Noncompetitive Blocker Site Research Articles

Inhibitory effects of carvone isomers on the GABAA receptor in primary cultures of rat cortical neurons.

Carvone is a natural terpene which can be purified as R-(-) or S-(+) enantiomers. There are many reports about its antibacterial, antifungal, and insecticide activities, and also of some effects on the nervous system, where both enantiomers showed different potencies. Considering that the GABA(A) receptor is a major insecticide target, we studied the pharmacological activity of both carvone enantiomers, and of thujone as a reference compound acting on the receptor, on native GABA(A) by determining their effects on benzodiazepine recognition sites using primary neuronal cultures. Both isomers were able to inhibit the GABA-induced stimulation of [(3)H]flunitrazepam binding, suggesting their interaction with the GABA(A) receptor as negative allosteric modulators. Their activity was comparable to that described for thujone in the present article, with the R-(-)-carvone being the more similar and potent stereoisomer. The different configuration of the isopropenyl group in position 5 thus seems to be significant for receptor interaction and the bicycle structure not to be critical for receptor recognition. The concentrations necessary to induce negative modulation of the receptor were not cytotoxic in a murine neuron culture system. These results confirm that, at least partially, the reported insecticidal activity of carvones may be explained by their interaction with the GABA(A) receptor at its noncompetitive blocker site.

Nereistoxin and cartap neurotoxicity attributable to direct block of the insect nicotinic receptor/channel.

Nereistoxin (NTX) (4-dimethylamino-1,2-dithiolane) is the naturally occurring prototype for cartap [the bis(thiocarbamate) derivative of the NTX dithiol], which is generally regarded as a proinsecticide reverting to NTX. The aim of this study is to define the target site(s) for dithiolanes and dithiol esters. The affinity of [(3)H]NTX was not suitable for binding assays with honeybee (Apis mellifera) head membranes. However, NTX and cartap are equally potent, direct-acting, and competitive displacers of [(3)H]thienylcyclohexylpiperidine binding at the noncompetitive blocker (NCB) site of the Apis nicotinic acetylcholine receptor (nAChR)/channel. NTX also binds at the Apis [(3)H]imidacloprid agonist site, but cartap does not. As candidate metabolic pathways, sequential N-desmethylation and S-oxidation of NTX progressively reduce its potency at the NCB site and toxicity to houseflies. A P450 inhibitor reduces the toxicity of NTX and enhances it with cartap. Surprisingly, cartap is not just a pro-NTX but instead directly induces inhibitory neurotoxicity by blocking the nAChR/channel, whereas NTX may have dual NCB and agonist targets.

Regional Modification of [3H]Ethynylbicycloorthobenzoate Binding in Mouse Brain GABAA Receptor by Endosulfan, Fipronil, and Avermectin B1a

[3H]Ethynylbicycloorthobenzoate ([3H]EBOB), a high affinity radioligand for the noncompetitive blocker site of the GABAA receptor, is used here for quantitative autoradiography to determine regional binding in mouse brain and the effects on this binding of administering toxic doses of endosulfan, fipronil, and avermectin B1a (AVM). Animals were euthanized 4–8 min after 1 LD50 or 2 LD50 doses of the two channel blockers and 32 min after 1 LD50 or 4 LD50 doses of the channel activator AVM. Specific binding of [3H]EBOB was determined for 20-μm brain sections as the difference in labeling on incubation with 2 nM [3H]EBOB either alone (total binding) or with 5 μM α-endosulfan (nonspecific binding). The highest specific labeling was observed for layers I and IV of the cerebral cortex, the globus pallidus, and the medial septal nucleus/nucleus of the vertical limb of the diagonal band. Dose-dependent inhibition by endosulfan was highest in the nucleus accumbens and least in the cerebellum and periaqueductal gray matter. Fipronil had much less effect on binding even at severely toxic doses. AVM increased [3H]EBOB binding in most regions and was the only one of the three agents inhibiting in vitro [3H]strychnine binding to the glycine receptor. In summary, the noncompetitive blocker site was strongly inhibited with dose dependence and regional selectively by α-endosulfan but was generally poorly inhibited or activated by fipronil and was activated by avermectin.

Mechanisms for selective toxicity of fipronil insecticide and its sulfone metabolite and desulfinyl photoproduct.

