Differences in structure and distribution of the molecular forms of acetylcholinesterase.

Two structurally distinct molecular forms of acetylcholinesterase are found in the electric organs of Torpedo californica. One form is dimensionally asymmetric and composed of heterologous subunits. The other form is hydrophobic and composed of homologous subunits. Sequence-specific antibodies were raised against a synthetic peptide corresponding to the COOH-terminal region (Lys560-Leu575) of the catalytic subunits of the asymmetric form of acetylcholinesterase. These antibodies reacted with the asymmetric form of acetylcholinesterase, but not with the hydrophobic form. These results confirm recent studies suggesting that the COOH-terminal domain of the asymmetric form differs from that of the hydrophobic form, and represent the first demonstration of antibodies selective for the catalytic subunits of the asymmetric form. In addition, the reactive epitope of a monoclonal antibody (4E7), previously shown to be selective for the hydrophobic form of acetylcholinesterase, has been identified as an N-linked complex carbohydrate, thus defining posttranslational differences between the two forms. These two form-selective antibodies, as well as panselective polyclonal and monoclonal antibodies, were used in light and electron microscopic immunolocalization studies to investigate the distribution of the two forms of acetylcholinesterase in the electric organ of Torpedo. Both forms were localized almost exclusively to the innervated surface of the electrocytes. However, they were differentially distributed along the innervated surface. Specific asymmetric-form immunoreactivity was restricted to areas of synaptic apposition and to the invaginations of the postsynaptic membrane that form the synaptic gutters. In contrast, immunoreactivity attributable to the hydrophobic form was selectively found along the non-synaptic surface of the nerve terminals and was not observed in the synaptic cleft or in the invaginations of the postsynaptic membrane. This differential distribution suggests that the two forms of acetylcholinesterase may play different roles in regulating the local concentration of acetylcholine in the synapse.

Structure/activity and molecular modeling studies of the lophotoxin family of irreversible nicotinic receptor antagonists.

Lophotoxin is a small cyclic diterpene that irreversibly inhibits agonist binding to nicotinic acetylcholine receptors by reacting covalently with Tyr190 in the alpha-subunits of the receptor. Structure/activity and molecular modeling studies were undertaken to investigate the structural and conformational features responsible for this unique biological activity. A total of 18 naturally occurring and 7 chemically modified analogues were evaluated for their ability to inhibit the binding of [125I]-alpha-bungarotoxin to nicotinic acetylcholine receptors on membranes prepared from Torpedo electric organ. When the toxins were incubated with the receptor for short durations they did not slow the initial rate of binding of [125I]-alpha-bungarotoxin, suggesting that they have relatively low reversible affinity. However, their ability to inhibit the equilibrium binding of [125I]-alpha-bungarotoxin increased progressively with longer incubation times, consistent with an irreversible mechanism of action. Comparison of active and inactive analogues allowed identification of a conserved pharmacophore that appeared to be required for irreversible inhibition of the receptor. This pharmacophore contains lactone oxygens and an electron-deficient epoxide that may mimic the acetate oxygens and quaternary ammonium group of acetylcholine, respectively. Computer modeling of the toxins using molecular mechanics and dynamics revealed that the toxins have restricted conformational mobility, thus allowing identification of a minimum-energy conformation. The results allow speculation concerning the site of covalent reaction between Tyr190 and the toxins, the normal function of Tyr190 in binding acetylcholine, and the bound conformation of acetylcholine.

Evidence for a carbocation intermediate during conversion of bipinnatin-A and -C into irreversible inhibitors of nicotinic acetylcholine receptors.

