2nd NATIONAL CONGRESS, ITALY
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BIOCHEMISTRY OF CELL MEMBRANES: RECEPTOR SITES AND ENZYMES: a Colloquium organized by G. H. de Haas, J. N. Hawthorne, J. Massoulit5 and G. Porcellati
Receptor Sites on Cell Membranes EVeRARD J. ARIENS and ANNA-MARIA SIMONIS Pharmacological Institute, University of Nijmegen, Nijmegen 6804, The Netherlands An understanding of drug action requires a molecular approach, since an active agent can only induce a pharmacodynamic effect in a biological object as the result of an interaction between its molecules and certain molecules in the biological object. The chemical properties of a drug, therefore, are determinants of its action and activity. Thus a relationship between chemical (structural) properties and action must exist (Ariens, 1971). The molecular sites of action of drugs, i.e. those molecules with which the active agent must interact in order to induce the effect considered, are called the specific receptors. The receptors are located in or on the target cells, which are not necessarily the cells in which the effect is generated. The parameter, considered as the effect, is to a certain degree arbitrary. For instance, the receptors for the convulsant agent strychnine are located in the central nervous system, but the convulsions are generated in the striated muscle. However, instead of the convulsions, the changes in the electroencephalogram may also be measured as the effect. As shown by binding studies, the receptors for various transmitters, drugs, hormones and hormonoids are located in the cell membranes (De Robertis, 1976). The receptors for acetylcholine (the cholinergic receptors) can be shown to be located on the outside of the membrane, since application of cholinergic agents under the cell membrane appears to be ineffective @el Castillo & Katz, 1955). For other receptor types, namely those for noradrenaline, histamine and 5-hydroxytryptamine, a location on the outside of the cell membrane is also probable, since these receptors are easily accessible for the quaternary form of the respective competitive blocking agents (Ariens, 1967, 1971). A location on the cell membrane has also been confirmed, by binding studies, for the receptors of various peptide hormones and adrenaline involved in the activation of adenylate cyclase. The drug-receptor interaction is, as a rule, reversible and is much more dynamic than the classical lock-and-key model suggests. It implies a mutual moulding of drug and receptor by intermolecular forces. In the receptor molecule, conformational changes are induced which trigger the sequence of biochemical and biophysical events leading to the effect (Ariens, 1964). For enzyme-substrate interaction the changes induced in the substrate molecule are essential. With regard to receptor isolation and identification, there is an essential difference between soluble receptors, e.g. the steroid-receptorproteins from the cytoplasm,and membrane-boundreceptors. There is a tight interrelation between the receptor molecule and the surrounding molecules of the membrane, and conformational changes induced in the receptor molecule also involve the surrounding molecules of the membrane. Separation of the receptor molecule from its surroundings may well disturb its specific conformation, and thus its specific receptor characteristics. Undoubtedly the isolation technique, especially the type of detergents (Maddy & Dunn, 1976) used as solubilizing agents, may influence the conformation of the isolated protein. Therefore the variation in binding constants reported for drug-isolated-receptor interactions does not mean that there is necessarily a variety of physiological receptor states or conformations. A special problem, which
Vol. 5
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BIOCHEMICAL SOCIETY TRANSACTIONS
applies to the soluble as well as the membrane-bound drug receptors, is that once the receptors have been isolated no pharmacological effects can be induced. This makes identification of the receptors very dficult. Analogously to the differentiation between the active site on an enzyme and the enzyme molecule, a distinction must be made between the receptor site and the receptor molecule. The molecules, not the sites, can be isolated. Since the structure-action relationship is based on interaction between the drug molecule and the receptor site, it may give information on the properties of this site. A certain degree of chemical complementarity must be assumed to exist between the drug and the specific receptor site with which it interacts. The structure-binding relationship therefore can be used as a tool for the identification of isolated receptors. One may expect that the structure-binding relationship for an isolated receptor, assuming that it is not to any degree denatured in the isolated form, will parallel the structureaction relationship except that for the isolated receptors there will be, as a rule, little difference between the action of agonists, partial agonists and competitive antagonists; all thesecompoundshavean affinityfor the receptor molecules, but only the first two classes have intrinsic activity. In order to isolate and identify receptors, use can also be made of agents that irreversibly bind in a selectiveway to the receptors