Fipronil, an N-phenylpyrazole with a trifluoromethylsulfinyl substituent, initiated the second generation of insecticides acting at the gamma-aminobutyric acid (GABA) receptor to block the chloride channel. The first generation includes the polychlorocycloalkanes alpha-endosulfan and lindane. In this study, we examine the mechanisms for selective toxicity of the sulfoxide fipronil and its sulfone metabolite and desulfinyl photoproduct relative to their target site interactions in vitro and ex vivo and the importance in fipronil action of biooxidation to the sulfone. Differences in GABA receptor sensitivity, assayed by displacement of 4'-ethynyl-4-n-[2, 3-3H2]propylbicycloorthobenzoate ([3H]EBOB) from the noncompetitive blocker site, appear to be a major factor in fipronil being much more toxic to the insects (housefly and fruit fly) than to the vertebrates (humans, dogs, mice, chickens, quail, and salmon) examined; in insects, the IC50s range from 3 to 12 nM for fipronil and its sulfone and desulfinyl derivatives, while in vertebrates, the IC50 average values are 1103, 175, and 129 nM for fipronil, fipronil sulfone, and desulfinyl fipronil, respectively. The insect relative to the vertebrate specificity decreases in the following order: fipronil > lindane > desulfinyl fipronil > fipronil sulfone > alpha-endosulfan. Ex vivo inhibition of [3H]EBOB binding in mouse brain is similar for fipronil and its sulfone and desulfinyl derivatives at the LD50 dose, but surprisingly, at higher doses fipronil can be lethal without detectably blocking the [3H]EBOB site. The P450 inhibitor piperonyl butoxide, acting in houseflies, increases the metabolic stability and effectiveness of fipronil and the sulfone but not those of the desulfinyl compound, and in mice it completely blocks the sulfoxide to sulfone conversion without altering the poisoning. Thus, the selective toxicity of fipronil and fipronil-derived residues is due in part to the higher potency of the parent compound at the insect versus the mammalian GABA receptor but is also dependent on the relative rates of conversion to the more persistent and less selective sulfone metabolite and desulfinyl photoproduct.

Structure-activity relationships of acyclic nicotinoids and neonicotinoids for insect nicotinic acetylcholine receptor/ion channel complex

The insect nicotinic acetylcholine (Ach) receptor (nAChR) is a target site for the neonicotinoid insecticides such as imidacloprid and its acyclic derivative acetamiprid. The structure–activity relationships of acetamiprid homologues and 3-pyridylmethylamines (known as the essential structural requirement of nicotinoid) are compared in terms of the affinity to the [3H]α-bungarotoxin (α-BGT) site (designated as Ach site) and the [3H]phencyclidine (PCP) site [designated as noncompetitive blocker (NCB) site] of the insect nAChR from the honeybee heads. Increasing the chain length of alkyl substituents (from methyl to n-butyl) on an amino nitrogen atom of acetamiprid homologue and 3-pyridylmethylamine reduces the potency as inhibitors of [3H]α-BGT binding, whereas it confers the enhanced potency as inhibitors of [3H]PCP binding in the insect nAChR. Scatchard analysis reveals that homologues of acetamiprid and 3-pyridylmethylamine having n-butyl substituents interact with the high-affinity binding site for [3H]PCP, which is considered to be the NCB site located in the ion channel of the insect nAChR. The interaction of acetamiprid homologues with the Ach or NCB site of nAChR is selective for insects, while that of the 3-pyridylmethylamines is effective for both insect and Torpedo [Tomizawa et al., J Pesticide Sci 21:412–418 (1996)]. The explorations in further structural modification of neonicotinoid compounds may facilitate development of new insecticides or probes for the ion channel of insect nAChR. Arch. Insect Biochem. Physiol. 37:17–23, 1998. © 1998 Wiley-Liss, Inc.

Novel photoactivatable agonist of the nicotinic acetylcholine receptor of potential use for exploring the functional activated state.

