Torpedo Receptor Research Articles

A New Peptide from the Venom of the Madagascar Cat-Eyed Snake Madagascarophis colubrinus Blocks Nicotinic Acetylcholine Receptor

—In screening the venoms of various snake species, we found that the venom of the Madagascar cat-eyed snake Madagascarophis colubrinus competes with α-bungarotoxin for binding to the nicotinic acetylcholine receptor from Torpedo californica. Using liquid chromatography, a peptide that inhibits the binding of α‑bungartoxin to this receptor was isolated from the venom and named macoluxin. The amino acid sequence of this 23-amino acid peptide was determined by automatic Edman degradation. Comparison with amino acid sequences of known proteins showed that the macoluxin sequence is homologous to the α-helical region of the sequence of snake venom metalloproteinases. The peptide was synthesized by solid-phase peptide synthesis, and the study of its biological activity showed that it inhibits the binding of α-bungarotoxin to the Torpedo receptor with an IC50 of 47 μM. Macoluxin also reversibly inhibited acetylcholine-induced currents in the muscle-type nicotinic acetylcholine receptor. This is the first data on the presence of a peptide that can inhibit the nicotinic acetylcholine receptor in the venom of rear-fanged snakes.

A New Peptide from the Venom of the Madagascar Cat-Eyed Snake <i>Madagascarophis colubrinus</i> Blocks Nicotinic Acetylcholine Receptor

In screening the venoms of various snake species, we found that the venom of the Madagascar cat-eyed snake Madagascarophis colubrinus competes with α-bungarotoxin for binding to the nicotinic acetylcholine receptor from Torpedo californica. Using liquid chromatography, a peptide, called macoluxin and inhibiting the binding of the toxin to the receptor, was isolated from the venom. The amino acid sequence of this 23-amino acid peptide was determined by automatic Edman degradation. Comparison with amino acid sequences of known proteins showed that the macoluxin sequence is homologous to the α-helical region of the sequence of snake venom metalloproteinases. The peptide was synthesized by solid-phase peptide synthesis, and the study of its biological activity showed that it inhibits the binding of α-bungarotoxin to the Torpedo receptor with an IC50 of 47 μM. Macoluxin also reversibly inhibited acetylcholine-induced currents in the muscle-type nicotinic acetylcholine receptor. This is the first data on the presence in the venom of rear fanged snakes of a peptide that can inhibit the nicotinic acetylcholine receptor.

The molecular mechanism of snake short-chain alpha-neurotoxin binding to muscle-type nicotinic acetylcholine receptors.

Bites by elapid snakes (e.g. cobras) can result in life-threatening paralysis caused by venom neurotoxins blocking neuromuscular nicotinic acetylcholine receptors. Here, we determine the cryo-EM structure of the muscle-type Torpedo receptor in complex with ScNtx, a recombinant short-chain α-neurotoxin. ScNtx is pinched between loop C on the principal subunit and a unique hairpin in loop F on the complementary subunit, thereby blocking access to the neurotransmitter binding site. ScNtx adopts a binding mode that is tilted toward the complementary subunit, forming a wider network of interactions than those seen in the long-chain α-Bungarotoxin complex. Certain mutations in ScNtx at the toxin-receptor interface eliminate inhibition of neuronal α7 nAChRs, but not of human muscle-type receptors. These observations explain why ScNtx binds more tightly to muscle-type receptors than neuronal receptors. Together, these data offer a framework for understanding subtype-specific actions of short-chain α-neurotoxins and inspire strategies for design of new snake antivenoms.

