X-ray diffraction studies of Ross and co-workers ( 1 25) have yielded some additional information on the state of the lipids in AChR-rich mem branes. The Fourier refinement of the small-angle data to a 1 3-A resolution indicated a characteristic bilayer profile, with maxima at 40 ± 3 A , as is found for the bulk lipid in artificial phospholipid systems, The high angle patterns revealed a broad 4. 6 ;\ band characteristic of interhydrocar bon chain spacings in lipids above the fluid-crystalline phase transition; the 4.2-A sharp reflection expected for crystalline lipid constituted a minor proportion of the signal above the lipid phase transition, which was calcu lated to occur at 2°C. Reinterpreting earlier nanosecond and fluorescence polarization studies (142) on the basis of the present knowledge on the chemical composition of AChR-rich membranes and the location of the probes used, the conclusion can be reached that these membranes have somewhat immobile protein constituents (see Dynamics of Receptors in the Membrane) embedded in a no-less-rigid lipid framework. The upper limit set for the time scale of the immobilization was about 0,7 p.sec (142). Depletion of cholesterol from the AChR membranes from Torpedo results in the reduction of the immobilization of the lipid moiety, as studied with fluorescence polarization techniques (B. Rivnay and F. J. Barrantes, unpublished observations). As far as the time scale of the lipid immobiliza tion is concerned, the electron spin resonance studies of Marsh & Barrantes (85) have revealed rotational correlation times of 50-70 and 30-50 nsec for the probe motion around and perpendicular to the long molecular axis, respectively. These values are 50-100 times longer than is found in typical bilayer membranes. The lateral translational motion in these immo bilized lipid regions will also be a factor of 50-100 times slower, with average frequencies of 105 sec-I. -

Reconstitution of Receptor Systems

Re-association of detergent-solubilized AChR-rich membranes after exten sive dialysis and addition of natural lipids has been reported (65). The re-association yielded closed vesicles (microsacs) of 300-1 000 A in diame ter, which retained 22Na+. Carbamoylcholine, an ACh analogue, ac celerated the rate of efflux of the ion in the time course of seconds to minutes, and this effect could be blocked with antagonists. Attempts to reconstitute the AChR-associated ionic permeability using the purified receptor and natural lipids have come up with variable results. High con centrations of agonist were needed to moderately affect the 22Na leak (92) or were ineffective on the 22Na+ influx (89).



30 I

A second approach has been to reconstitute the AChR system into black lipid membranes. De Robertis et al (48, 107) pioneered in this and other areas of the same field, employing proteolipids extracted from excita ble membranes with organic solvents; this enabled the incorporation of the extracted material directly into the membrane-forming solution. Tran sient changes in artificial membrane conductance were elicited by high concentrations of agonist. Similar effects, however, were observed when negatively charged lipids were used instead of the complete proteolipids (106). In view of the remarkable properties of the proteolipids and associ ated lipids from excitable tissues, in particular their high affinity, and in some cases stereospecific binding characteristics (see 48, 82, 107), their effects on ionic permeability certainly deserve further investigation. Other studies, attempting the incorporation of detergent-solubilized, purified AChR (of varying degrees of purity) into black lipid membranes, have been reviewed recently (26). The pessimism with which the current state of research in this area is contemplated is amply justifiable: The re-incorpo ration of the AChR into bilayers is seldom reproducible and is nonquantita tive, and the transient conductance events induced by ligands are qualitatively and quantitatively different from the physiologically occurring ones. A third approach to the problem of reconstituting the AChR system has been introduced recently. In one study (147) purified AChR in the presence of detergent was injected into the subphase of a lipid monolayer formed at the air-water interface with erythrocyte lipids. The amount of protein incorporated was measured by labeling the AChR with radioactive a-toxin. Only 3.5% of the receptor injected into the subphase was trans ferred to the monolayer, producing an increase in the surface pressure of 5-10 dyn/cm2• In a subsequent study ( 1 10) synthetic lipids were used. Monolayers formed with cholesterol incorporated more AChR than those made with phospholipids. It has not been attempted to ascertain the effect of cholinergic ligands on these reconstituted systems. The results of reconstitution experiments on this the most extensively studied receptor system have been far from successful. In my opinion, the contact with certain membrane lipids that detergents alter by substitu tion is essential to the normal functioning of the ion-translocation machin ery. This hypothesis is substantiated by the combined information given above, all of which indicates that the AChR system is more delicate than membrane-bound enzymes, such as sarcoplasmic reticulum ATPase, even in terms of simple re-incorporation into the bilayer. The deleterious contact with detergents might also hinder a hypothetical "coupling" between the recognition subunit and the channel (see below). Along these lines the successful restoration of l3-adrenergic receptor-adenylate cyclase activity

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by somatic hybridization has opened up one of the most challenging ave nues of research on this subject ( l 05). RECEPTOR-LIGAND INTERACTIONS