The lophotoxins are naturally occurring antagonists of nicotinic acetylcholine receptors. These toxins are small diterpenes that irreversibly inhibit nicotinic receptors by specific covalent modification of Tyr190 in the alpha-subunits of the receptor. The naturally occurring lophotoxin analogs, bipinnatin-A and -C, are inactive protoxins. Activation of these toxins occurs spontaneously in buffer and involves replacement of the C2 acetate ester with a hydroxyl group. The mechanism involved in conversion of the inactive bipinnatins into their biologically active solvolysis products was investigated in this study. Solvolysis of bipinnatin-A in buffer containing [18O]water demonstrated that the C2 hydroxyl of the biologically active solvolysis product originated from the solvent. The rates of solvolysis of bipinnatins-A and -C were not affected by sodium azide. However, in the presence of azide, solvent products decreased and new azide-containing products appeared. Thus azide acted as a nucleophile after a rate-limiting step, such as the formation of a carbocation intermediate. The kaz/ks values for bipinnatin-A (2900 M-1) and bipinnatin-C (1450 M-1) suggest that the carbocation intermediates are relatively stable. Compounds capable of spontaneously generating carbocations may represent a novel new class of active-site-directed affinity reagents that can be applied to other receptors and enzymes.

Chemical syntheses and biological activities of lactam analogues of alpha-conotoxin SI.

Bicyclization represents an effective method for the introduction of conformational constraints into small, biologically important peptides. Several strategies have been developed for the preparation of bicyclic lactam analogues of alpha-conotoxin SI, a 13-residue peptide neurotoxin found in cone snail venom. Four analogues of the natural regioisomer of alpha-conotoxin SI were designed and synthesized, each with one of the two paired cysteines of the parent peptide being replaced by a side-chain lactam bridged glutamic acid/lysine pair. Solid-phase lactamization was studied to determine rates of formation of the two possible loops and to document the extent of dimerization and higher oligomerization. Radioligand binding assays were carried out on all synthesized peptides, including the naturally occurring two-disulfide form, in order to determine their affinities for nicotinic acetylcholine receptors (nAChRs). Replacement of the Cys(2)-Cys(7) loop of alpha-conotoxin SI with a lactam bridge resulted in complete loss of activity, whereas replacement of the Cys(3)-Cys(13) disulfide loop resulted in a approximately 60-fold reduction in affinity for one orientation and a approximately 70-fold increase in affinity for the other. The two active lactam analogues retain the selectivity exhibited by the naturally occurring peptide for the alpha/delta subunit of nAChRs, as judged by competition experiments with the curariform antagonist metocurine.

Irreversible inhibitors of nicotinic acetylcholine receptors: isolation and structural characterization of the biologically active solvolysis products of bipinnatin-A and bipinnatin-C.

The lophotoxins irreversibly inhibit nicotinic acetylcholine receptors by covalent modification of Tyr190 in the alpha-subunits of the receptor. Previous studies have shown that the naturally occurring lophotoxin analogs bipinnatin-A, -B, and -C are actually inactive protoxins and that their ability to irreversibly inhibit nicotinic receptors is enhanced by preincubation in buffer. However, the ability of lophotoxin to irreversibly inhibit nicotinic receptors does not appear to be enhanced by preincubation in buffer. These observations led to the current effort to isolate and determine the structures of biologically active bipinnatins. Disappearance of the lophotoxins from solution followed a simple first-order exponential decay function. Lophotoxin, however, was approximately 40-fold more stable then bipinnatin-A, -B, or -C. Solvolysis of the bipinnatins, but not of lophotoxin, resulted in production of an equimolar amount of acetic acid at a rate similar to the rate of solvolysis, suggesting that the initial event in solvolysis of these toxins involves hydrolysis of an acetate ester. Proton NMR and fast-atom bombardment mass spectroscopy were used to confirm the structures of the active solvolysis products of bipinnatin-A and -C. Their structures and the relative pH insensitivity of the solvolysis reaction suggest that biological activation of the bipinnatins may proceed through an SN1 type of substitution reaction involving elimination of acetate followed by reaction of a carbocation intermediate with solvent.

In vitro interactions of agonists and antagonists with beta-adrenergic receptors.

Neurotransmitters and hormones mediate their effects through interaction with specific receptors. A complete understanding of the effects of these chemical signals requires detailed knowledge, at the molecular level, of agonist/receptor interactions. It is likely that agonists and antagonists interact with the same site on a receptor. Agonists, however, are by definition different from antagonists in that agonists are responsible for transducing information across the cell membrane, ultimately resulting in a biological response, while antagonists appear to act through passive occupancy of receptors. Implicit in this concept is the idea that these fundamental differences between agonists and antagonists arise from the sequelae induced by agonist-specific interactions with receptors.