The nicotinic acetylcholine receptor (AChR) exhibits at least four different conformational states varying in affinity for agonists such as acetylcholine (ACh). Photoaffinity labeling has been previously used to elucidate the topography of the AChR. However, to date, the photosensitive probes used to explore the cholinergic binding site photolabeled only closed or desensitized states of the receptor. To identify the structural modifications occurring at the ACh binding site on allosteric transition associated with receptor activation, we have investigated novel photoactivatable 4-diazocyclohexa-2,5-dienone derivatives as putative cholinergic agonists. Such compounds are fairly stable in the dark and generate highly reactive carbenic species on irradiation. In binding experiments using AChRs from Torpedo marmorata, these ligands had affinities for the ACh binding site in the micromolar range and did not interact with the noncompetitive blocker site (greater than millimolar affinity). Irreversible photoinactivation of ACh binding sites was obtained with the ligand 1b (up to 42% at 500 microM) in a protectable manner. In patch-clamp studies, 1b was shown to be a functional agonist of peripheral AChR in TE 671 cells, with the interesting property of exhibiting no or very little desensitization even at high concentrations.

Metaldehyde Molluscicide Action in Mice: Distribution, Metabolism, and Possible Relation to GABAergic System

Two proposed modes of toxic action of the molluscicide metaldehyde are considered here by studies in orally treated mice of symptomatology, acetaldehyde and metaldehyde concentrations in tissues, and characteristics of the γ-aminobutyric acid (GABA) receptor complex. An earlier proposal that metaldehyde-induced poisoning is due to acetaldehyde liberation is not supported by either their different symptomatology or tissue analyses. Unmetabolized metaldehyde is excreted to the extent of 8% and the urinary metabolites from [13C]metaldehyde and [13C]acetate are consistent with primary degradation of metaldehyde via acetaldehyde and acetate. Poisoning signs are initiated at ∼12 ppm metaldehyde in brain, progressing to tonic convulsions and death at ∼40 ppm and lessened by diazepam and phenobarbital without greatly altering the tissue levels of metaldehyde. Possible involvement of the GABAergic system in metaldehyde toxicity is suggested by the lowered levels of GABA in the brain of poisoned mice determined by (1) earlier chemical analysis of the whole brain (Homeida and Cooke,J. Vet. Pharmacol. Ther.5,77 (1982)) and (2) the presently observed reduced effect of endogenous inhibitors (primarily GABA) in preventing binding of [3H]ethynylbicycloorthobenzoate ([3H]EBOB) to brain membranes. Metaldehyde (1 mM) does not: (1) inhibit [3H]EBOB bindingin vitroat the noncompetitive blocker site, (2) alter the coupled effects on [3H]EBOB binding of GABA as an inhibitor (acting at the GABA recognition site) and the amidine steroid RU 5135 in reversing this inhibition (acting primarily as a competitive antagonist), or (3) inhibit glutamic acid decarboxylase activity. Thus, the toxicity of metaldehyde is not due to degradation to acetaldehyde but instead may result from disrupting the GABAergic system by a mechanism which remains to be determined.

Affinity probes for the GABA-gated chloride channel: Selection of 5 e- tert-Butyl-2 e-[4-(substituted-ethynyl)phenyl]-1,3-dithianes and optimization of linker moiety

The noncompetitive blocker (NCB) site of the γ-aminobutyric acid (GABA)-gated chloride channel is the target for many important insecticides and potent convulsants. This site is specifically blocked by 3H ethynylbicycloorthobenzoate ( 3H EBOB) and other trioxabicyclooctane radioligands and might be suitable for affinity probes with an appropriate heterocyclic substituent and linker moiety. Optimal potency at the NCB site is achieved with 5 e- tert-butyl-2 e-[4-(substituted-ethynyl)phenyl]-1,3-dithianes compared with analogs in which the butyldithiane portion is replaced with butyldithiane-sulfoxide or -sulfone, n-propyltrioxabicyclooctane or dioxatricyclododecene. Three positions were examined for coupling the linker and dithiane: C-2 of the dithiane; a branched substituent within the alkynyl moiety; the terminus of a straight chain extension from the ethynyl group, which proved to be the best. Optimized linkers for addition to the ethynylphenyldithiane to achieve appropriate length and fit within the active site, i.e. receptor potency, are CH 2OCH 2C(O)SCH 2CH 2(SH or NH 2) and the corresponding thiolates and amides. Several compounds with these spacers block the chloride channel, measured as inhibition of 3H EBOB binding, at 4–50 nM.

Drosophila GABA-gated chloride channel: modified [3H]EBOB binding site associated with Ala-->Ser or Gly mutants of Rdl subunit.