A Word on Energetics

The native conformation of proteins is held together by the sum of a large number of small energy interactions. We ignore the extent to which these forces might be different in proteins located partly in the hydrophobic domain of lipid bilayers and partly exposed to the extracellular space, i.e. in typical integral membrane proteins, as most receptors are. The study of receptor-ligand interactions could shed some light on this point, bearing in mind that the forces that keep the protein structure together are of the same magnitude as those operating in such interactions. Unfortunately, the information available on this topic in most cases provides a misleading picture of the energetics of the physiologically relevant interactions. The study of AChR-toxin association, for instance, characterized by endo thermic exergonic reactions with !J.. G = 1 2 kcal/mol and !J.. H = 1 7 kcal (83), has little if any relevance to the energetics of the interaction of the AChR with ACh or its analogues. Although we lack direct thermodynamic information on the latter process, the evidence available on the acetylcho linesterase-ligand interactions (see 1 37), the parallel trends followed by the pharmacological activity of compounds acting on the enzyme and the AChR (93, 1 37), and experimental data on the energetics of agonist activity in vivo ( 1 37), all point to standard free energy changes of a few kilocalories being operative in the physiologically significant case. If the reaction mechanisms postulated from some electrophysiological experi ments prove to be correct, and activation of the AChR in vivo is mediated through the binding of a second ACh molecule (e.g. 3), then one can expect to find standard free energy changes in the range typically observed in the free energy coupling of two ligands [� 1 . 5 kcal/mol (145)] . These elementary and still hypothetical considerations on the thermodynamics of receptor-ligand interactions point to an apparent paradox that probably applies to all receptor systems: Molecules exhibiting full receptor activation (agonists) can be, and often are, "worse" ligands than those being effective blockers (i.e. antagonists). All that is expected of the latter, after all, is that they be accurately recognized and that they shift the equilibrium population of receptors to an energetically stable level. Ligands designed to trigger the physiological effect, on the other hand, have to go beyond this step, and often rapidly (e.g. neurotransmitters). To some extent nature probably has lubricated their way by imposing smaller energy barriers in their path.


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Structural Aspects

Although one cannot expect many common features to be shared by ligands as dissimilar as a complex polypeptide hormone and a small biogenic amine, the first step in all receptor-ligand interactions (the recognition process) must necessarily center on specific structure identification. Each individual ligand contains a message (topochemical pattern) to be conveyed to the target cell. The code of this message lies within the very structure of the ligand, and hence the decoding ability of receptors, a form of pattern recognition, constitutes one of the expressions of biological specificity at the molecular level. How and when was this specificity acquired? At present we can only surmise that in receptor systems it probably resulted from a "buberian dialogue" between ligands and "protoreceptors" throughout the lengthy course of phylogenetic evolution. Discrimination between mus carinic and nicotinic ligands in cholinergic receptors (see 93), for instance, is absent in Echinoderms; its presence in bivalve mollusks most likely reflects the optimization of subtle modifications in the structure of the AChR recognition site, which have been preserved ever since in genetic patterns. A common trend becomes apparent from the few examples given: Molec ular recognition is the result of many small contributions, none of which is dominant. This simple corollary constitutes the structural replica of the corresponding thermodynamic description of protein-ligand interac tions (145). COMPLEMENTARITY BETWEEN LIGAND AND RECOGNITION SITE Various structural techniques have been applied to the study of pharmacologically active ligands in an attempt to gain an insight into (a) the spatial distri bution of invariant groups for a family of related drugs, from which the essential features defining the molecular details of the pharmacophore re gion could ideally be inferred, and (b) the structure of the pharmacophore recognition site, i.e. the recognition site on the receptor. The former of these two aims has led to the design of chemically modified ligands or completely synthetic analogues, which can be used to further refine struc tural information on the system. This is accomplished by combining data derived, for example, from crystallographic or quantum mechanical calcu lations with spectroscopic studies of the ligand in solution, and parallel measurements of the biological activity and binding affinities of native and modified ligands. Complementing data in this manner is particularly critical in the evaluation of theoretical calculations from orbital theory in quantum mechanics. Aside from the more classical estimates of preferred molecular conformations obtained from energy profile computation, quan tum mechanical data in principle can yield information on the stabilization


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energy involved in the formation of molecular complexes, perhaps the most relevant application of orbital theory [specifically superdelocalizabil ity calculations (see 62)] to ligand-receptor interactions. The reactivity indices obtained in this manner acquire meaningfulness when a correlation can be established with the pharmacological activity (e.g. potency) of a ligand. Such correspondence is seldom found in the literature (see 62, 1 20). The structure-activity relationships derived from the knowledge of the absolute configuration of ACh, catecholamines, and other biogenic amines from X-ray crystallography in combination with pharmacological determi nations, which the reader may find illustrated in an excellent mono graph ( 1 37), have had a considerable impact in the development of molecu lar pharmacology. The same approach promises to be of applicability to the case of the larger polypeptide hormones. The studies of Blundell and co-workers ( 1 1 2, 1 29) on the pancreatic hormone insulin have already provided substantial evidence on the detailed three-dimensional structure of the hormone and the residues putatively involved in the recognition process. Insulin appears to be a very favorable case from several points of view; in particular, the same residues having a direct participation in the binding process seem to be involved in the triggering of the subsequent biological effect (47). This is in contrast with other hormones, such as adrenocorticotrophin, which apparently possesses separate regions in its molecule for binding and activating the target receptor. How stringent are the structural requirements that a receptor "active site" imposes on its ligands? This point is amply illustrated in X-ray, nuclear magnetic resonance, circular dichroism, and other types of studies performed on analgesic substances. The corresponding receptor, operation ally defined for many years as the "opioid receptor" because of its capacity to react with morphine-like drugs (codeine, heroin), has recently been found to respond to naturally occurring peptides (enkephalines and en dorphines) (68, 74). This receptor is highly stereo-selective; virtually all active compounds-i.e. agonists-are isomers with the structure of (-)-morphine. In spite of the wide chemical diversity of these ligands, conformation and chirality dictate their pharmacological effect. It is also likely that within a series of opioid drugs, potency can be ascribed to similar, invariant regions of the molecule. A balance of rather rigid regions (e.g. with fixed torsion angles) and flexible parts of the molecule seems to be essential for awarding potency to these ligands. The reader is referred to the extensive studies ofPortoghese and co-workers for further illustration of this point ( 1 1 1). As far as the relationship between opioid ligands and the natural analgesic substances is concerned, it is precisely the information derived from crystallographic studies that enabled a structural homology