Interactions of beta-adrenergic receptors with a membrane protein other than the stimulatory guanine nucleotide-binding protein.

Beta-adrenergic receptors on membranes prepared from rat lung, wild-type S49 lymphoma cells, and the adenylate cyclase-deficient variant of S49 lymphoma cells (cyc-) bind the agonist [3H]hydroxybenzylisoproterenol ([3H]HBI) with high affinity and this binding of [3H]HBI can be inhibited by GTP. Membranes prepared from these tissues were incubated with the agonist [3H]HBI or the antagonist [125I]iodopindolol ([125I]IPIN), labeled receptors were solubilized with digitonin, and the apparent molecular sizes of the ligand-bound receptor complexes were determined by high-performance size-exclusion chromatography. Results with all three tissues demonstrated that receptors labeled with [125I]IPIN were retained by the size-exclusion columns longer than receptors labeled with [3H]HBI. Thus, the apparent molecular size of soluble beta-adrenergic receptors from rat lung, wild-type S49 cells, and cyc- S49 cells was larger when receptors were occupied with an agonist rather than an antagonist. The results suggest that receptors, including those on cyc- S49 cells, interact with a membrane protein, presumably a guanine nucleotide-binding protein. Since cyc- S49 cells do not contain a functional stimulatory guanine nucleotide-binding protein, but do contain a functional inhibitory guanine nucleotide-binding protein, an interaction between beta-adrenergic receptors and the inhibitory guanine nucleotide-binding protein may be responsible for the apparent increase in the molecular size of the receptor after occupation of the receptor with an agonist.

Interaction of beta-adrenergic receptors with the inhibitory guanine nucleotide-binding protein of adenylate cyclase in membranes prepared from cyc- S49 lymphoma cells.

beta-Adrenergic receptors on membranes prepared from L6 myoblasts, wild-type S49 lymphoma cells, and an adenylate cyclase-deficient variant (cyc-) of S49 lymphoma cells bind the agonist [3H]hydroxybenzylisoproterenol ([3H]HBI) with high affinity. In each case the agonist [3H]HBI is associated with a larger complex than is the antagonist [125I]iodopindolol, and the binding of [3H]HBI can be inhibited by GTP. These observations suggest that there is an agonist-dependent association of the receptor with a guanine nucleotide-binding protein. The goal of the present experiments was to investigate the possibility that an interaction of beta-adrenergic receptors with the inhibitory guanine nucleotide-binding protein of adenylate cyclase was responsible for these observations. Treatment of S49 cells with pertussis toxin decreased the extent of pertussis toxin-catalyzed [32P]ADP-ribosylation of a 41,000-dalton protein, measured in vitro, and decreased the inhibition of adenylate cyclase activity observed in the presence of somatostatin or analogues of GTP. Isoproterenol-stimulated adenylate cyclase activity was potentiated following treatment of wild-type S49 cells and L6 myoblasts with pertussis toxin. Although the ability of receptors on membranes prepared from L6 myoblasts to bind the agonist [3H]HBI was not affected by treatment of cells with pertussis toxin, treatment of cyc- S49 cells with pertussis toxin markedly decreased the ability of receptors to bind [3H]HBI. The observed inhibition of the binding of the agonist [3H]HBI to beta-adrenergic receptors on membranes prepared from cyc- S49 cells after treatment with pertussis toxin could be explained by an interaction between beta-adrenergic receptors and the inhibitory guanine nucleotide-binding protein. Such an interaction may represent a mechanism through which stimulation of the activity of adenylate cyclase by beta-adrenergic receptors can be regulated or through which beta-adrenergic receptors can affect the activity of cyclic AMP-independent cellular processes.

Distinctions in ligand binding sites on the nicotinic acetylcholine receptor.