The non-competitive blocker site of the GABA-gated chloride ion channel in normal susceptible strains of Drosophila melanogaster and simulans binds 4-n-[3H]propyl-4'-ethynylbicycloorthobenzoate ([3H]EBOB) at specific sites with KdS of 1.6-1.9 nM and BmaxS of 171-181 fmol/mg protein. This specific binding of [3H]EBOB is strongly inhibited by: a large number and variety of insecticidal channel blockers at 20 nM (lindane, alpha-endosulfan, dieldrin, 12-ketoendrin, fipronil, and a representative bicycloorthobenzoate and dithiane) or 200 nM (picrotoxinin); the insecticidal channel activators avermectin and moxidectin at 20 nM; muscimol at 30 microM and GABA at 300 microM. Cyclodiene resistance in D. melanogaster has been attributed to a mutation resulting in an Ala302-->Ser replacement in the Rdl GABA receptor subunit and in D. simulans to an homologous Ala-->Ser or Gly replacement. These mutations are shown here to greatly reduce [3H]EBOB binding, i.e. lower affinity and apparent number of binding sites. The Ala-->Ser replacement with both melanogaster and simulans almost always reduces the potency in inhibiting [3H]EBOB binding of each of eight channel blockers and of muscimol and GABA. The Ala-->Gly replacement in D. simulans is generally less effective than the Ala-->Ser modification in reducing sensitivity to the channel blockers and to muscimol and GABA. The channel activators avermectin and moxidectin usually retain their inhibitory potency in the Rdl subunit mutants. Thus, it appears that replacement of Ala by Ser generally modifies the non-competitive blocker site and its coupling to the GABA-recognition site with less effect on the channel activator site. In contrast, the Ala-->Gly replacement has less impact in protecting the chloride channel from the action of insecticidal blockers. Each of the resistant strains has the same level of resistance to the lethal action of the five channel blockers examined but none to avermectins and muscimol.

GABA-gated chloride channel: Binding site for 4′-ethynyl-4- n-[2,3- 3H 2]propylbicycloorthobenzoate ([ 3H]EBOB) in vertebrate brain and insect head

4′-Ethynyl-4- n-[2,3- 3H 2]propylbicycloorthobenzoate ([ 3H]EBOB) is an improved radioligand for the noncompetitive blocker site of the vertebrate and fly γ-aminobutyric acid (GABA)-gated chloride channel. It binds with high affinity ( K d 1–4 n M) to a single, saturable site in the GABA-gated chloride channel of human, dog, mouse, and chicken brain assayed at 37°C, of fish brain at 12°C, and of adult house fly and fruit fly heads at 22°C with specific binding of 51–93%. The B max's (pmol/mg protein) are 0.9 – 5.7 for the vertebrates and 0.2 – 0.4 for the insects. Association and dissociation rates fall in the ranges of 0.012 to 0.063 min −1 n M −1 for k +1 and 0.006 to 0.046 min −1 for k −1. The vertebrate preparations are much more sensitive than the insect preparations to GABA and two GABA mimetics (3-aminopropanesulfonic acid and muscimol). Considerable specificity is evident between vertebrates and insects in the inhibitory potencies of various insecticides with the following average selectivity ratios (IC 50 vertebrates/IC 50 flies): t-butylbicyclophosphorothionate and 4- t-butyl-1-ethynyl-2,6,7-trioxabicyclo[2.2.2]octane, 0.04 – 0.18; α-endosulfan, 12-ketoendrin, toxaphene, picrotoxinin, and 2 eq-(4-bromophenyl)-5- t-butyl-1,3-dithiane-2,2-dioxide, 3–8; lindane, 130; and avermectin B 1a and moxidectin, 615–>714. It therefore appears that receptor-site specificity may contribute to species selectivity.

The noncompetitive blocker [3H]chlorpromazine labels three amino acids of the acetylcholine receptor gamma subunit: implications for the alpha-helical organization of regions MII and for the structure of the ion channel.