305

between chemical groups in each type of molecule to be drawn (6 1). The tetrapeptide Tyr-D-Ala-Gly-Phe was shown to possess common three-di mentional features with nearly 40 analgesics acting on the receptor. More recently, a preliminary crystallographic study of the (Leu5)-enkephalin molecule has appeared ( 1 34), and the proposal has been made that two intramolecular hydrogen bonds partially "rigidify" the otherwise flexible pentapeptide in a specific spatial arrangement. The crystallographic structure of two almost identical hormones, prosta glandins E2 and F2a [they differ only in the saturation of the C(9) 0(2) bond], also provides a reasonable explanation for their marked differ ences in biological activity (they have little if any affinity for each other's receptor). In addition to their common hair-pin conformation, X-ray stud ies have shown disimilar ring geometries (77). A curious case of three-dimensional homology has been observed be tween insulin and relaxin, the latter a polypeptide hormone synthesized in the corpus luteum and involved in most mammals in the dilation of the symphysis pubis before parturition. These two hormones share only five common amino acids in identical positions. However, when their three dimentional maps are compared ( 1 6), a remarkable similarity is apparent. The suggestion has been made that a common ancestor, an insulin-like molecule, evolved into the present hormones by gene duplication followed by accepted mutations ( 1 6). The experimental finding is undoubtedly of great interest in relation to the problem of evalution of ligand and receptor structure. RELATIONSHIP BETWEEN LIGAND

(AND RECEPTOR) CONFORMATION AND

The inference has been made from the X-ray studies on insulins that the complementary surfaces of ligand and recognition site contact via many hydrophobic interactions ( 1 1 2), this being a property common to many hormone receptor systems ( 1 1 2). In the particular case of the glucagon receptor, this type of interaction may stabilize the otherwise very flexible peptide, which exists in solution as an equilibrium population of conformers with little retention of structure (mainly as random coil), in a preferred helical conformation when bound to the receptor ( 1 29). The a-helical region of the hormone has been identified recently around Trp-25 by using a combination of optically detected magnetic resonance and phosphorescence spectroscopy ( 124). It has also been postulated that glucagon binds to two "regulatory" subunits of the receptor arranged as a dimer with a twofold axis, attending to the symmetry properties of the glucagon helices ( 1 29). A similar twofold symmetry has been proposed for the active conformation of the luteinizing hormone (79), in analogy with the case of 2,3-diphosphoglycerate, the allosteric effector of hemogloRECOGNITION


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bin. In the case of insulin it has been suggested that the region of the molecule that is involved in dimerization plays a fundamental role in site site interactions on the receptor (46, 47). Multiple points of contact and stabilization of ligand structure upon binding therefore seem to be a com mon trend with large polypeptide hormones. These facts taken together point to the presence of many energetically equivalent contributions in the formation of the recognition complex and have obvious implications for the kinetics of binding. Furthermore, regardless of the invariability of a given group in a family of related ligands, no group or atom thereof is all important (i.e. dominant) in the complex with the receptor. As Weber puts it when explaining specificity of binding in the context of protein evolution (145): "If protein specificity of binding is the result of many independent effects none of which is dominant, the resultant property, the binding energy, will have a normal distribution with respect to those changes in the enzyme [here the receptor] that give different weights to the individual interactions, as example of Liapunoff's theorem. . . . Evolu tion, playing on the protein modifications has finally adjusted the binding energy to the optimal value for each particular case." Multiplicity of contacts, rather rigorous spatial configurations, large numbers of energetic contributions . . . . . . should one also expect the same in the case of neurotransmitter systems? The kinetics of such systems, as is shown below, probably dictates other sets of rules: lower energy barriers, small and flexible ligands, and correspondingly simpler topogra phy in the receptor recognition site (e.g. 93). Antagonists may not necessar ily adhere to such rules, as shown by the example of the AChR-a neurotoxin complex. Extremely low equilibrium dissociation constants characterize the tight complex for both short and long toxins (32, 54, 55, 1 1 5). The three-dimensional map at 2.7-A resolution of a similar type of quasi-irreversible receptor blocker, erabutoxin-b, shows this 62-amino acid-long peptide as a rigid disk-shaped molecule with one protruding loop, the single chain twisted into five strands of antiparallel ,a-sheet (8 1 , 1 38). The multiple structural features revealed by chemical modification studies as being relevant to the binding of toxins (36) appear to have physical representation in the crystal structure. Upon inspection of this structure it seems far more likely that one "disk" (i.e. the extended surface of the toxin) sits on the receptor surface establishing the numerous contacts that lend it its quasi-irreversibility, than that a lock-and-key fit exists be tween a protrusion in the toxin, penetrating 20 A into a cleft in the receptor ( 1 3 8). A COUPLING PROCESS MEDIATES BINDING AND RESPONSE In some recep tor systems a process intervenes between ligand recognition and the trans duction of the biological response. This process is of importance to both