Ligand-gated ion channels possess intrinsic binding sites for noncompetitive inhibitors that differ substantially in ligand specificity and structural characteristics from most binding sites found on globular proteins. We have used the nicotinic acetylcholine receptor to examine the characteristics of such diverse sites because the high-affinity binding site in the proximity of the ion channel has unusual binding interactions and ligand specificity, whereas the site of agonist activation exhibits classical structure-activity characteristics. Noncompetitive inhibitors that bind to the former site show a wide degree of structural variation and appear to associate at separate loci and in distinct orientations in the vicinity of the channel. The receptor structure appears to provide a large domain with multiple hydrophobic crevices that can bind noncompetitive inhibitors, yet binding of these inhibitors is mutually exclusive. The mutually exclusive behavior suggests that association of a single ligand is sufficient to prevent access of additional ligands to distinct sites. This could occur either by physical occlusion to the site of binding or by formation of a conformational state that will not allow entry of additional noncompetitive inhibitors.

Actions of neurotoxins (bungarotoxins, neosurugatoxin and lophotoxins) on insect and nematode nicotinic acetylcholine receptors.

Neurotoxins of natural origin have proved to be of considerable value in the isolation and characterization of vertebrate muscle and neuronal nicotinic acetylcholine receptors (nAChRs). To date, they have been used less extensively in studies of invertebrate nAChRs. Here we examine how a variety of neurotoxins (the snake toxins alpha-bungarotoxin, alpha-BGT, and kappa-bungarotoxin, kappa-BGT, the molluscan toxin, neosurugatoxin, and the soft coral toxins, lophotoxin and bipinnatin-B) can be used to characterize nAChRs in an insect, Periplaneta americana, and in a parasitic nematode, Ascaris suum. The agonist profiles of these nAChRs are distinct, but the most striking differences are in the actions of antagonists. Whereas the insect nAChR is blocked by both alpha- and kappa-bungarotoxins, the nematode receptor is only blocked by kappa-BGT. Neosurugatoxin blocks nAChRs in both species, but the lophotoxins which block all nAChRs investigated to date are much less effective on the Ascaris muscle receptor.

Lophotoxin is a slow binding irreversible inhibitor of nicotinic acetylcholine receptors.

Lophotoxin and the bipinnatins are members of the lophotoxin family of marine neurotoxins, which covalently react with Tyr190 in the alpha-subunits of the nicotinic acetylcholine receptor. Bipinnatin-A, -B, and -C are protoxins that have been shown to spontaneously convert from inactive to active toxins during preincubation in buffer. However, in this report, we show that preincubation of lophotoxin did not result in an increase in the subsequent rate of irreversible inhibition of nicotinic receptors. Thus, unlike the bipinnatins, lophotoxin does not appear to be an inactive protoxin. Lophotoxin preferentially inhibited one of the two acetylcholine-binding sites on the receptor, and this preference resulted from both a higher reversible affinity and a faster rate of irreversible inhibition at this site. Association of 125I-alpha-bungarotoxin in the presence of lophotoxin was analyzed to obtain the apparent reversible association and dissociation rate constants for lophotoxin. The apparent association rate constant of lophotoxin was approximately 10(6)-fold slower than expected for a diffusion-limited interaction, indicating that lophotoxin is a slow binding irreversible inhibitor. The kinetic constants that describe the interaction of lophotoxin with the receptor did not change in the presence of dibucaine, suggesting that, unlike agonists, the slow apparent association of lophotoxin does not result from a slow transition of the receptor to a desensitized conformation.

Properties of beta-adrenergic receptors of cultured mammalian cells. Interaction of receptors with a guanine nucleotide-binding protein in membranes prepared from L6 myoblasts and from wild-type and cyc- S49 lymphoma cells.