Labeling studies of Torpedo marmorata nicotinic acetylcholine receptor with the noncompetitive channel blocker [3H]chlorpromazine have led to the initial identification of amino acids plausibly participating to the walls of the ion channel on the alpha, beta, and delta subunits. We report here results obtained with the gamma subunit, which bring additional information on the structure of the channel. After photolabeling of the membrane-bound receptor under equilibrium conditions in the presence of agonist and with or without phencyclidine (a specific ligand for the high-affinity site for noncompetitive blockers), the purified labeled gamma subunit was digested with trypsin, and the resulting fragments were fractionated by HPLC. Sequence analysis of peptide mixtures containing various amounts of highly hydrophobic fragments showed that three amino acids are labeled by [3H]chlorpromazine in a phencyclidine-sensitive manner: Thr-253, Ser-257, and Leu-260. These residues all belong to the hydrophobic and putative transmembrane region MII of the gamma subunit. Their distribution along the sequence is consistent with an alpha-helical organization of this segment. The [3H]chlorpromazine-labeled amino acids are conserved at homologous positions in the known sequences of other ligand-gated ion channels and may, thus, play a critical role in ion-transport mechanisms.

The noncompetitive blocker [ 3H]chlorpromazine labels segment M2 but not segment M 1 of the nicotinic acetylcholine receptor α-subunit

The membrane bound acetylcholine receptor from Torpedo marmorata was photolabeled by the noncompetitive channel blocker [ 3H]chlorpromazine under equilibrium conditions in the presence of the agonist carbamoylcholine. The radioactivity incorporated into the AChR subunits was reduced by addition of phencyclidine, a specific ligand for the high-affinity site for noncompetitive blockers. The α-subunit was purified digested with trypsin and/or CNBr and the resulting fragments fractionated by HPLC. Sequence analysis resulted in the identification of Ser-248 as a major residue labeled by [ 3H]chlorpromazine in a phencyclidine-sensitive manner. This residue is located in the hydrophobic and putative transmembrane segment M2 of the α-subunit, a region homologous to that containing the chlorpromazine-labeled Ser-262 in the δ-chain [1] and the Ser-254 and Leu-257 in the β-chain [2]. Extended sequence analysis of the hydrophobic segment M 1 further showed that no labeling occurred in this region.

Photoaffinity labeling of the acetylcholine binding sites on the nicotinic receptor by an aryldiazonium derivative

p-(Dimethylamino)benzenediazonium fluoroborate (DDF) behaves, in the dark, as a reversible competitive antagonist of the electrical response of Electrophorus electricus electroplaque to acetylcholine and of the acetylcholine-gated single-channel currents recorded in the C2 mouse cell line. This chemically stable but highly photoreactive compound binds irreversibly to the acetylcholine receptor when irradiated by visible light. In vivo, it irreversibly blocks the postsynaptic response of E. electricus electroplaque to agonists. In vitro, it reduces the alpha-bungarotoxin-binding capacity of acetylcholine receptor rich membrane fragments prepared from Torpedo marmorata electric organ. Once reversibly bound to the T. marmorata acetylcholine receptor, this ligand can be selectively photodecomposed by an energy-transfer reaction involving a tryptophan residue(s) of the protein. By use of reagent concentrations that are below the dissociation constant at equilibrium, up to 60% of the agonist-binding sites are covalently labeled. Under these conditions the alpha subunit of the acetylcholine receptor is preferentially labeled, and this labeling is partially prevented by agonists or competitive antagonists. This protective effect is substantially increased by prior incubation with phencyclidine, a compound known to prevent the binding of DDF at the level of the high-affinity site for noncompetitive blockers [Kotzyba-Hibert, F., Langenbuch-Cachat, J., Jaganathen, J., Goeldner, M. P., & Hirth, C. G. (1985) FEBS Lett. 182, 297-301]. The incorporation of about one molecule of label in an agonist/competitive antagonist protectable manner per alpha-bungarotoxin-binding site suffices to fully block alpha-bungarotoxin binding to the membrane-bound receptor. Thus, DDF behaves as a monovalent photoaffinity label of the acetylcholine-binding site.

Structure of the high-affinity binding site for noncompetitive blockers of the acetylcholine receptor: [3H]chlorpromazine labels homologous residues in the beta and delta chains.