307

hormonal and neurotransmitter receptor functions. Research in specific areas has spearheaded the advance in our knowledge on this topic: the coupling of the AChR-recognition site to the ion-translocation machinery (the channel, ion modulator, or ionophore); and the coupling of several types of hormonal receptor to adenylate cyclase. In both cases, the nature of the coupling remains one of today's most important open questions in the field. The hormone case is particularly puzzling for the following reasons: (a) The receptor binding sites face the extracellular phase, the catalytic active sites the cytoplasmic phase of the membrane; (b) at least in the adipocyte, seven different hormones (t3-catecholamines, glucagon, TSH, LH, secretin, growth hormone, and ACTH) (2 1 , 42), for which effects mediated through the production of 3 I ,5 I -cAMP are documented, outnumber and compete for coupling with the cyclase molecules; (c) the lifetime of receptor-hormone complexes greatly exceeds that of the activa tion-deactivation cycle of the enzyme; and (d) in the case of morphologi cally polarized cells (e.g. the epithelial ones, having apical, lateral, and basal membranes), receptors and cyclase may be located at opposite poles of the cell (42). The first of these points does not really constitute an obstacle to physical coupling; the integral nature of these membrane pro teins suffices to guarantee the possibility of contact between the partners. Of course, the existence of a third, unknown linkage factor cannot be excluded; following on from the initial studies of Rodbell ( 1 22, and refer ences therein), growing evidence shows that a guanyl nucleotide-binding protein is involved. As regards the numerical discrepancy between receptors and cyclase, the mobile receptor hypothesis (42) has obvious implications for smoothing out these differences. The common pool of enzymes supply ing a second messenger (i.e. 3 I ,5 ' -cAMP) to different receptors apparently is able to fulfill its role because of its unspecificity, or rather universality, which applies not only to the various receptors within one cell but also to the correct coupling with foreign receptors. This is exemplified in the elegant experiments of Orly & Schramm ( l 05), in which the t3-adrenergic receptor from turkey erythrocytes (in which the cyclase was chemically inactivated) was successfully coupled with the adenylate cyclase from eryth roleukaemia cells (lacking the receptor) via cell fusion; within minutes, hormone-dependent catalytic activity was restored in the hybrid. The apparent discrepancies between the lifetimes and absolute numbers of hormone receptors and cyclase may be related to the often invoked cooperative interactions. Whereas hormones such as gonadotropins, prolac tin, calcitonin, and growth hormone show no interaction between indepen dent binding sites, insulin, t3-adrenergic catecholamines, thyroid-stimulat ing hormone, and nerve growth factor have been claimed to possess negative cooperativity (see 46, and references therein). Nevertheless, cooperativity of binding does not necessarily imply a similar type of behavior on cyclase


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activation and vice versa; vasopressin, for instance, binds non-cooperatively while activating the cyclase in a negatively cooperative fashion. The oppo site situation is observed with other high-affinity polypeptide hormones; negative cooperativity of binding would facilitate the dissociation of tightly bound hormone-receptor complexes at high ligand concentration and pro tect the cyclase from acute changes beyond tolerable levels in the circulating hormone concentration. Desensitization of either the receptor or the cy clase, well documented for example in the /3-adrenergic system, could also affect the number of functionally active liganded receptor-cyclase com plexes. An additional factor to be taken into account is the observed cellular internalization of some hormone-receptor complexes (42, 1 30), which might bear upon desensitization. This topic is covered in a recent mono graph ( 1 36) together with an elaborate treatment of the intriguing phenom enon of "single-hit" activation of the biological response, i.e. maximal hormone-mediated activation with minimal receptor occupancy. As is the case with the AChR, perturbation of the membrane framework (e.g. deter gent or phospholipase treatment) has profound effects on the coupling process, abolishing in many instances the hormonal response. The specific involvement of lipids in the coupling process has been considered (see 88). Fluorescence studies with the dansyl-derivative DNETMA ( 1 46) consti tuted one of the first pieces of experimental evidence pointing to the exis tence of "secondary sites" in addition to the AChR-recognition site (37) (see also 32, 54, 1 35). On the basis of the analogy with regulatory enzymes, Changeux and co-workers (who incidentally developed this line of research) envisage the AChR as a regulatory protein and postulate the coupling of the recognition unit with the ion-translocating unit via allosteric mecha nisms (32, 1 3 5). This avenue of research has entered a phase of rapid acceler ation now that the "secondary sites" have been attributed more firmly as corresponding to the site of action of local anaesthetics (32, 53, 1 35). Important advances have been made on the mode of action of these sub stances, which act as non-competitive antagonists of the AChR system. In particular, the developments leading to the observation of single-channel events (see below) permitted the recording of channel blockage in the presence of local anaesthetics (99). One interpretation of these findings is that the channel itself is the locus of anaesthetic action (99). In parallel, several laboratories are currently attempting the isolation of the putative molecular entity directly involved in ion translocation; histrionicotoxin, a toxin extracted from a frog, appears to constitute an appropriate medium for its characterization (53, 54), as were a-toxins for the AChR recognition site. It remains to be seen whether the ion translocation unit is in fact a distinct molecular entity, different from the AChR and physically coupled



