Broad-spectrum, non-opioid analgesic activity by selective modulation of neuronal nicotinic acetylcholine receptors.

Development of analgesic agents for the treatment of severe pain requires the identification of compounds that are devoid of opioid receptor liabilities. A potent (inhibition constant = 37 picomolar) neuronal nicotinic acetylcholine receptor (nAChR) ligand called ABT-594 was developed that has antinociceptive properties equal in efficacy to those of morphine across a series of diverse animal models of acute thermal, persistent chemical, and neuropathic pain states. These effects were blocked by the nAChR antagonist mecamylamine. In contrast to morphine, repeated treatment with ABT-594 did not appear to elicit opioid-like withdrawal or physical dependence. Thus, ABT-594 may be an analgesic that lacks the problems associated with opioid analgesia.

P2X7-related modulation of pathological nociception in rats.

Growing evidence supports a role for the immune system in the induction and maintenance of chronic pain. ATP is a key neurotransmitter in this process. Recent studies demonstrate that the glial ATP receptor, P2X7, contributes to the modulation of pathological pain. To further delineate the endogenous mechanisms that are involved in P2X7-related antinociception, we utilized a selective P2X7 receptor antagonist, A-438079, in a series of in vivo and in vitro experiments. Injection of A-438079 (10-300 micromol/kg, i.p.) was anti-allodynic in three different rat models of neuropathic pain and it attenuated formalin-induced nocifensive behaviors. Using in vivo electrophysiology, A-438079 (80 micromol/kg, i.v.) reduced noxious and innocuous evoked activity of different classes of spinal neurons (low threshold, nociceptive specific, wide dynamic range) in neuropathic rats. The effects of A-438079 on evoked firing were diminished or absent in sham rats. Spontaneous activity of all classes of spinal neurons was also significantly reduced by A-438079 in neuropathic but not sham rats. In vitro, A-438079 (1 microM) blocked agonist-induced (2,3-O-(4-benzoylbenzoyl)-ATP, 30 microM) current in non-neuronal cells taken from the vicinity of the dorsal root ganglia. Furthermore, A-438079 dose-dependently (0.3-3 microM) decreased the quantity of the cytokine, interleukin-1beta, released from peripheral macrophages. Thus, ATP, acting through the P2X7 receptor, exerts a wide-ranging influence on spinal neuronal activity following a chronic injury. Antagonism of the P2X7 receptor can in turn modulate central sensitization and produce antinociception in animal models of pathological pain. These effects are likely mediated through immuno-neural interactions that affect the release of endogenous cytokines.

Discovery of P2X7 receptor-selective antagonists offers new insights into P2X7 receptor function and indicates a role in chronic pain states.

ATP-sensitive P2X(7) receptors are localized on cells of immunological origin including peripheral macrophages and glial cells in the CNS. Activation of P2X(7) receptors leads to rapid changes in intracellular calcium concentrations, release of the proinflammatory cytokine interleukin-1beta and following prolonged agonist exposure, the formation of cytolytic pores in plasma membranes. Both the localization and functional consequences of P2X(7) receptor activation indicate a role in inflammatory processes. The phenotype of P2X(7) receptor gene-disrupted mice also indicates that P2X(7) receptor activation contributes to ongoing inflammation. More recently, P2X(7) receptor knockout data has also suggested a specific role in inflammatory and neuropathic pain states. The recent discovery of potent and highly selective antagonists for P2X(7) receptors has helped to further clarify P2X receptor pharmacology, expanded understanding of P2X(7) receptor signaling, and offers new evidence that P2X(7) receptors play a specific role in nociceptive signaling in chronic pain states. In this review, we incorporate the recent discoveries of novel P2X(7) receptor-selective antagonists with a brief update on P2X(7) receptor pharmacology and its therapeutic potential.

A-85380 [3-(2(S)-azetidinylmethoxy) pyridine]: in vitro pharmacological properties of a novel, high affinity alpha 4 beta 2 nicotinic acetylcholine receptor ligand.