or, better still, to specific receptor sites on the receptors. Such agents are only rarely available. The reactive molecules, e.g. alkylating agents, used for such affinity labelling have a tendency also to bind irreversibly to various unspecific sites on proteins. On the basis of the postulated complementarity between drug molecules and their specific receptor sites, structure-action-relationship studies can be used for receptor-site mapping. Interesting results are thus obtained with regard to various membrane-active agents, such as acetylcholine, histamine and noradrenaline, and their respective competitive antagonists. An agonist and an antagonist have an affinity for a ‘common’ receptor, but only the agonist has intrinsic activity. The interaction of the agonist molecule with the receptor results in a change to the activated (A) state or conformation, whereas the competitive antagonist, on binding, keeps the receptor in the non-activated (N)state. With partial agonists only a fraction of the drug-receptor interactionsresults in activation; the intrinsic activity depends on the size of this fraction (Ariens, 1964). Taking into account the complementarity between drugs and their receptor sites, the agonist and competitive antagonist should be chemically related. This often appears not to be the case. There is little or no chemical relationship between an agonist and the corresponding antagonist. There is, however, much similarity between the chemical structures of the various types of competitive antagonist (Ariens & Simonis, 1967). The various agonists are highly polar hydrophilic molecules with clear-cut differences in structure, for instance charge distribution. Competitive antagonists, on the other hand, are predominantly hydrophobic in nature. They have as a rule hydrophobic double-ring systems, located at a certain (three to five atoms) distance from an amino or ammonium group. It is essentially the hydrophobic groups that cause the high affinity for the receptors and they therefore cannot be bound to the polar receptor sites complementaryto the highly polar agonists (Ariens & Simonis, 1967). Introduction of centres of asymmetry into various moieties of the drugs, for instance cholinergic and anticholinergicagents, indicatesthat in the binding ofagonists and the corresponding antagonists,essentiallydifferent chemical groups and different receptor sites are involved. The activity ratio for the optical isomers of the cholinergicagent acetyl-8-methylcholine (200) indicates an intricate relationship between the choline moiety and the receptor site. Elimination of the onium group results in a total loss of activity. On the other hand, the ratio for the optical isomers of the anticholinergic benzilic acid ester of 8-methylcholine (5/6) indicates that there is a large degree of structural freedom and therefore only a loose binding of the choline moiety to the receptor site occurs. For the cyclohexylphenylglycollicacid ester of choline, in which the centre of asymmetry is located in the hydrophobic acidic moiety, the ratio is 60, which indicates an intricate relationship between this moiety and the receptor site. For the cyclohexylphenylglycollic acid ester of p-methylcholine, in which there are two centres of asymmetry,
1977
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the ratios for the pairs of isomers that differ in their centres of asymmetry in the choline moiety, are 4 and 2, whereas for the pairs of isomers that differ in their centres of asymmetry in the hydrophobic acidic moiety, the ratios are 100 and 50. Replacement of the choline moiety of the cyclohexylpheny1glycollic acid ester of choline by neopentyl alcohol gives a compound that is still a potent anticholinergic agent even though there is no onium or amino group present; the activity ratio for the optical isomers of this compound, with the centre of asymmetry in the hydrophobic acidic moiety, is 100 (Ariens & Simonis, 1967). These observations clearly indicate that the hydrophobicmoieties of anticholinergicagents, by binding to accessory hydrophobic areas, strongly contribute to their affinity for cholinergic receptors. Anticholinergic agents apparently bind to accessory binding areas which are functionally related to the receptor sites for the cholinergic agonists and thus block the activation of the receptor by the agonist. They interfere with the binding of the agonist to its receptor sites without actually occupying the receptor site in the strict sense. There is a ‘dualism’ in the receptor sites. On the basis of this concept, the lack of structural relationship between agonists and the corresponding competitive antagonists, the existence of structural relationships between competitive antagonists blocking different types of agonist receptors, the existence of competitive antagonists blocking the receptor for different agonists, and the dependence of the selectivity of the action of the competitive antagonists on the steric structure of the hydrophobic moieties in the molecule (Harms & Nauta, 1960; Rekker et al., 1971) are understandable (Ariens & Simonis, 1976). The receptor sites for agonists and competitive antagonists are not ‘common’ as suggested by the original ‘one receptor site’ concept. The receptor sites for the drugs do not necessarily have to be located on the