309

to it, or merely a subunit of the latter. Recent reports identify a 43,000mol-wt polypeptide found in AChR-rich membranes as the one conferring local anaesthetic sensitivity, and by extension channel properties, to the AChR ( 1 35). In summary, it has been established beyond doubt that functional cou pling takes place between recognition and gating units in neurotransmitter and hormonal systems; physical coupling between discrete molecular con stituents carrying these functions has not yet been unambiguously demon strated in any case. Dynamic Aspects GROSS ELECTRICAL MANIFESTATIONS OF RECEPTOR-LIGAND INTERAC TION IN VIVO The AChR-mediated conductance increase occurring in postsynaptic membranes of cholinergic synapses is by far the most exten sively studied epiphenomenon of receptor-ligand interactions. The natural neurotransmitter ACh elicits the ion permeability changes that are respon sible for the conductance increase. Synchronous and massive liberation of ACh, triggered by the nerve action-potential, depolarizes the chemically excitable membrane (57, 1 37). The end-plate potential (about 0.2-1 fLA in amplitude) is the electrical manifestation of the physiologically relevant event leading to muscle contraction. In the absence of nerve stimulus ACh is also liberated; discrete quanta of about 10,000 ACh molecules are spontaneously released (57, 73), generating miniature end-plate currents of 2-5 nA within 50--300 J.Lsec. Transient local concentrations of 100300 fLM ACh would build up during this period, declining thereafter in a few milliseconds. Aside from these two transient events, a steady ACh associated depolarization of the muscle membrane of only 40 fLV has been observed (7 1 ) under special conditions and interpreted as resulting from the leakage of about 106 ACh molecules per sec (and per end-plate), which would maintain concentrations of 20--40 nM in the synaptic cleft. The factors governing these macroscopic electrical responses have been extensively covered in recent reviews (57, 1 37). The discussion that now follows concentrates on the developments leading to the characterization of the common elementary electrical event responsible for the above macro scopic behavior of chemically excitable membranes.

Ev idence in favor of discrete channels as opposed to carrier mechanisms in the AChR system dates from Katz & Miledi's (70) observations on voltage noise and those of Anderson & Stevens (5) on current fluctuations under voltage-clamp conditions (see 57). The stochastical variations in these elecEXTRACTING SIGNALS FROM MACROSCOPIC LIGAND-INDUCED NOISE


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trical parameters as induced by ACh or its analogues have been attributed to the "elementary event," that is the opening and closure of ion channels, which adds up linearly and appears superimposed on the steady conduc tance increase. The spontaneous, microscopic deviations from the steady current would thus reflect the thermal fluctuations in the equilibrium dis tribution of these individual two-state devices, i.e. the channels. The proba bilistic nature of channel open-close transitions is implicit in this interpretation. For this reason, statistical analysis has been applied to the study of these fluctuations, a subject covered in detail in various mono graphs (5, 38, 57, 1 00). Two pieces of information result from fluctuation analysis: an estimate of the mean duration of the elementary event-the channel lifetime-and the current contribution of such event. The channel lifetime is characteristic for each agonist and varies with temperature and membrane potential; typical values for ACh at -80 mV and 1 2°C are about 3 msec (57). The amplitude information on the elementary current pulse is contained in the relationship i (T2I I; the mean single-channel conductance, 'Y, follows then from 'Y = il( V v;,q), where V and v;,q are the holding and resting potential, respectively. Values of 20-30 pS have been obtained for this parameter (5, 39, 98, 1 00). =

-

The shape of the elementary current event must be assumed to be able to extract information from fluctuation analysis. Direct recording of the uni tary AChR-mediated current event has eliminated the ambiguity caused by this assumption; as shown in Figure 2, individual AChR-channel events appear as all-or-none, rectangular pulse-like elementary wave forms of a constant amplitude (a few picoamperes), which under special circumstances persist for several milliseconds. In concurrence with the results given by power spectral or autocorrelation function analysis, the observed channel open state is characterized by its random occurrence with exponentially distributed durations. The system can be treated as a Markov process with discrete states in continuous time. Each individual channel has the same probability of closing in a given period of time, regardless of its previous history. Variations in this quantity over a l O-fold range as a function of the nature of the agonist (39, 84) and over an e-fold range for every 80-mV change in membrane potential are observed. The voltage dependence is explained on the basis of differences in the dipole moment of open and closed channels, respectively (38, 84). How does this microscopic event relate to the macroscopic depolarization of the synapse? By using the experimental value of 25 pS for the elementary channel conductance and recent estimates of channel number per end plate [about 4 X 1 07 (55, 87), a maximal conductance of 1 mS could be expected to develop if all channels were activated simultaneously. The DIRECT OBSERVATION OF ELEMENTARY SINGLE-CHANNEL EVENTS



311

""��"f"' �� I

Figure 2 Direct recording of current pulses flowing through individual AChR-associated channels using the patch-clamp method (98). Basically, this technique consists of positioning a semimicropipette onto a small area on the muscle membrane (-10 f.Lm2). The effect is twofold: The imput impedance of such small area is much higher than that of the whole fiber, and secondly, on the average not more than one channel is open at any given time. ACh (500 nM) was applied to the denervated muscle fiber maintained at -80 mV holding potential (voltage clamp) at 10°C. Note approximately constant pulse amplitude (downward deflection-inward current) but varying duration of pulses. Calibration marks correspond to 200 msec and 4 pA, respectively. (Original illustration courtesy of E. Neher and B. Sakmann, Max-Planck-Institut flir Biophysikalische Chemie.)

observed peak conductance, -4 p.S (38, 57, 1 37), indicates that only 0.4% of the channels are open under such conditions. As an offspring of the patch technique for single-channel recording, "microscopic" noise, arising from a few AChR-associated channels in small areas of perisynaptic and synaptic regions, can be recorded (Figure 3).

Figure 3 "Microscopic" noise recorded with an extracellular patch electrode of the type used for single-channel measurements (98) filled with 500 nM ACh. Normal frog muscle. ( left) The noise generated by a few single channels in the perisynaptic region of a normal end-plate. When the same electrode is moved toward the synaptic region (right) the noise becomes much larger because of the superimposition of several contributing AChR-associated channels. Both parts of the recording have been taken under identical instrumental conditions. Calibration marks represent 100 msec and 4 pA, respectively. Temperature, lOoC. (Unpub lished results from J. Patlak, B. Sakmann, and E. Neher, Max-Planck-Institut flir Bio physikalische Chemie.)