The radiolabeled agonist [3H]hydroxybenzylisoproterenol ([3H]HBI) and antagonist [125I]iodopindolol ([125I]IPIN) were used to investigate the properties of beta-adrenergic receptors on membranes prepared from L6 myoblasts and S49 lymphoma cells. The high affinity binding of (-)-[3H]HBI to membranes prepared from L6 myoblasts was stereoselectively inhibited by the active isomers of isoproterenol and propranolol. The density of receptors determined with (-)-[3H]HBI was less than that determined with [125I]IPIN. The binding of (-)-[3H]HBI was inhibited by guanine nucleotides, suggesting an agonist-mediated association of the receptor with a guanine nucleotide-binding protein, presumably the stimulatory guanine nucleotide-binding protein (Ns) of adenylate cyclase. Results obtained in studies with membranes prepared from wild-type S49 lymphoma cells and the adenylate cyclase-deficient variant (cyc-) were similar to those obtained in experiments carried out with membranes prepared from L6 myoblasts. Thus, the high affinity binding of (-)-[3H]HBI to membranes prepared from wild-type and cyc- S49 lymphoma cells was stereoselectively inhibited by the active isomers of isoproterenol and propranolol, and was inhibited by GTP. Moreover, the density of sites determined with (-)-[3H]HBI was less than that determined with [125I]IPIN. These results suggest either that cyc- cells contain a partially functional Ns, or alternatively, that the inhibitory guanine nucleotide-binding protein (Ni) is capable of interacting with beta-adrenergic receptors.

Actions of a coral toxin analogue (bipinnatin-B) on an insect nicotinic acetylcholine receptor.

The lophotoxin analogue, bipinnatin-B, is a potent neurotoxin isolated from the gorgonian coral Pseudopterogorgia bipinnata. When tested on the cell body of an identified motor neurone, the fast coxal depressor motor neurone (Df) in the cockroach metathoracic ganglion, bipinnatin-B, at concentrations of 10 micronM,partially blocked nicotine-induced depolarization. Blockade of the response to nicotine was almost complete at 30 micronM bipinnatin-B, and was partially reversible on rebathing the preparation in normal saline. Responses of the same neurone to GABA were unaffected by 30 micronM bipinnatin-B.

Evaluation of models for analysis of radioligand binding data.

In the presence of agonists, many neurotransmitter receptors interact with regulatory components, resulting in the formation of a ternary complex composed of agonist, receptor, and a regulatory component, and in biphasic or shallow dose response curves for inhibition of the binding of a radiolabeled antagonist by agonists. Complex dose response curves are often analyzed using equations that describe a model that assumes the presence of two independent populations of receptors (two-independent-receptor model) or equations that describe a model that assumes the reversible interaction of agonist-occupied receptors with a regulatory component (ternary complex model). In this study, the ability of these models to provide good estimates of the concentration of the regulatory component and of the affinities involved in formation of the ternary complex was evaluated. Dose response curves were generated by a computer using an equation that describes the ternary complex model. Analysis of the dose response curves with the two-independent-receptor model resulted in good estimates of the concentration of the regulatory component and of the affinity of the receptor for the agonist. Reliable estimates of the other affinity constants that relate to formation of the ternary complex could not be obtained. Analysis of the dose response curves with the ternary complex model resulted in good estimates of the concentration of the regulatory component and of the affinity constants that relate to formation of the ternary complex. Random error was added to the data points that made up the dose response curves to simulate error observed in experiments with biological systems. Analysis of the dose response curves that contained random error with the two-independent-receptor model yielded results similar to those obtained from analysis of dose response curves that did not contain random error. Analysis of the dose response curves that contained random error with the ternary complex model resulted in good estimates of the concentration of the regulatory component and of the affinity of the receptor for the agonist. However, reliable estimates of the other affinity constants involved in formation of the ternary complex could not be obtained. Thus, caution should be exercised when interpreting results of the analysis of dose response curves with either the two-independent-receptor model or the ternary complex model.

An analog of lophotoxin reacts covalently with Tyr190 in the alpha-subunit of the nicotinic acetylcholine receptor.