The in vitro pharmacological properties of a novel cholinergic channel ligand, A-85380 [3-(2(S)-azetidinylmethoxy)pyridine], were examined using tissue preparations that express different putative nAChR subtypes. In radioligand binding studies, A-85380 is shown to be a potent and selective ligand for the human alpha 4 beta 2 nAChR subtype (Ki = 0.05 + 0.01 nM) relative to the human alpha 7 (Ki = 148 +/- 13 nM) and the muscle alpha 1 beta 1 dg subtype expressed in Torpedo electroplax (Ki = 314 +/- 12 nM). The R-enantiomer of A-85380, A-159470, displays little enantioselectivity towards the alpha 4 beta 2 and alpha 1 beta 1 delta gamma subtypes but does not display 12-fold enantioselectivity towards the alpha 7 subtype (Ki = 1275 +/- 199 nM). (+)- and(-)-Epibatidine display similar potencies at the human human alpha 4 beta 2 (Ki = 0.04 +/- 0.02 nM and 0.07 +/- 0.02 nM, respectively), human alpha 7 (Ki = 16 +/- 2 nM and 22 +/- 3 nM, respectively) and muscle alpha 1 beta 1 delta gamma g (Ki = 2.5 +/- 0.9 nM and 5.7 +/- 1.0 nM, respectively) nAChRs. Functionally, A-85380 is a potent activator of cation efflux through the human alpha 4 beta 2 (EC50 = 0.7 +/- 0.1 microM) and ganglionic (EC50 = 0.8 +/- 0.09 microM) subtypes, effects that are attenuated by pretreatment with mecamylamine (10 microM). Further, A-85380 can activate (EC50 = 8.9 +/- 1.9 microM) currents through channels formed by injection of the human alpha 7 subunit into Xenopus oocytes, effects that are attenuated by pretreatment with the alpha 7 nAChR antagonist, methyllycaconitine (10 nM). In all cases, A-85380 is more potent than (-)-nicotine but less potent than (+/-)-epibatidine. In neurotransmitter release studies, A-85380 stimulates the release of dopamine with an EC 50 value of 0.003 +/- 0.001 microM which is equipotent to (+/-)-epibatidine, and 20-fold more potent than (-)-nicotine (EC50 = 0.04 +/- 0.009 microM). Thus, A-85380 displays a profile of robust activation of a number of nAChR subtypes with substantially less affinity for [125I] alpha-BgT sites than [3H](-)-cytisine sites, suggesting that it may serve as a more selective pharmacologic probe for the alpha 4 beta 2 subtype relative to the alpha 7 and alpha 1 beta 1 delta g nAChRs than (+/-)-epibatidine.

Novel 3-Pyridyl ethers with subnanomolar affinity for central neuronal nicotinic acetylcholine receptors.

Recent evidence indicating the therapeutic potential of cholinergic channel modulators for the treatment of central nervous system (CNS) disorders as well as the diversity of brain neuronal nicotine acetylcholine receptors (nAChRs) have suggested an opportunity to develop subtype-selective nAChR ligands for the treatment of specific CNS disorders with reduced side effect liabilities. We report a novel series of 3-pyridyl ether compounds which possess subnanomolar affinity for brain nAChRs and differentially activate subtypes of neuronal nAChRs. The synthesis and structure-activity relationships for the leading members of the series are described, including A-85380 (4a), which possesses ca.50 pM affinity for rat brain [(3)H]-(-)-cytisine binding sites and 163% efficacy compared to nicotine to stimulate ion flux at human alpha4beta2 nAChR subtype, and A-84543 (2a), which exhibits 84-fold selectivity to stimulate ion flux at human alpha4beta2 nAchR subtype compared to human ganglionic type nAChRs. Computational studies indicate that a reasonable superposition of a low energy conformer of 4A with (S)-nicotine and (-)-epibatidine can be achieved.