surface of the receptor proteins. This is, however, likely for the relatively polar sites for the agonist agents, although these receptors may also be located at, and even be constituted by, the interface, where the polar groups of the protein interact with the polar groups of the membrane lipids, e.g. the phosphate and choline groups in phosphatidic acid and sphingosidic acid. Similarly the receptor sites for the hydrophobic moieties of the various competitive antagonists of membrane-active agents may well consist of the interfacebetween the hydrophobicsurfaceof the protein and the lipid chains in the membrane. The extremely high affinity constants, lo9 and 1O1O, for competitive antagonists (e.g. anticholinergic and antihistaminic agents), which are mainly the result of the hydrophobic groups, can hardly be expected to exist on a mere protein surface. Lipoproteins would be more suitable in this respect. The fact that the lipid molecules facing the hydrophobic surface of the protein are to a large extent in a quasi-crystalline form makes the high degree of stereospecificity, especially with regard to centres of asymmetry in the hydrophobic moiety, of the antagonists understandable. Drugreceptor interaction then results in changes in the interface characteristics. Isolation of complete receptors as molecular entities is impossible. Incorporation of the receptorprotein fraction into a membrane may be required for receptor reconstitution (De Robertis, 1976). The interaction between the receptor protein and the membrane lipids will strongly influence the conformation of the protein and thus the action of drugs on, and their affinity for, the receptor protein. The receptor molecule must be assumed to occur in different states, an activated (A) and a non-activated (N) form, depending on the presence of the agonist or competitive antagonist. Kasai & Changeux (1971) postulated that even in the absence of drugs there is a dynamic equilibrium between the different functional states of the cholinergic receptor. The simplest concept in this respect is the ‘dual receptor concept’: the occurrence in the cell membrane of an equilibrium between the receptors in the A and those in the N form (conformation). The A form contributes to the effect, and the N form does not. An agonist shifts the equilibrium towards the A conformation and the competitive antagonist shifts it towards the N conformation. The agonist has a relatively high a6nity for the receptors in the A conformation, and the competitive antagonist for those in the N conformation, thus to a certain degree locking the receptors
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in the respective states. Partial agonists have an a f i i t y for both states, the ratio of the affinities determining the intrinsic activity. As long as the rate constants for the equilibrium are large enough, this model will result in phenomena similar to those expected for competition between drugs based on the ‘one receptor site’ concept. The receptor sites for agonist and antagonist, although one receptor protein is involved, are therefore different, which is in accord with the idea of dualism in receptor sites deduced from the structure-action relationship outlined above. Taking into account the fact that the receptor molecules are embedded in the lipid membrane structure, one may expect that the behaviour of such protein molecules, for instance their tendency for aggregation or segregation, strongly depends on the hydrophobic and hydrophilic properties respectively of the protein surface, especially of that part of the receptor molecule in contact with the lipids in the membrane. The receptor proteins, although predominantly hydrophobic (De Robertis, 1975), have an amphiphilic character. Binding of the agonists, usually strongly polar agents, may change the conformation and thus shift the equilibrium of the receptor population towards a more hydrophilic state, i.e. the A form, whereas binding of hydrophobic antagonistic agents may shift it towards the hydrophobic state, i.e. the N form. The change in the tertiary structure involved may well result in changes in the quaternary structure of the proteins, and thus in the degree and type of aggregation among receptor molecules or among receptor molecules and other proteins in the membrane. This receptor-activation-migration-aggregation model, which fits quite well with the fluid-mosaic concept for the membrane structure (Singer & Nicolson, 1972). Various membrane-active drugs clearly change the protein distribution in membranes and the type of receptor-protein aggregation in solutions of isolated receptors. The proteins in the luminal cell membrane of the toad bladder aggregate to a mosaic pattern under the influence of the antidiuretic hormone (Kachadorian et al., 1975). The protein-walled pores thus formed may well account for the increased water permeability induced by this hormone. The various receptor proteins for the different hormones activating adenylate cyclase are supposed to aggregate with the enzyme molecule under the influence of the various hormones, thus activating the enzyme in an allosteric way (Cuatrecasas et al., 1975; Jacobs, 1976). Also the mode of aggregation among receptor proteins may be changed under the influence of drugs. In those cases in which proteinwalled