Lophotoxin and lophotoxin analog-1 are natural diterpenes from coral that inhibit nicotinic acetylcholine receptors by covalent reaction with the acetylcholine recognition sites on the alpha-subunits. Although both toxins contain potentially reactive epoxides and alpha,beta-unsaturated aldehydes, the mechanism of their covalent reaction with the receptor is not known. The role of the alpha,beta-unsaturated aldehyde in analog-1 was investigated by reduction of the aldehyde to an alcohol with [3H]NaBH4. The reduced [3H]analog-1 bound selectively and covalently to the alpha-subunit of the receptor. Covalent binding was inhibited by agonists and antagonists, but not by noncompetitive allosteric inhibitors. The apparent dissociation constant of the reduced [3H]analog-1 was approximately 1.5 x 10(-6) M. These results demonstrate that the alpha,beta-unsaturated aldehyde in analog-1 is not an absolute requirement for covalent reaction with the receptor. Receptors were treated with the reduced-[3H]analog-1, and the labeled alpha-subunits were isolated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and digested with staphylococcal V8 protease. A labeled 20-kDa V8 protease fragment was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase high performance liquid chromatography and subjected to sequence analysis. A peptide beginning at Ser173 was identified, and the label appeared in the 18th step corresponding to Tyr190. This assignment was confirmed by digestion of the labeled 20-kDa V8 protease fragment with cyanogen bromide, followed by purification of the labeled cyanogen bromide peptide on reverse-phase high performance liquid chromatography. A peptide beginning at Lys179 was identified, and the label appeared in the 12th step, again corresponding to Tyr190. Tyr190 may react with the coral toxin by nucleophilic addition at one of the carbons associated with an epoxide, and may form part of the alkylammonium-binding subsite of the acetylcholine recognition site.

Determinants involved in the affinity of alpha-conotoxins GI and SI for the muscle subtype of nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors from muscle contain two functionally active and pharmacologically distinct acetylcholine-binding sites located at the alpha/gamma and alpha/delta subunit interfaces. The alpha-conotoxins are competitive antagonists of nicotinic receptors and can be highly site-selective, displaying greater than 10,000-fold differences in affinities for the two acetylcholine-binding sites on a single nicotinic receptor. The higher affinity site for alpha-conotoxins GI, MI, and SI is the alpha/delta site on mouse muscle-derived BC3H-1 receptors. However, alpha-conotoxins GI and MI exhibit higher affinity for the other site (alpha/gamma site) on nicotinic receptors from Torpedo californica electric organ. alpha-Conotoxin SI does not distinguish between the two acetylcholine-binding sites on Torpedo receptors. In this study, alpha-conotoxins [K10H]SI and [K10N]SI displayed wild-type affinity for the two acetylcholine-binding sites on BC3H-1 receptors but a 10-20-fold decrease in apparent affinity at one of the two acetylcholine-binding sites on Torpedo receptors. alpha-Conotoxin [P9K]SI displayed a selective and dramatic increase in the apparent affinity for the alpha/delta site of BC3H-1 receptors and for the alpha/gamma site of Torpedo receptors. alpha-Conotoxin [R9A]GI displayed a reduction in affinity for both acetylcholine-binding sites on BC3H-1 receptors, although the extent of its selectivity for the alpha/delta site was retained. alpha-Conotoxin [R9A]GI also displayed a loss of affinity for the two acetylcholine-binding sites on Torpedo receptors, but its site-selectivity was apparently abolished. These results indicate that positions 9 and 10 in alpha-conotoxins GI and SI are involved in complex species- and subunit-dependent interactions with nicotinic receptors.

Irreversible inhibition of nicotinic acetylcholine receptors by the bipinnatins. Toxin activation and kinetics of receptor inhibition.

Bipinnatin-A, -B, and -C belong to a family of naturally occurring marine neurotoxins known as the lophotoxins. The lophotoxins are unique in that they irreversibly inhibit nicotinic acetylcholine receptors by forming a covalent bond with a tyrosine residue at position 190 in the alpha-subunit of the receptor. In this study, we show that the inhibitory activity of the bipinnatins against the nicotinic receptor increased with preincubation of the toxins in aqueous buffer prior to incubation with the receptor. The parent species of the bipinnatins displayed little, if any, affinity for the nicotinic receptor. Preincubation of the toxins appeared to produce a single, relatively stable, active toxin species that irreversibly inhibited the two acetylcholine-binding sites on the nicotinic receptor with two distinguishable apparent pseudo first-order rates. The difference in the rates of irreversible inhibition of the two binding sites on the receptor was exploited to selectively inhibit one site for the pharmacological investigation of the other. The bipinnatins preferentially inhibited the binding site near the alpha/delta-subunit interface that displays low affinity for metocurine and high affinity for acetylcholine. The bimolecular reaction constants for the interaction of the bipinnatins with the nicotinic receptor decreased in the order bipinnatin-B > bipinnatin-A > bipinnatin-C for both acetylcholine-binding sites. The ratio of the bimolecular reaction constants for the two binding sites on the receptor was not the same for the three bipinnatins. This indicates that the reaction of the bipinnatins with the nicotinic receptor is sensitive to differences in the structure of the two acetylcholine-binding sites. The bipinnatins may be useful in the design of novel drugs for the nicotinic receptor that exclusively inhibit one of the two binding sites and for the investigation of structural differences between the two acetylcholine-binding sites of the receptor.