Identification and initial structure-activity relationships of (R)-5-(2-azetidinylmethoxy)-2-chloropyridine (ABT-594), a potent, orally active, non-opiate analgesic agent acting via neuronal nicotinic acetylcholine receptors.

New members of a previously reported series of 3-pyridyl ether compounds are disclosed as novel, potent analgesic agents acting through neuronal nicotinic acetylcholine receptors. Both (R)-2-chloro-5-(2-azetidinylmethoxy)pyridine (ABT-594, 5) and its S-enantiomer (4) show potent analgesic activity in the mouse hot-plate assay following either intraperitoneal (i.p.) or oral (p.o.) administration, as well as activity in the mouse abdominal constriction (writhing) assay, a model of persistent pain. Compared to the S-enantiomer and to the prototypical potent nicotinic analgesic agent (+/-)-epibatidine, 5 shows diminished activity in models of peripheral side effects. Structure-activity studies of analogues related to 4 and 5 suggest that the N-unsubstituted azetidine moiety and the 2-chloro substituent on the pyridine ring are important contributors to potent analgesic activity.

Structure-activity studies on 2-methyl-3-(2(S)-pyrrolidinylmethoxy) pyridine (ABT-089): an orally bioavailable 3-pyridyl ether nicotinic acetylcholine receptor ligand with cognition-enhancing properties.

2-Methyl-3-(2(S)-pyrrolidinylmethoxy)pyridine, ABT-089 (S-4), a member of the 3-pyridyl ether class of nicotinic acetylcholine receptor (nAChR) ligands, shows positive effects in rodent and primate models of cognitive enhancement and a rodent model of anxiolytic activity and possesses a reduced propensity to activate peripheral ganglionic type receptors. The profiles of S-4, its N-methyl analogue, and the corresponding enantiomers across several measures of cholinergic channel function in vitro and in vivo are presented, together with in vitro metabolism and in vivo bioavailability data. On the basis of its biological activities and favorable oral bioavailability, S-4 is an attractive candidate for further evaluation as a treatment for cognitive disorders.

Sodium dodecyl sulfate- and carbamylcholine-induced changes in circular dichroism spectra of acetylcholine receptor synthetic peptides.

The effect of sodium dodecyl sulfate (SDS) on the conformation of acetylcholine receptor alpha-subunit synthetic peptides was investigated by circular dichroism. In the presence of SDS (0.01-0.02%), the affinity of a 173-204 32 residue peptide and a 172-227 56 residue peptide for the competitive antagonist alpha-bungarotoxin increases about 10-fold to the nanomolar range. Circular dichroism spectroscopy of these peptides revealed significant changes in the secondary structure of the peptides in the presence of SDS at concentrations below the critical micelle concentration. It is concluded that SDS induces a conformation of the peptides that is conductive to high affinity binding. Carbamylcholine, an acetylcholine analog, produced small but significant changes in the spectrum of the 173-204 peptide. This change could be the result of agonist-induced conformational changes in this region of the acetylcholine receptor alpha-subunit or to changes in the asymmetric environments of aromatic chromophores in the binding site. These studies demonstrate that synthetic peptides alone are capable of retaining significant functional activity and contain significant secondary structure.

Structural and conformational similarity between synthetic peptides of curaremimetic neurotoxins and rabies virus glycoprotein.

Antibodies were raised in rabbits against synthetic peptides corresponding to loop 2, the 'toxic' loop reacting with the acetylcholine-binding site on the nicotinic acetylcholine receptor, of curaremimetic neurotoxins and the structurally similar segment of the rabies virus glycoprotein. Some of the antibodies cross-reacted with the corresponding peptides confirming the structural similarity between the neurotoxin and glycoprotein peptides. A polyclonal antibody raised against a 29 residue glycoprotein peptide (175-203) in the presence of 0.1% sodium dodecyl sulfate reacted with native alpha-bungarotoxin and rabies virus. Circular dichroism spectroscopy of the 29 residue glycoprotein peptide and a 20 residue king cobra loop 2 peptide (25-44) revealed these peptides to be conformationally similar and composed predominantly of beta sheet structure. These results show the rabies glycoprotein segment is structurally and conformationally similar to neurotoxin loop 2. This similarity may confer on the glycoprotein the capability of interacting with the neurotoxin-binding site on the acetylcholine receptor.