The membrane-bound acetylcholine receptor from Torpedo marmorata was photolabeled by the noncompetitive channel blocker [3H]chlorpromazine under equilibrium conditions in the presence of the agonist carbamoylcholine. The amount of radioactivity incorporated into all subunits was reduced by addition of phencyclidine, a specific ligand for the high-affinity site for noncompetitive blockers. The labeled beta chain was purified and digested with trypsin or CNBr, and the resulting fragments were fractionated by high-performance liquid chromatography. Sequence analysis resulted in the identification of Ser-254 and Leu-257 as residues labeled by [3H]chlorpromazine in a phencyclidine-sensitive manner. These residues are located in the hydrophobic and potentially transmembrane segment M II of the beta chain, a region homologous to that containing the chlorpromazine-labeled Ser-262 in the delta chain [Giraudat, J., Dennis, M., Heidmann, T., Chang, J. Y., & Changeux, J.-P. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2719-2723]. These results show that homologous regions of different receptor subunits contribute to the unique high-affinity site for noncompetitive blockers, a finding consistent with the location of this site on the axis of symmetry of the receptor molecule.

Structure of the high-affinity binding site for noncompetitive blockers of the acetylcholine receptor: serine-262 of the delta subunit is labeled by [3H]chlorpromazine.

The membrane-bound acetylcholine receptor from Torpedo marmorata was photolabeled by the noncompetitive channel blocker [3H]chlorpromazine under equilibrium conditions in the presence of agonist. Incorporation of radioactivity into all subunits occurred and was reduced by addition of phencyclidine, a specific ligand for the high-affinity site for noncompetitive blockers. The delta subunit was purified and digested with trypsin, and the resulting fragments were fractionated by reversed-phase HPLC. The labeled peptide could not be purified to homogeneity because of its marked hydrophobic character, but a combination of differential CNBr subcleavage and cosequencing of partially purified fragments enabled us to identify Ser-262 as being labeled by [3H]chlorpromazine. The labeling of this particular residue was prevented by phencyclidine and thus took place at the level of, or in proximity to, the high-affinity site for noncompetitive blockers. Ser-262 is located in a hydrophobic and potentially transmembrane segment termed MII.

Interactions of charatoxins and nereistoxin with the nicotinic acetylcholine receptors of insect cns and Torpedo electric organ

AbstractInteractions of charatoxin (4‐methylthio‐1,2‐dithiolane; ChTX) and four openchain analogs as well as nereistoxin (NTX) with acetylcholine (ACh) receptors were studied using biochemical assays on the Torpedo electric organ and honey bee brain receptors and using electrophysiological assays on the response of the cell body of the fast coxal depressor motoneuron (Df) of the cockroach Periplaneta americana to ACh. The actions of ChTXs were complex. Except for ChTX Xl, they all potentiated the ACh‐induced current in Periplaneta neurons, but at higher concentrations all ChTXs, except for ChTX XII, caused voltage‐dependent block of this current. All CHTXs inhibited binding of [3H]perhydrohistrionicotoxin in the presence of ACh to the highaffinity noncompetitive blocker site on the Torpedo receptor, but all, except for ChTX XI, potentiated its binding in absence of ACh. The actions of ChTXs on the honey bee brain receptor were quite different from those on the Torpedo receptor. They inhibited, or had no effect on, [125I]α‐bungarotoxin (α‐BGT) binding to the Torpedo receptor, but all ChTXs, except for ChTX I, potentiated its binding to the honey bee receptor. It is suggested that the action of ChTXs on nicotinic ACh‐receptors resulted from binding to lowaffinity noncompetitive blocker site. On the other hand, NTX was more potent than ChTXs on nicotinic ACh‐receptors, and some similarities were noted between the actions of NTX on Torpedo and honey bee receptors NTX had a weak agonistlike effect in both cases and possibly bound to the ACh binding sites as well as the high‐affinity noncompetitive blocker site. Thus the mechanisms of action of ChTXs and NTX on nicotinic ACh‐receptors are different, and there are also differences in the responses to these toxins between receptors of insect central nervous system and Torpedo electric organ.

Demonstration and affinity labeling of a stereoselective binding site for a benzomorphan opiate on acetylcholine receptor-rich membranes from Torpedo electroplaque.