Onchidal: a naturally occurring irreversible inhibitor of acetylcholinesterase with a novel mechanism of action.

Onchidal has been identified as the major lipid-soluble component of the defensive secretion of the mollusc Onchidella binneyi, and it has been proposed as the compound responsible for the chemical protection of Onchidella [Bioorg. Chem. 7:125-131 (1978)]. In support of this hypothesis, we now report that onchidal can be found in several different species of Onchidella and that it is toxic to fish. Because onchidal is an acetate ester similar to acetylcholine, its ability to interact with nicotinic acetylcholine receptors and acetylcholinesterase was investigated. Although onchidal did not prevent the binding of 125I-alpha-bungarotoxin to nicotinic acetylcholine receptors, it inhibited acetylcholinesterase in a progressive, apparently irreversible, manner. The apparent affinity of onchidal for the initial reversible binding to acetylcholinesterase (Kd) was approximately 300 microM, and the apparent rate constant for the subsequent irreversible inhibition of enzyme activity (kintact) was approximately 0.1 min-1. Onchidal was a substrate for acetylcholinesterase, and approximately 3250 mol of onchidal were hydrolyzed/mol of enzyme irreversibly inhibited. The calculated kcat for onchidal was 325 min-1. Irreversible inhibition resulted from either onchidal itself or a reactive intermediate in the enzyme-catalyzed hydrolysis of onchidal, rather than from the hydrolysis products of onchidal. Irreversible inhibition of enzyme activity was prevented by coincubation with reversible agents that either sterically block (edrophonium and decamethonium) or allosterically modify (propidium) the acetylcholine binding site. Enzyme activity was not regenerated by incubation with oxime reactivators; therefore, the mechanism of irreversible inhibition does not appear to involve acylation of the active site serine. Because onchidal contains a potentially reactive alpha,beta-unsaturated aldehyde, irreversible inhibition of acetylcholinesterase may result from formation of a novel covalent bond between the toxin and the enzyme. Thus, this novel toxin could potentially be exploited in the design of a new class of anticholinesterase insecticides and in the identification of amino acids that contribute to the binding and hydrolysis of acetylcholine.

alpha-Conotoxins selectively inhibit one of the two acetylcholine binding sites of nicotinic receptors.

Muscle subtypes of the nicotinic acetylcholine receptor contain two acetylcholine binding sites that can be distinguished pharmacologically. The affinities of several alpha-conotoxins for the two acetylcholine binding sites on nicotinic receptors from BC3H1 cells and Torpedo electric organ were investigated. alpha-Conotoxins MI, GI, and SIA each inhibited the binding of 125I-alpha-bungarotoxin to nicotinic acetylcholine receptors on BC3H1 cells with two distinct and independent affinities, which differed by > 10,000-fold. The affinities of alpha-conotoxins SI and SII were significantly lower and the differences in the affinities of each of these toxins for the two sites were < 400-fold. alpha-Conotoxins MI, GI, SIA, and SI had higher affinity for the acetylcholine binding site near the alpha/delta subunit interface of nicotinic receptors from BC3H1 cells. However, when assessed using nicotinic receptors from Torpedo electric organ, alpha-conotoxin MI displayed higher affinity for the acetylcholine binding site near the alpha/gamma subunit interface. These observations suggest that species variations in the sequences of the gamma and delta subunits resulted in a dramatic reversal of the relative affinities of the alpha-conotoxins for each acetylcholine binding site. Some of the practical implications of these observations are discussed.