The role of neuronal nicotinic acetylcholine receptors in antinociception: effects of ABT-594.

ABT-594, a nicotinic acetylcholine receptor agonist, has antinociceptive effects in rat models of acute thermal, persistent chemical, and neuropathic pain. Direct injection of ABT-594 into the nucleus raphe magnus (NRM) is antinociceptive in a thermal threshold test and destruction of serotonergic neurons in the NRM attenuates the effect of systemic ABT-594. However, lidocaine-inactivation of the NRM prevents the antinociceptive effect of systemic (-)-nicotine but not that of systemic ABT-594.

In vitro neuroprotective properties of the novel cholinergic channel activator (ChCA), ABT-418.

Recent literature has shown that compounds interacting with neuronal nicotinic acetylcholine receptors (nAChRs) have the potential to be neuroprotective both in vitro and in vivo. ABT-418 is a novel ChCA that selectively stimulates discrete subtypes of the nAChRs and exhibits cognitive enhancing activity. In the present study, the neuroprotective effects of ABT-418 and (-)-nicotine, as measured by the release of lactate dehydrogenase (LDH) into the media, were investigated in a glutamate (Glu)-induced cytotoxicity assay using either primary rat cortical neurons or a human differentiated cell line, IMR 32. The neuroprotection elicited by ABT-418 and (-)-nicotine is both time and concentration dependent with an optimal concentration of 10 microM and an optimal pretreatment time of 2 h. ABT-418 remained neuroprotective and not cytotoxic to rat cortical cells following subacute exposure for 7 days. Protection appears to be mediated via an interaction with nAChRs, possibly the alpha 7 subtype, since the neuroprotection was prevented by alpha-bungaratoxin (alpha-Bgt) and methyllycaconitine (MLA), both selective alpha 7 antagonists. Removal of extracellular Ca2+ prevented the neuroprotective effects of ABT-418 and (-)-nicotine, consistent with the known ability of alpha 7 nAChRs to modulate calcium dynamics. These data support the idea that ABT-418 not only enhances cognition, but may possibly slow the progression of the neurodegenerative process.

Reduced nicotinic receptor-mediated antinociception following in vivo antisense knock-down in rat.

Pharmacological activation of neuronal nicotinic acetylcholine receptors can produce non-opioid antinociception in rodents. However, multiple nAChR subtypes exist, the most abundant of which contain alpha4 and beta2 subunits. The purpose of the present study was to investigate the role of alpha4-containing nAChRs in mediating nicotinic antinociception using an in vivo antisense strategy. Both i.c.v. infusion and repeated bolus injections into the cerebral aqueduct of an antisense oligonucleotide against the alpha4 subunit significantly attenuated the antinociceptive effects of the nAChR agonist A-85380 in the paw withdrawal test of acute thermal pain. Rats treated with a scrambled oligonucleotide displayed a full antinociceptive response to A-85380, while discontinuing antisense treatment restored the antinociceptive effects of the nicotinic agonist. Double immunohistochemical labeling revealed near-complete overlap of expression of the serotonin marker tryptophan hydroxylase and the alpha4 nAChR subunit in the dorsal raphe nucleus. The expression of alpha4-containing nAChRs by serotonergic neurons in the dorsal raphe offered a means to address nonspecific alpha4 knock-down, i.e., oligonucleotide-induced neurotoxicity. Immunohistochemical detection of alpha4 expression was reduced by nearly 50% in the dorsal raphe of antisense-treated rats as compared to either saline or missense-treated controls. In contrast, the expression of tryptophan hydroxylase, as well as, the alpha7 nAChR subunit in antisense-infused rats was similar to that observed in saline- and missense-treated controls. The results of these studies suggest that alpha4-containing nAChRs, possibly expressed by serotonergic neurons, are involved in nicotinic-mediated analgesia. However, these data do not eliminate the possibility that other nicotinic subunit combinations may also play a role in antinociception produced by nAChR activation.