pores are present in a membrane in the normal state, their permeability to, for instance, ions may well change as a result of a charge rearrangement in the receptor molecules constituting the pores. The proteins may switch from an open to a closed conformation. Again there may be an equilibrium between these conformations, shifted by the agonist to (usually) the opcn conformation and by the competitive antagonists to the closed conformation. In the dual-receptor-site model, although there is only one receptor protein, the receptor sites for agonist molecules and for the corresponding antagonist molecules are distinct functionally interdependent entities. Therefore affinity constants for, e.g., an agonist derived from displacement experiments may well depend on whether agonist or antagonist molecules are displaced. The dual-receptor-site concept undoubtedly opens intriguing perspectives for the interpretation of the action of drugs, hormones etc. on membrane receptors. Ariens, E. J. (1964)Mol. Pharmacol. 1, 119-286 Ariens, E.J. (1967)Ann. N. Y. Acad. Sci. 139,606-631 Ariens, E.J. (1971)Drug Design 1, 1-157 Ariens, E.J. & Simonis, A. M. (1967)Ann. N.Y. Acad. Sci. 144, 842-868 Ariens, E.J. & Simonis, A. M. (1976)in Beta-Adrenoceptor Blocking Agents (Saxena, P. R. & Forsyth, R. P.,eds.), pp. 3-27, North-Holland Publishing Co., Amsterdam Cuatrecasas,P., Hollenberg, M. D., Chang, K. J. &Bennett, V.(1975)Recent Prog. Horm. Res. 31,37-94
Del Castillo, J. & Katz, B. (1955)J. Physiol. (London) 128,157-181 Pe Robertis, E.(1975)Rev. Physiol. Biochem. Pharmacol. 73, 9-38 1977
2nd NATIONAL CONGRESS, ITALY
51 1
De Robertis, E. (1976) in The Structural Basis of Membrane Function (Hatefi, Y . & DjavadiOhaniance, L., eds.), pp. 339-361, Academic Press, New York, San Francisco and London Harms, A. F. & Nauta, W. Th. (1960) J. Med. Pharm. Chem. 2,57-77 Jacobs, S . & Cuatrecasas, P. (1976) Biochim. Biophys. Acta 433,482-495 Kachadorian, W. A., Wade, J. B. & DiScala, V. A. (1975) Science 190, 67-69 Kasai, M. & Changeux, J. P. (1971) J. Membr. Biol. 6, 24-32 Maddy, A. H. & Dunn, M. J. (1976) in Biochemical Analysis of Membranes (Maddy,A. H., ed.), pp. 177-196, Chapman and Hall, London Rekker, R. F., Timmerman, H., Harms, A. F. & Nauta, W. Th. (1971) Arzneim-Forsch. 21, 688-691 Singer, S . J. & Nicolson, G. L. (1972) Science 175,720-731
Purification and Characterization of the Cholinergic Receptor Protein in its Membrane-Bound and Detergent-Soluble Forms from the Electric Organ of Torpedo marmorata AND&
SOBEL AND JEAN-PIERRE CHANGEUX
Laboratoire de Neurobiologie Moldculaire, Institut Pasteur, 25 Rue du Dr. Roux, 75724 Paris Cedex 15, France
The cholinergic receptor protein has been purified from electric organs of the electric fishes Electrophorus electricus and Torpedo by affinity chromatography through a column made up of a cholinergic ligand covalently bound to Sepharose [for review see Karlin (1974) and Changeux (1975)l. However, it has been shown that a prolonged incubation of the solubilized cholinergic receptor with a cholinergic agonist (Weber et al., 1975), as well as the passage through an affinity column (Sugiyama & Changeux, 1975), may change the molecular properties of the receptor protein. In the present communication, we describe a simple and rapid method for the purification of large amounts of acetylcholine-receptor-rich membranes as well as of acetylcholine-receptor protein, without the use of an affinity-chromatography step. It has been shown previously (Cohen et al., 1972; Nickel & Potter, 1973; Duguid & Raftery, 1973a,b), that the fresh Torpedo electric organ can be fractionated after homogenization by a differential centrifugation followed by an ultracentrifugation in a sucrose gradient, in order to yield membrane fragments particularly rich in the cholinergic-receptor protein. These membranes have a specific radioactivity of 2-3 pmol of 3H-labelled a-toxin-binding sites/g of protein and most likely represent pieces of subsynaptic membrane. The original fractionation method of Cohen et al. (1972) has been improved in order to obtain larger quantities of completely pure subsynaptic membranes. All the purification steps were carried out in the presence of 0.1 mwphenylmethanesulphonyl fluoride as a proteinase inhibitor and 0.02 % NaN, to prevent bacterial growth. The freshly dissected electric organ from Torpedo marmorata was homogenized in distilled water containing NaN, nd phenylmethanesulphonyl fluoride, and then submitted to successive differential centrifugations. A first low-speed centrifugation (7000g, 10min) of the homogenate gave a supernatant S1 and a pellet PI,which was homogenized again and re-centrifuged in the same conditions to yield the S2 supernatant. Supernatants S1 and S2 were mixed to form supernatant SIF2,which was centrifuged at 10OOOgfor 2h to pellet the membranes (P,). P3was resuspended in 32 % sucrose and the resulting membrane suspension (El) was layered on top of a discontinuous sucrose gradient (3437.5-41.5 %) and centrifuged at lOOOOOg for 6 h. The band at 37.5 %sucrose was collected, diluted and centrifuged to pellet the membranes, which were again resuspended in 32% sucrose (E2). Suspension Ez was layered on top of a continuous (35-43 %) sucrose gradient and centrifuged at 1OOOOOg for 6h. The cholinergic-receptor-
Vol. 5