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PERTURBATION STUDIES IN VIVO The electrical parameters of the excit able postsynaptic membrane are obviously the ones more amenable to control by the electrophysiologist. The potential across the membrane, for instance, can be fixed ("clamped") at the desired level and suddenly "jumped" to a new level within time periods comparatively shorter than some of the processes leading to re-equilibration of the system.3 Adams (2) exploited these properties by applying voltage jumps to measure conduc tance kinetics in the neuromuscular junction. The relaxation technique was further employed to study the voltage-sensitive components of the channel open-closed transitions (3, 78, 95, 97, 1 32). Basically the same conclusion as in fluctuation analysis or single-channel conductance mea surements was reached: When only a small fraction of the channels are open the kinetics of this process are dominated by a single exponential relaxation, whose rate constant is attributed to the mean channel lifetime. Figure 4 shows examples of voltage-jump relaxations in the single-cell preparation of Electrophof1Js electroplax, together with a novel type of relaxation measurement introduced by Lester and his colleagues (78, 95). The latter technique takes advantage of the photochemical transitions of a probe, bis-Q, which acts as a powerful agonist on the AChR in its trans-isomer form ( 1 5). After prolonged exposure to 320 nm of light, the probe exists predominantly in its cis, inactive form. Concentration jumps can be evoked thus by flash irradiation at 420 nm, which produces the reverse (cis --> trans) isomerization (Figure 4). The opposite isomerization (trans --> cis) generates a complex spectrum of relaxations (95). This is unfortunate, since the induced concentration-jump otherwise constitutes a close approximation to the natural relaxation of the AChR system fol lowing the nearly instantaneous removal of ACh in the declining phase of the neurally evoked postsynaptic currents. A further potential advan tage of the method is that it is less prone to be affected by desensitiza tion artifacts, given the short times needed to produce the concentration transients. In any event, this technique, together with the novel single channel recording (98), will certainly be of great help in understanding the mechanisms of the ion-translocation processes, particularly if it proves 3 The now classical technique of chemical relaxation kinetics (52) consists of subjecting a system, originally at equilibrium, to an enforced sudden perturbation in an intensive thermo dynamic variable and following the system's evolution towards a new equilibrium. The tempo ral response of this evolution is characterized by a discrete spectrum of relaxation times and associated amplitudes. Usually the dependence of a physical parameter (fluorescence,

transmission) upon the concentration of the reactants is followed, and the resulting spectra

of relaxations is analyzed in terms of theoretical reaction mechanisms. For small enough perturbations, the reaction-free energies for each step are a function of all the net reaction advancements, and reaction rates are characterized by linearized first-order equations.


313

f lash

�

+

:C:::::-.. : --:::


__

_ _

Figure 4 Relaxations associated with jumps of membrane voltage and of agonist concentra tion at Electrophorus electroplaques. Three voltage clamp episodes, taken at intervals of 0.5 sec. Traces show agonist-induced currents in the presence of the photoisomerizable com pound bis-Q (3,3 ' -bis[a-(trimethylammonium)-methyl]azobenzene, 400 nM) (15). At the start of the trial, the solution contained 60 nM trans-bis-Q (a potent agonist); the remaining bis-Q was in the inactive cis-form. Fifteen milliseconds after the start of each episode the voltage was jumped from +5 1 to - 1 50 mY. The agonist-induced current increased along an exponential time course. For the first two episodes the voltage-jump relaxations superim pose; the rate constant is 0. 1 msec-1. About half-way through the second episode, a flash light (Xe are, duration 0.5 msec) increases the trans-bis-Q concentration to about 240 nM. Following the concentration jump, the conductance increases exponentially to a much larger value; the rate constant in this case was 0. 1 7 msec-1. The third voltage jump (lower trace) occurred in the higher bis-Q concentration; the rate constant and final conductance equal those for the concentration-jump relaxation. Calibration marks: 24 msec and 24 mA/cm2• Temperature, goC. (Original record kindly provided by H. A. Lester, M. M. Nass, and M. E. Krouse, California Institute of Technology.)

possible to combine them with physical signals reporting on receptor state in vivo. The Present Melting Point: Converging Towards Molecular Mechanisms

The application of a variety of techniques to the study of the AChR system in vivo and the recent availability of suitable preparations and probes for its characterization in vitro have resulted in the formulation of questions critical to the definition of the basic problems. Not unexpect edly, the questions are the same, since they concern the elementary steps of the receptor-ligand interactions at the molecular level. Although a two-state (open-closed) model of an idealized channel at first glance might be contem plated in accounting for the AChR-associated ion translocation, such sim plification yields an insufficient description of the reality. The reasons for this inadequacy are fully substantiated in a recent review (40). The three-state reaction mechanism postulated on the basis of fluctuation analysis (5, 84) closely resembles the one originally formulated by del Castillo & Katz (45) by analogy with enzyme-substrate reactions: SINGLE-CHANNEL EVENTS, NOISE, AND MODELS

3 14

BARRANTES

..... R + nL ""'

ki

..... RLn .... /3

.::0..