Stable expression and pharmacological properties of the human alpha 7 nicotinic acetylcholine receptor.

The alpha 7 neuronal nicotinic acetylcholine receptor subtype forms a Ca(2+)-permeable homooligomeric ion channel sensitive to alpha-bungarotoxin in Xenopus oocytes. In this study, we have stably and functionally expressed the human alpha 7 cDNA in a mammalian cell line, HEK-293 and examined its pharmacologic properties. [125I] alpha-Bungarotoxin bound to transfected cells with a Kd value of 0.7 nM and a Bmax value of 973 pmoL/mg protein. No specific binding was detected in untransfected cells. Specific binding could be displaced by unlabeled alpha-bungarotoxin (Ki = 0.5 nM) and an excellent correlation was observed between binding affinities of a series of nicotinic cholinergic ligands in transfected cells and those in the human neuroblastoma IMR-32 cell line. Additionally, cell surface expression of alpha 7 receptors was detected by fluorescein isothiocyanate-conjugated alpha-bungarotoxin in transfected cells. Whole cell currents sensitive to blockade by alpha-bungarotoxin, and with fast kinetics of activation and inactivation, were recorded from transfected cells upon rapid application of (-)-nicotine or acetylcholine with EC50 values of 49 microM and 155 microM respectively. We conclude that the human alpha 7 subunit when expressed alone can form functional ion channels and that the stably transfected HEK-293 cell line serves as a unique system for studying human alpha 7 nicotinic receptor function and regulation, and for examining ligand interactions.

Erysodine, a competitive antagonist at neuronal nicotinic acetylcholine receptors.

Erysodine, an erythrina alkaloid related to dihydro-beta-erythroidine, was found to be a more potent inhibitor of [3H]cytisine binding at neuronal nicotinic acetylcholine receptors but a less potent inhibitor of [125I]alpha-bungarotoxin binding at muscle-type nicotinic acetylcholine receptors than dihydro-beta-erythroidine. Erysodine was a competitive, reversible antagonist of (-)-nicotine-induced dopamine release from striatal slices and inhibited (-)-nicotine-induced 86Rb+ efflux from IMR-32 cells. Erysodine was equipotent with dihydro-beta-erythroidine in the dopamine release assay but 10-fold more potent in the 86Rb+ efflux assay, suggesting differential subtype selectivity for these two antagonists. Erysodine, systemically administered to mice, entered the brain and significantly attentuated nicotine's hypothermic effects and its anxiolytic-like effects in the elevated plus-maze test. There was greater separation between antagonist and toxic doses for erysodine than for dihydro-beta-erythroidine, perhaps because of erysodine's greater selectivity for neuronal receptors. In rats, erysodine prevented both the early developing decrease and the late-developing increase in locomotor activity produced by (-)-nicotine. The potent and competitive nature of erysodine's antagonism together with its ability to enter the brain after systemic administration suggest that erysodine may be a useful tool in characterizing neuronal nicotinic acetylcholine receptors.

Functional characterization of the novel neuronal nicotinic acetylcholine receptor ligand GTS-21 in vitro and in vivo.