507
BIOCHEMISTRY OF CELL MEMBRANES: RECEPTOR SITES AND ENZYMES: a Colloquium organized by G. H. de Haas, J. N. Hawthorne, J. Massoulit5 and G. Porcellati
Receptor Sites on Cell Membranes EVeRARD J. ARIENS and ANNA-MARIA SIMONIS Pharmacological Institute, University of Nijmegen, Nijmegen 6804, The Netherlands An understanding of drug action requires a molecular approach, since an active agent can only induce a pharmacodynamic effect in a biological object as the result of an interaction between its molecules and certain molecules in the biological object. The chemical properties of a drug, therefore, are determinants of its action and activity. Thus a relationship between chemical (structural) properties and action must exist (Ariens, 1971). The molecular sites of action of drugs, i.e. those molecules with which the active agent must interact in order to induce the effect considered, are called the specific receptors. The receptors are located in or on the target cells, which are not necessarily the cells in which the effect is generated. The parameter, considered as the effect, is to a certain degree arbitrary. For instance, the receptors for the convulsant agent strychnine are located in the central nervous system, but the convulsions are generated in the striated muscle. However, instead of the convulsions, the changes in the electroencephalogram may also be measured as the effect. As shown by binding studies, the receptors for various transmitters, drugs, hormones and hormonoids are located in the cell membranes (De Robertis, 1976). The receptors for acetylcholine (the cholinergic receptors) can be shown to be located on the outside of the membrane, since application of cholinergic agents under the cell membrane appears to be ineffective @el Castillo & Katz, 1955). For other receptor types, namely those for noradrenaline, histamine and 5-hydroxytryptamine, a location on the outside of the cell membrane is also probable, since these receptors are easily accessible for the quaternary form of the respective competitive blocking agents (Ariens, 1967, 1971). A location on the cell membrane has also been confirmed, by binding studies, for the receptors of various peptide hormones and adrenaline involved in the activation of adenylate cyclase. The drug-receptor interaction is, as a rule, reversible and is much more dynamic than the classical lock-and-key model suggests. It implies a mutual moulding of drug and receptor by intermolecular forces. In the receptor molecule, conformational changes are induced which trigger the sequence of biochemical and biophysical events leading to the effect (Ariens, 1964). For enzyme-substrate interaction the changes induced in the substrate molecule are essential. With regard to receptor isolation and identification, there is an essential difference between soluble receptors, e.g. the steroid-receptorproteins from the cytoplasm,and membrane-boundreceptors. There is a tight interrelation between the receptor molecule and the surrounding molecules of the membrane, and conformational changes induced in the receptor molecule also involve the surrounding molecules of the membrane. Separation of the receptor molecule from its surroundings may well disturb its specific conformation, and thus its specific receptor characteristics. Undoubtedly the isolation technique, especially the type of detergents (Maddy & Dunn, 1976) used as solubilizing agents, may influence the conformation of the isolated protein. Therefore the variation in binding constants reported for drug-isolated-receptor interactions does not mean that there is necessarily a variety of physiological receptor states or conformations. A special problem, which
Vol. 5
508
BIOCHEMICAL SOCIETY TRANSACTIONS
applies to the soluble as well as the membrane-bound drug receptors, is that once the receptors have been isolated no pharmacological effects can be induced. This makes identification of the receptors very dficult. Analogously to the differentiation between the active site on an enzyme and the enzyme molecule, a distinction must be made between the receptor site and the receptor molecule. The molecules, not the sites, can be isolated. Since the structure-action relationship is based on interaction between the drug molecule and the receptor site, it may give information on the properties of this site. A certain degree of chemical complementarity must be assumed to exist between the drug and the specific receptor site with which it interacts. The structure-binding relationship therefore can be used as a tool for the identification of isolated receptors. One may expect that the structure-binding relationship for an isolated receptor, assuming that it is not to any degree denatured in the isolated form, will parallel the structureaction relationship except that for the isolated receptors there will be, as a rule, little difference between the action of agonists, partial agonists and competitive antagonists; all thesecompoundshavean affinityfor the receptor molecules, but only the first two classes have intrinsic activity. In order to isolate and identify receptors, use can also be made of agents that irreversibly bind in a selectiveway to the receptors or, better still, to specific receptor sites on the receptors. Such agents are only rarely available. The reactive