_ _

k2

a

where R, RL, and R* L are the receptors in the resting, liganded, and active states, respectively, and n is the number of ligand molecules needed to open the channel. This sequential binding-isomerization scheme has dominated the conceptualization of the process; its intuitive strength, bio chemical roots, and lack of measurable physical signals have practically ruled out the consideration of the (formally feasible) alternative scheme where the spontaneous isomerization between open and closed states exists in the absence of ligand and preceeds the binding step. Probabilistic consid erations are also invoked to exclude the latter scheme (e.g. 38, 40) and to favor the biliganded state as the one finally leading to the open conforma tion of the AChR (3). Since both reaction mechanisms predict two relax ations, and only one is observed under normal conditions by all types of electrical measurements, the distinction between these two (and many more complicated) schemes is not yet clear. This uncertainty in deciding between kinetic schemes extends to the inability to determine whether binding ( k2 ([L] + 1 ) � (a + /3) or isomerization (the reverse condition) constitutes the rate-limiting step in the above reaction. Indirect information on the binding step in vitro has been obtained by monitoring the displacement of CaH from the detergent-solubilized AChR by using the probe murexide and absorption spectroscopy ( 1 0 1). The bimo lecular association rate constant calculated for ACh was in the order of 107 M-I sec- I. The concentration dependence of the fast reaction followed with intrinsic fluorescence on the membrane-bound AChR ( 1 3; and see below) was also compatible with a binding reaction of agonist to the (pre sumably) resting state of the AChR; the on-rate was of the same order. What inferences can be made from these simple facts on the characteristics of the binding of ACh in vivo? The most straightforward is that unless an unknown rate-enhancement mechanism operates in the living synapse, binding must necessarily constitute the rate-limiting step in the reaction leading to the channel activation. Similar constraints are also apparent in the rationalization of electrophysiological data (3, 1 32). In view of the previously discussed rotational immobilization of the AChR in its mem brane-bound form, enhancement in association rates might have been ob served in going from the solubilized ( 1 0 1 ) to the membrane-bound state ( 1 3). Only a 1 0-fold reduction of the dissociation rate constant is observed. This implies that the angular constraint factors invoked to account for the drastic decrease in association rates of ligands to macromolecules ( 1 3 1 ) as compared t o the maximum diffusion controlled rate predicted for such a pair from the Smoluchowski formalism (-8 X 1 07 M - I sec- I) are not operative in this case. The often invoked reduction of dimensionality factors



315

of Adam & Delbrlick (1) can be rejected as rate-enhancement agents on the same grounds, with the additional comment that guided diffusion (5 1 , 1 2 1) would add little in vivo, given the end-plate geometry and high density of the AChR. The ionic effects stressed by some authors (1 37, and refer ences therein) are in fact second order (5 1), since they can not account for the gap between the on-rates observed in vitro and the maximal attain able values needed in putative models where channel kinetics are rate limiting. Furthermore, an elementary calculation based on available experi mental data suggests that binding is in fact rate limiting. Thus, it was shown above from the observed conductances of the single channel event and the miniature end-plate current (25 pS and 4 p.S, respectively), and from the measured number of toxin sites per end-plate [-4 X 1 07 (see 87)J, that only about 0.4% of the available channels (assuming a conserva tive 1 : 1 ratio of toxin sites per channel, given the uncertainties) would be open at the peak of the macroscopic electrical event. Assuming that every liganded AChR corresponds to an open channel state, and using the recently calculated values of - 1 mM for the transient ACh concentra tion in the synaptic cleft (73, 87) and the value of 50 p.sec for the rise time of the miniature end-plate currents (57, 1 37, and references therein), a first-order association rate constant in the order of 107 M- l sec-1 results. CONFORMATIONAL STATES OF THE ACHR AND TRANSITIONS THEREOF

Identification of discrete conformational states in macromolecules by non destructive techniques is, to some extent, an indulgent connivance. Its validity holds, however, if one considers that such states represent averages over the population and over the accessible time domain. Recent experi mental work in this field is beginning to unravel some kinetic pathways of the neurotransmitter receptor and its effectors, which might have rele vance to the physiological ones. In particular, some of these studies point to the existence of states identifiable with those postulated in vivo. One of the factors that made these studies possible is the availability of suitable probes for physical investigation, among which fluorescent probes have received the most attention. Since the introduction by Weber and co-workers ( 1 46) of a dansyl-choline analogue, a variety of bifunctional ligands carrying a fluorescent moiety and a receptor-directed chemical group have been designed (Figure 5). These probes, in combination with appropriate spectroscopic detection, have enabled the application of steady state and rapid kinetic techniques for the characterization of reaction path ways and intermediates in the AChR-ligand interactions (37, 63, 64, 66, 1 1 3). In addition to the use of fluorescent markers, intrinsic fluorescence of the membrane-bound AChR has been employed recently to measure the kinetics of nonfluorescent agonists interacting with the AChR ( 1 2, 1 3, 22). The latter, in a way minimal perturbation, studies provided one