The interaction of an optically pure benzomorphan opiate, (-)-N-allyl-N-normetazocine [(-)-ANMC], with the nicotinic acetylcholine receptor from Torpedo electroplaque was studied by using radioligand binding and affinity labeling. The binding was complex with at least two specific components having equilibrium dissociation constants of 0.3 microM and 2 microM. The affinity of the higher affinity component was decreased by carbamoylcholine but not by alpha-bungarotoxin. The effect of carbamoylcholine was not blocked by alpha-bungarotoxin. In comparison, the affinity of [3H]phencyclidine, a well-characterized ligand for a high-affinity site for noncompetitive blockers on the acetylcholine receptor, is increased by carbamoylcholine and the increase is blocked by alpha-bungarotoxin. The binding of (-)-[3H]ANMC was inhibited by a number of other benzomorphans, with (-) isomers being 4- to 5-fold more potent than (+) isomers. Phencyclidine inhibits the binding of (-)-[3H]ANMC to its high-affinity site by a mechanism that is not competitive. UV-catalyzed affinity labeling indicated that the high-affinity-binding site for (-)-[3H]ANMC is at least partially associated with the delta subunit. Tryptic degradation of the Torpedo marmorata delta chain suggested that (-)-ANMC labeled a 16,000-dalton COOH-terminal portion of the subunit. In contrast, 5-azido-[3H]trimethisoquin, a photoaffinity label of the high-affinity site for noncompetitive blockers, labels a 47,000-dalton NH2-terminal fragment of the delta subunit. These results suggest that (-)-[3H]ANMC binds to sites completely distinct from the binding sites for acetylcholine. The high-affinity-binding site for (-)-ANMC and that for phencyclidine and 5-azidotrimethisoquin are allosterically coupled but are regulated differently and are probably physically distinct.

Selective labeling of the delta subunit of the acetylcholine receptor by a covalent local anesthetic.

A radioactive photoaffinity derivative of the potent local anesthetic trimethisoquin, 5-azido[3H]trimethisoquin, was used to label the acetylcholine receptor from Torpedo marmorata electric organ. The product labeled the 66 000-dalton (delta) subunit of the receptor with the selectivity expected for an affinity label of the site for noncompetitive blockers. That is, the labeling was enhanced by cholinergic agonists and inhibited by other noncompetitive blockers. The 40 000-dalton (alpha)( subunit of the receptor was labeled in a manner consistent with the attachment of 5-azido[3H]trimethisoquin to an acetylcholine binding site as the incorporation of radioactivity into the alpha chain was inhibited by cholinergic agonists and antagonists, such as carbamylcholine, d-tubocurarine, and alpha-bungarotoxin. The reversible binding of [3H]phencyclidine, a potent noncompetitive blocker, to acetylcholine receptor rich membranes resembled qualitatively and quantitatively the 5-azido[3H]trimethisoquin labeling of the delta subunit and was inhibited by the prior covalent labeling of the membranes with nonradioactive 5-azidotrimethisoquin. Thus, 5-azido[3H]-trimethisoquin labels at least a portion of the binding site for noncompetitive blockers at the level of the delta subunit. The functional significance of this site and the use of 5-azidotrimethisoquin in the study of acetylcholine receptor structure and function are discussed.

Ultraviolet light-induced labeling by noncompetitive blockers of the acetylcholine receptor from Torpedo marmorata.

Reversible ligands were attached covalently to membrane-bound acetylcholine receptor from Torpedo marmorata by a method which is generally applicable and does not require the synthesis of specially designed molecules. UV irradiation of the receptor in the presence of [3H]trimethisoquin, [3H]phencyclidine, or [3H]perhydrohistrionicotoxin resulted in the labeling of the binding site(s) for these noncompetitive blockers of the permeability response. The labeling of the delta chain was enhanced by carbamoylcholine, and this increase was blocked by snake alpha-toxins. The effect of carbamoylcholine on [3H]trimethisoquin binding was more pronounced than with the other two noncompetitive blockers; in all instances, the labeling was abolished by unlabeled histrionicotoxin. These three compounds therefore interact with the high-affinity site for noncompetitive blockers. Incorporation of radioactivity also occurred into the alpha chain but either was insensitive to cholinergic effectors or decreased in the presence of carbamoylcholine (or snake alpha-toxin), probably as a result of an interaction with the acetylcholine-binding site. In contrast to the other noncompetitive blockers tested, [3H]chlorpromazine heavily labeled the four receptor polypeptides (alpha, beta, gamma, delta), and this labeling also was enhanced by carbamoylcholine and decreased by histrionicotoxin. These data indicate a contribution of the delta chain to the binding site(s) of several well-characterized noncompetitive blockers and suggest that other receptor polypeptides may also contribute to this binding.