molecules, e.g. alkylating agents, used for such affinity labelling have a tendency also to bind irreversibly to various unspecific sites on proteins. On the basis of the postulated complementarity between drug molecules and their specific receptor sites, structure-action-relationship studies can be used for receptor-site mapping. Interesting results are thus obtained with regard to various membrane-active agents, such as acetylcholine, histamine and noradrenaline, and their respective competitive antagonists. An agonist and an antagonist have an affinity for a ‘common’ receptor, but only the agonist has intrinsic activity. The interaction of the agonist molecule with the receptor results in a change to the activated (A) state or conformation, whereas the competitive antagonist, on binding, keeps the receptor in the non-activated (N)state. With partial agonists only a fraction of the drug-receptor interactionsresults in activation; the intrinsic activity depends on the size of this fraction (Ariens, 1964). Taking into account the complementarity between drugs and their receptor sites, the agonist and competitive antagonist should be chemically related. This often appears not to be the case. There is little or no chemical relationship between an agonist and the corresponding antagonist. There is, however, much similarity between the chemical structures of the various types of competitive antagonist (Ariens & Simonis, 1967). The various agonists are highly polar hydrophilic molecules with clear-cut differences in structure, for instance charge distribution. Competitive antagonists, on the other hand, are predominantly hydrophobic in nature. They have as a rule hydrophobic double-ring systems, located at a certain (three to five atoms) distance from an amino or ammonium group. It is essentially the hydrophobic groups that cause the high affinity for the receptors and they therefore cannot be bound to the polar receptor sites complementaryto the highly polar agonists (Ariens & Simonis, 1967). Introduction of centres of asymmetry into various moieties of the drugs, for instance cholinergic and anticholinergicagents, indicatesthat in the binding ofagonists and the corresponding antagonists,essentiallydifferent chemical groups and different receptor sites are involved. The activity ratio for the optical isomers of the cholinergicagent acetyl-8-methylcholine (200) indicates an intricate relationship between the choline moiety and the receptor site. Elimination of the onium group results in a total loss of activity. On the other hand, the ratio for the optical isomers of the anticholinergic benzilic acid ester of 8-methylcholine (5/6) indicates that there is a large degree of structural freedom and therefore only a loose binding of the choline moiety to the receptor site occurs. For the cyclohexylphenylglycollicacid ester of choline, in which the centre of asymmetry is located in the hydrophobic acidic moiety, the ratio is 60, which indicates an intricate relationship between this moiety and the receptor site. For the cyclohexylphenylglycollic acid ester of p-methylcholine, in which there are two centres of asymmetry,
1977
2nd NATIONAL CONGRESS, ITALY
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the ratios for the pairs of isomers that differ in their centres of asymmetry in the choline moiety, are 4 and 2, whereas for the pairs of isomers that differ in their centres of asymmetry in the hydrophobic acidic moiety, the ratios are 100 and 50. Replacement of the choline moiety of the cyclohexylpheny1glycollic acid ester of choline by neopentyl alcohol gives a compound that is still a potent anticholinergic agent even though there is no onium or amino group present; the activity ratio for the optical isomers of this compound, with the centre of asymmetry in the hydrophobic acidic moiety, is 100 (Ariens & Simonis, 1967). These observations clearly indicate that the hydrophobicmoieties of anticholinergicagents, by binding to accessory hydrophobic areas, strongly contribute to their affinity for cholinergic receptors. Anticholinergic agents apparently bind to accessory binding areas which are functionally related to the receptor sites for the cholinergic agonists and thus block the activation of the receptor by the agonist. They interfere with the binding of the agonist to its receptor sites without actually occupying the receptor site in the strict sense. There is a ‘dualism’ in the receptor sites. On the basis of this concept, the lack of structural relationship between agonists and the corresponding competitive antagonists, the existence of structural relationships between competitive antagonists blocking different types of agonist receptors, the existence of competitive antagonists blocking the receptor for different agonists, and the dependence of the selectivity of the action of the competitive antagonists on the steric structure of the hydrophobic moieties in the molecule (Harms & Nauta, 1960; Rekker et al., 1971) are understandable (Ariens & Simonis, 1976). The receptor sites for agonists and competitive antagonists are not ‘common’ as suggested by the original ‘one receptor site’ concept. The receptor sites for the drugs do not necessarily have to be located on the surface of the receptor proteins. This is, however, likely for the relatively polar sites for the