(2.4)-Dimethoxybenzylidene anabaseine dihydrochloride (GTS-21), a compound that interacts with rat neuronal nicotinic acetylcholine receptors (nAChRs), was evaluated using human recombinant nAChRs in vitro and various pharmacokinetic and behavioral models in rodents, dogs and monkeys. GTS-21 bound to human alpha 4 beta 2 nAChR (K1-20 nM) 100-fold more potently than to human alpha 7 nAChR, and was 18- and 2-fold less potent than (-)-nicotine at human alpha 4 beta 2 and alpha 7 nAChR, respectively. Functionally. GTS-21 stimulated [5H]dopamine release from rat striatal slices with an EC50 of 10 +/- 2 microM (250-fold less potent and 70% as efficacious as (-)-nicotine), an effect blocked by the nAChR antagonist dihydro-beta-erythroidine. However, GTS-21 did not stimulate human alpha 4 beta 2 nor human ganglionic nAChRs significantly. In vivo, GTS-21 had no adverse effect on dog blood pressure (< or = 2.5 micromol/kg i.v. bolus infusion), in marked contrast with (-)-nicotine, GTS-21 (-62 micromol/kg.s.e.) also did not cross-discriminate significantly with (-)-nicotine in rats and did not reduce temperature or locomotion in mice. Neither was it active in the elevated plus maze anxiety model (0.19-6.2 micromol/kg.IP) in normal mice. However, GTS-21 did improve learning performance of monkeys in the delayed matching-to-sample task (32-130 nmol/kg.i.m.).

Pharmacological characterization of P2X7 receptors in rat peritoneal cells.

OBJECTIVE AND DESIGN: P2X(7) receptor activation by ATP results in the release of IL-1beta and IL-18. Prolonged stimulation can lead to pore formation and cell death. In this study we pharmacologically characterized P2X(7) receptors on rat peritoneal cells (RPC) and on 1321N1 cells transfected with rat P2X(7) receptor (1321rP2X(7)-11). MATERIALS AND METHODS: RPC were isolated from rats by lavage. P2X(7) agonist induced pore formation in RPC was measured by EtBr uptake. P2X(7)-stimulated pore formation and Ca(++) influx in 1321rP2X(7)-11 cells were measured by a fluorometric imaging plate reader. The effects of pyridoxal phosphate-6-azo phenyl -2'-4'-disulfonic acid (PPADS) on pore formation and Ca(++) influx were examined in both RPC and 1321rP2X(7)-11. P2X(7)-mediated IL-1beta release in RPC and the effect of PPADS were determined. RESULTS: RPC express functional P2X(7) receptors that were activated by ATP analogs with a rank order of potency of 2'- 3'-O-(4-Benzoylbenzoyl) adenosine 5'-triphosphate (BzATP) > ATP > alpha,beta-methylene ATP. Activation of P2X(7) receptors by BzATP was inhibited by PPADS. Similar results were also obtained in 1321rP2X(7)-11 cells. Activation of P2X(7) receptors on RPC resulted in IL-1 beta secretion, which was inhibited by PPADS. CONCLUSIONS: RPC express functional P2X(7) receptors that form pores and mediate the release of IL-1beta.

Effects of mutations of Torpedo acetylcholine receptor alpha 1 subunit residues 184-200 on alpha-bungarotoxin binding in a recombinant fusion protein.

Residues between positions 184 and 200 of the Torpedo acetylcholine receptor alpha 1 subunit were changed by oligonucleotide-directed mutagenesis in a recombinant fusion protein containing residues 166-211. Amino acids were substituted with residues present in the snake alpha subunit, with an alanine, or with a functionally dissimilar residue. The competitive antagonist alpha-bungarotoxin bound to the fusion protein with high apparent affinity (IC50 = 3.2 x 10(-8) M), and binding was competed by agonists and antagonists. Mutation of His-186, Tyr-189, Tyr-190, Cys-192, Cys-193, Pro-194, and Asp-195 greatly reduced or abolished alpha-bungarotoxin binding, while mutation of Tyr-198 reduced binding, indicating these residues play an important role in binding either through functional interaction with neurotoxin residues or by stabilizing the conformation of the binding site. Molecular modeling of acetylcholine receptor residues 184-200 and knowledge of both neurotoxin and receptor residues essential for binding allow analysis of possible structure-function relationships of the interaction of alpha-bungarotoxin with this region of the receptor.