3 16

BARRANTES

", CH) CH2 CH, N / ;::: C H) CH3 H1N N N ,O / �N NO,


Ibl

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lei

�H3 / CH,CH3 HC (CH2 13 N

'CH,CH3

�H

lei

H3CO� VN�CI If I

Figure 5 Some fluorescent probes used in the study of receptor-ligand interactions. In addi tion to the more direct approach with intrinsic fluorescence spectroscopy, extrinsic markers have found wider applicability in this area of research. The current strategies associated with the use of such probes are simultaneously listed. Active-site-directed probes, conveniently derivatized with pharmacologically active chemical groups, are designed with the aim of characterizing the recognition site environment, solvent exposure, local segmental motion in the vicinity of the site, etc, and learning about the mechanism of interaction with the receptor. Examples of this type of probes are (a) the dansyl-derivatives like DNETMA (146) (where X CH2, n = I, i.e. I-dimethylaminonaphthalene-S-sulphonamido-ethyl-tri methylammonium) and others where the choline moiety was separated from the fluorophore by n varying from 2 to 6 (143). (b) Pyrene derivatives. The rationale behind their introduction for the study of the AChR was the information gained on other "large" macromolecules where the rotational relaxation times was investigated; the need to match long tumbling times with correspondingly long excited-state fluorescence lifetimes can be satisfied with this type of probes (14). One of the smallest chromophores available, covalently linked to the choline-like quaternary ammonium group ( c), was synthesized to circumvent the most acute problem in the study of the AChR with extrinsic markers: the bulkiness and hydropho bicity of the chromophore moiety conferring antagonistic or mixed agonist-local anaesthetic pharmacological activity on the probes (see 1 3). Probe ( c) is 2-[4-(7-nitro)-2, l , 3-benzodioxazo lylamino]-ethyl-trimethylammonium (NBD-choline; W. Stender and F. J. Barrantes, unpub lished data). ( d ) Bis-quaternary ammonium compounds such as DAP [bis 3-(amino pyridinium)-1 . lO-decane] have been used both in the study of the acetylcholinesterase and the AChR (86, I I S). There are few examples of active-site-directed probes in other receptor systems. An exception is the J3-adrenergic blocker probe 9-acridinopropanolol (e) introduced by Levitzki and colleagues (8). Ligands of recognized pharmacological activity have been derivatized with fluorescent markers. Advantage is taken of the biological specificity of the ligand; insulin (130), a-neurotoxin (6, 7, 9), and melanocyte stimulating hormone (140), covalently coupled to dansyl-, rhodamine, or fluorescein, are examples of this series. Indirect immunofluorescence techniques, where fluorescent-labeled antibodies to, for example, the AChR (2S) or the luteinizing hormone (67) were used, also find application. A larger number of fluorescent probes, not directly interacting with the receptor recognition site, or alternatively sensing the environment of the receptor, are exemplified by quinacrine (/) (63, 64), RbH (127), and ethidium bromide (1 13). Quinacrine, an antimalaria drug with local anaesthetic like activity in the AChR system, has been employed in kinetic studies with the membrane bound AChR. RbH has been used to explore the ionic environment, and in particular the effect of Ca2+ on the AChR (127, 128). =


��I,.\Io,v�� I


o

54

128 192 C ha n n e l

,

,

"

,

� � �_"

� _ _-_-��

,

317

j

256

Figure 6 Kinetics of agonist-induced intrinsic fluorescence changes in the membrane-bound AChR from Torpedo. (Left) Experimental traces of six averaged stopped-flow records ob tained upon mixing 6.5 ,..M suberyldicholine (a potent agonist) with the AChR-rich mem branes at low temperature (4 ° C), and theoretical curve fitting the data to a three-exponential decay. The eigenvalues obtained are 1 5, 1.4, and 0. 14 sec- l. Analysis was carried out by using the method of modulating functions of G. Striker (see 13, and references therein). Calibration bar in the upper right quadrant corresponds to 50 msec for the first trace, and to 400 msec for the second and third traces. ( Middle panel) Deviation between the experimen tal and fitted curves. (Right panel) Autocorrelation function of the error, an additional verification of the quality of the theoretical fit.

of the first pieces of experimental evidence on the existence of states of the AChR reported by an intrinsic physical signal. More importantly, direct access to the binary AChR-agonist system (as opposed to AChR agonist-probe ternary systems) was possible. The physical states revealed by the protein fluorescence could be attributed to functional states known to occur in vivo. Figure 6 shows the kinetics of the intrinsic fluorescence induced by a potent agonist, suberyldicholine. The studies with extrinsic fluorescence markers are in this respect complementary to the latter ones, not only because they rely in some cases on the aforementioned ternary complexes, but also because only probes with partial (see Figure 5) or mixed agonist-local anaesthetic pharmacological activity have been ob tained to date. Stopped-flow studies with quinacrine (64) or ethidium bro mide ( 1 1 3) reflect these limitations, but provide useful information on alternative pathways, which the AChR probably follows in the presence of modifiers, as outlined in the lower half of the scheme below. RB - - - - - - - - - - -DB ' 1 ,//1

1

I 1

I 1

I I

MRA----- - --MDA '

I

I

:

"

..}k,.,

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/

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I

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1

I

'- ' R - - - - - - - - - - - -D / J ...... "./ I II ' RA - - - - - - - - --DA/ I I .... . I ..... I I : -·· .. RA/" ;

' '-

,

,

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MRB - - - - - - - ,- - - -MDB

(1)


318

BARRANTES

Here the normal AChR-agonist interactions, of which the binding step of agonists (A) to the desensitized state D (66) or the resting form R ( 1 3) has been identified in vitro, constitute the "ceiling" of a provisional frame, which summarizes the kinetic studies. Reactions involving antago nists (blockers, B) are also included. Both in vivo (99) and in vitro (64, 66, 1 1 3) modifiers (M) alter the "normal" (upper half) cycles: either by plugging the open, activated state R* (99), by accelerating desensitization (64, 96, 1 37), or by inducing metaphilic effects (see 37). Such modified pathways are indicated in the lower half of the diagram. Probably many, if not all, of the poles in this scaffolding will have to be rebuilt several times before the edifice is finally completed. ACKNOWLEDGMENTS I am deeply indebted to my wife, without whose continuous help and encouragement this work would not have been completed. I also extend my thanks to the various colleagues who provided helpful comments and illustrative material. Literature Cited

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