agonist agents, although these receptors may also be located at, and even be constituted by, the interface, where the polar groups of the protein interact with the polar groups of the membrane lipids, e.g. the phosphate and choline groups in phosphatidic acid and sphingosidic acid. Similarly the receptor sites for the hydrophobic moieties of the various competitive antagonists of membrane-active agents may well consist of the interfacebetween the hydrophobicsurfaceof the protein and the lipid chains in the membrane. The extremely high affinity constants, lo9 and 1O1O, for competitive antagonists (e.g. anticholinergic and antihistaminic agents), which are mainly the result of the hydrophobic groups, can hardly be expected to exist on a mere protein surface. Lipoproteins would be more suitable in this respect. The fact that the lipid molecules facing the hydrophobic surface of the protein are to a large extent in a quasi-crystalline form makes the high degree of stereospecificity, especially with regard to centres of asymmetry in the hydrophobic moiety, of the antagonists understandable. Drugreceptor interaction then results in changes in the interface characteristics. Isolation of complete receptors as molecular entities is impossible. Incorporation of the receptorprotein fraction into a membrane may be required for receptor reconstitution (De Robertis, 1976). The interaction between the receptor protein and the membrane lipids will strongly influence the conformation of the protein and thus the action of drugs on, and their affinity for, the receptor protein. The receptor molecule must be assumed to occur in different states, an activated (A) and a non-activated (N) form, depending on the presence of the agonist or competitive antagonist. Kasai & Changeux (1971) postulated that even in the absence of drugs there is a dynamic equilibrium between the different functional states of the cholinergic receptor. The simplest concept in this respect is the ‘dual receptor concept’: the occurrence in the cell membrane of an equilibrium between the receptors in the A and those in the N form (conformation). The A form contributes to the effect, and the N form does not. An agonist shifts the equilibrium towards the A conformation and the competitive antagonist shifts it towards the N conformation. The agonist has a relatively high a6nity for the receptors in the A conformation, and the competitive antagonist for those in the N conformation, thus to a certain degree locking the receptors
VOI. 5
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in the respective states. Partial agonists have an a f i i t y for both states, the ratio of the affinities determining the intrinsic activity. As long as the rate constants for the equilibrium are large enough, this model will result in phenomena similar to those expected for competition between drugs based on the ‘one receptor site’ concept. The receptor sites for agonist and antagonist, although one receptor protein is involved, are therefore different, which is in accord with the idea of dualism in receptor sites deduced from the structure-action relationship outlined above. Taking into account the fact that the receptor molecules are embedded in the lipid membrane structure, one may expect that the behaviour of such protein molecules, for instance their tendency for aggregation or segregation, strongly depends on the hydrophobic and hydrophilic properties respectively of the protein surface, especially of that part of the receptor molecule in contact with the lipids in the membrane. The receptor proteins, although predominantly hydrophobic (De Robertis, 1975), have an amphiphilic character. Binding of the agonists, usually strongly polar agents, may change the conformation and thus shift the equilibrium of the receptor population towards a more hydrophilic state, i.e. the A form, whereas binding of hydrophobic antagonistic agents may shift it towards the hydrophobic state, i.e. the N form. The change in the tertiary structure involved may well result in changes in the quaternary structure of the proteins, and thus in the degree and type of aggregation among receptor molecules or among receptor molecules and other proteins in the membrane. This receptor-activation-migration-aggregation model, which fits quite well with the fluid-mosaic concept for the membrane structure (Singer & Nicolson, 1972). Various membrane-active drugs clearly change the protein distribution in membranes and the type of receptor-protein aggregation in solutions of isolated receptors. The proteins in the luminal cell membrane of the toad bladder aggregate to a mosaic pattern under the influence of the antidiuretic hormone (Kachadorian et al., 1975). The protein-walled pores thus formed may well account for the increased water permeability induced by this hormone. The various receptor proteins for the different hormones activating adenylate cyclase are supposed to aggregate with the enzyme molecule under the influence of the various hormones, thus activating the enzyme in an allosteric way (Cuatrecasas et al., 1975; Jacobs, 1976). Also the mode of aggregation among receptor proteins may be changed under the influence of drugs. In those cases in which proteinwalled pores are present in a membrane in the normal state, their permeability to, for instance, ions