Binding sites for alpha-bungarotoxin and the noncompetitive inhibitor phencyclidine on a synthetic peptide comprising residues 172-227 of the alpha-subunit of the nicotinic acetylcholine receptor.

The binding of the competitive antagonist alpha-bungarotoxin (alpha-Btx) and the noncompetitive inhibitor phencyclidine (PCP) to a synthetic peptide comprising residues 172-227 of the alpha-subunit of the Torpedo acetylcholine receptor has been characterized. 125I-alpha-Btx bound to the 172-227 peptide in a solid-phase assay and was competed by alpha-Btx (IC50 = 5.0 x 10(-8) M), d-tubocurarine (IC50 = 5.9 X 10(-5)M), and NaCl (IC50 = 7.9 x 10(-2)M). In the presence of 0.02% sodium dodecyl sulfate, 125I-alpha-Btx bound to the 56-residue peptide with a KD of 3.5 nM, as determined by equilibrium saturation binding studies. Because alpha-Btx binds to a peptide comprising residues 173-204 with the same affinity and does not bind to a peptide comprising residues 205-227, the competitive antagonist and hence agonist binding site lies between residues 173 and 204. After photoaffinity labeling, [3H]PCP was bound to the 172-227 peptide. [3H]PCP binding was inhibited by chlorpromazine (IC50 = 6.3 x 10(-5)M), tetracaine (IC50 = 4.2 x 10(-6)M), and dibucaine (IC50 = 2.7 x 10(-4)M). Equilibrium saturation binding studies in the presence of 0.02% sodium dodecyl sulfate showed that [3H]PCP bound at two sites, a major site of high affinity with an apparent KD of 0.4 microM and a minor low-affinity site with an apparent KD of 4.6 microM. High -affinity binding occurred at a single site on peptide 205-227 (KD = 0.27 microM) and was competed by chlorpromazine but not by alpha-Btx.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies against an alpha-bungarotoxin-binding peptide of the alpha-subunit of the acetylcholine receptor.

Polyclonal and monoclonal antibodies were raised against a peptide comprising residues 173-204 of the alpha-subunit of the acetylcholine receptor. The polyclonal and pooled monoclonal antibodies inhibited up to 50% of 125I-alpha-bungarotoxin binding to peptide 173-204. Some of the antibodies recognized native receptor but did not significantly affect alpha-bungarotoxin binding. Epitope mapping revealed that the antibodies are directed against residues 183-194 indicating this region is a major determinant of toxin binding. This region is most likely conformationally constrained in the native receptor.

Synthetic peptides of neurotoxins and rabies virus glycoprotein behave as antagonists in a functional assay for the acetylcholine receptor.

Peptides of portions of loop 2 (the "toxic" loop) of snake venom curare-mimetic neurotoxins (alpha-bungarotoxin and king cobra toxin b) and of a structurally similar region of the rabies virus glycoprotein were synthesized. The effect of the peptides on carbachol-induced 22Na+ flux into BC3H-1 cells, which contain nicotinic acetylcholine receptors on their surfaces, was measured. Both the neurotoxin and glycoprotein peptides inhibited ion transport with IC50 values of 10(-4) M to 7 x 10(-7) M. The most effective peptides correspond to neurotoxin loop 2 and inhibited 22Na+ flux in the micromolar range comparable to the competitive antagonist d-tubocurarine. These findings show that neurotoxin loop 2 and the corresponding rabies virus glycoprotein segment interact with the agonist binding site of teh acetylcholine receptor and that short synthetic peptides representing portions of larger molecules by themselves can exert a biological effect on a large macromolecular complex like the acetylcholine receptor.