may well change as a result of a charge rearrangement in the receptor molecules constituting the pores. The proteins may switch from an open to a closed conformation. Again there may be an equilibrium between these conformations, shifted by the agonist to (usually) the opcn conformation and by the competitive antagonists to the closed conformation. In the dual-receptor-site model, although there is only one receptor protein, the receptor sites for agonist molecules and for the corresponding antagonist molecules are distinct functionally interdependent entities. Therefore affinity constants for, e.g., an agonist derived from displacement experiments may well depend on whether agonist or antagonist molecules are displaced. The dual-receptor-site concept undoubtedly opens intriguing perspectives for the interpretation of the action of drugs, hormones etc. on membrane receptors. Ariens, E. J. (1964)Mol. Pharmacol. 1, 119-286 Ariens, E.J. (1967)Ann. N. Y. Acad. Sci. 139,606-631 Ariens, E.J. (1971)Drug Design 1, 1-157 Ariens, E.J. & Simonis, A. M. (1967)Ann. N.Y. Acad. Sci. 144, 842-868 Ariens, E.J. & Simonis, A. M. (1976)in Beta-Adrenoceptor Blocking Agents (Saxena, P. R. & Forsyth, R. P.,eds.), pp. 3-27, North-Holland Publishing Co., Amsterdam Cuatrecasas,P., Hollenberg, M. D., Chang, K. J. &Bennett, V.(1975)Recent Prog. Horm. Res. 31,37-94
Del Castillo, J. & Katz, B. (1955)J. Physiol. (London) 128,157-181 Pe Robertis, E.(1975)Rev. Physiol. Biochem. Pharmacol. 73, 9-38 1977
2nd NATIONAL CONGRESS, ITALY
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De Robertis, E. (1976) in The Structural Basis of Membrane Function (Hatefi, Y . & DjavadiOhaniance, L., eds.), pp. 339-361, Academic Press, New York, San Francisco and London Harms, A. F. & Nauta, W. Th. (1960) J. Med. Pharm. Chem. 2,57-77 Jacobs, S . & Cuatrecasas, P. (1976) Biochim. Biophys. Acta 433,482-495 Kachadorian, W. A., Wade, J. B. & DiScala, V. A. (1975) Science 190, 67-69 Kasai, M. & Changeux, J. P. (1971) J. Membr. Biol. 6, 24-32 Maddy, A. H. & Dunn, M. J. (1976) in Biochemical Analysis of Membranes (Maddy,A. H., ed.), pp. 177-196, Chapman and Hall, London Rekker, R. F., Timmerman, H., Harms, A. F. & Nauta, W. Th. (1971) Arzneim-Forsch. 21, 688-691 Singer, S . J. & Nicolson, G. L. (1972) Science 175,720-731
Purification and Characterization of the Cholinergic Receptor Protein in its Membrane-Bound and Detergent-Soluble Forms from the Electric Organ of Torpedo marmorata AND&
SOBEL AND JEAN-PIERRE CHANGEUX
Laboratoire de Neurobiologie Moldculaire, Institut Pasteur, 25 Rue du Dr. Roux, 75724 Paris Cedex 15, France
The cholinergic receptor protein has been purified from electric organs of the electric fishes Electrophorus electricus and Torpedo by affinity chromatography through a column made up of a cholinergic ligand covalently bound to Sepharose [for review see Karlin (1974) and Changeux (1975)l. However, it has been shown that a prolonged incubation of the solubilized cholinergic receptor with a cholinergic agonist (Weber et al., 1975), as well as the passage through an affinity column (Sugiyama & Changeux, 1975), may change the molecular properties of the receptor protein. In the present communication, we describe a simple and rapid method for the purification of large amounts of acetylcholine-receptor-rich membranes as well as of acetylcholine-receptor protein, without the use of an affinity-chromatography step. It has been shown previously (Cohen et al., 1972; Nickel & Potter, 1973; Duguid & Raftery, 1973a,b), that the fresh Torpedo electric organ can be fractionated after homogenization by a differential centrifugation followed by an ultracentrifugation in a sucrose gradient, in order to yield membrane fragments particularly rich in the cholinergic-receptor protein. These membranes have a specific radioactivity of 2-3 pmol of 3H-labelled a-toxin-binding sites/g of protein and most likely represent pieces of subsynaptic membrane. The original fractionation method of Cohen et al. (1972) has been improved in order to obtain larger quantities of completely pure subsynaptic membranes. All the purification steps were carried out in the presence of 0.1 mwphenylmethanesulphonyl fluoride as a proteinase inhibitor and 0.02 % NaN, to prevent bacterial growth. The freshly dissected electric organ from Torpedo marmorata was homogenized in distilled water containing NaN, nd phenylmethanesulphonyl fluoride, and then submitted to successive differential centrifugations. A first low-speed centrifugation (7000g, 10min) of the homogenate gave a supernatant S1 and a pellet PI,which was homogenized again and re-centrifuged in the same conditions to yield the S2 supernatant. Supernatants S1 and S2 were mixed to form supernatant SIF2,which was centrifuged at 10OOOgfor 2h to pellet the membranes (P,). P3was resuspended in 32 % sucrose and the resulting membrane suspension (El) was layered on top of a discontinuous sucrose gradient (3437.5-41.5 %) and centrifuged at lOOOOOg for 6 h. The band at 37.5 %sucrose was collected, diluted and centrifuged to pellet the membranes, which were again resuspended in 32% sucrose (E2). Suspension Ez was layered on top of a continuous (35-43 %) sucrose gradient and centrifuged at 1OOOOOg for 6h. The cholinergic-receptor-
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