The molecular mechanism of snake short-chain α-neurotoxin binding to muscle-type nicotinic acetylcholine receptors

Bites by elapid snakes (e.g. cobras) can result in life-threatening paralysis caused by venom neurotoxins blocking neuromuscular nicotinic acetylcholine receptors. Here, we determine the cryo-EM structure of the muscle-type Torpedo receptor in complex with ScNtx, a recombinant short-chain α-neurotoxin. ScNtx is pinched between loop C on the principal subunit and a unique hairpin in loop F on the complementary subunit, thereby blocking access to the neurotransmitter binding site. ScNtx adopts a binding mode that is tilted toward the complementary subunit, forming a wider network of interactions than those seen in the long-chain α-Bungarotoxin complex. Certain mutations in ScNtx at the toxin-receptor interface eliminate inhibition of neuronal α7 nAChRs, but not of human muscle-type receptors. These observations explain why ScNtx binds more tightly to muscle-type receptors than neuronal receptors. Together, these data offer a framework for understanding subtype-specific actions of short-chain α-neurotoxins and inspire strategies for design of new snake antivenoms.

A triad of residues is functionally transferrable between 5-HT3 serotonin receptors and nicotinic acetylcholine receptors

Cys-loop receptors are pentameric ligand-gated ion channels that facilitate communication within the nervous system. Upon neurotransmitter binding, these receptors undergo an allosteric activation mechanism connecting the binding event to the membrane-spanning channel pore, which expands to conduct ions. Some of the earliest steps in this activation mechanism are carried out by residues proximal to the binding site, the relative positioning of which may reflect functional differences among members of the Cys-loop family of receptors. Herein, we investigated key side-chain interactions near the binding site via mutagenesis and two-electrode voltage-clamp electrophysiology in serotonin-gated 5-HT3A receptors (5-HT3ARs) and nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus laevis oocytes. We found that a triad of residues aligning to Thr-152, Glu-209, and Lys-211 in the 5-HT3AR can be exchanged between the homomeric 5-HT3AR and the muscle-type nAChR α-subunit with small functional consequences. Via triple mutant cycle analysis, we demonstrated that this triad forms an interdependent network in the muscle-type nAChR. Furthermore, nAChR-type mutations of the 5-HT3AR affect the affinity of nicotine, a competitive antagonist of 5-HT3ARs, in a cooperative manner. Using mutant cycle analyses between the 5-HT3A triad, loop A residues Asn-101 and Glu-102, β9 residue Lys-197, and the channel gate at Thr-257, we observed that residues in this region are energetically linked to the channel gate and are particularly sensitive to mutations that introduce a net positive charge. This study expands our understanding of the differences and similarities in the activation mechanisms of Cys-loop receptors.

Non-competitive Inhibition of Nicotinic Acetylcholine Receptors by Ladybird Beetle Alkaloids.

Ladybird beetles (Family Coccinellidae) secrete an alkaloid rich venom from their leg joints that protects them from predators. Coccinellines, the major venom constituents, are alkaloids composed of three fused piperidine rings that share a common nitrogen atom. Although many coccinellines have been isolated and chemically characterized, their pharmacological properties are essentially unknown. Using radioligand binding and functional assays we investigated the actions of several coccinellines on skeletal muscle and α7 nicotinic acetylcholine receptors (nAChRs). The alkaloids were shown to displace the specific binding of tritiated piperidyl-N-(1-(2-thienyl)cyclohexyl)-3,4-piperidine ([(3)H]-TCP), which has been shown to bind deep within the ion channel of the electric fish (Torpedo) muscle nAChR. The stereoisomers precoccinelline and hippodamine (whose nitrogens are predicted to be ionized at physiological pH) and their respective analogs N-methyl-precoccinelline and N-methyl-hippodamine (whose quaternary nitrogens are permanently charged) displayed similar IC50s for inhibition of [(3)H]-TCP binding. However, the corresponding precoccinelline and hippodamine N-oxides, coccinelline and convergine (which have an electronegative oxygen bonded to an electropositive nitrogen) displayed significantly higher binding IC50s. Finally, exochomine, a dimeric coccinelline containing the hippodamine structure, displayed the highest IC50 (lowest affinity) for displacing specific [(3)H]-TCP binding. The presence of a desensitizing concentration (10(-3) M) of carbachol (CCh) had little or no effect on the affinity of the Torpedo nAChR for the three coccinellines tested. High concentrations of the coccinellid alkaloids did not affect binding of [(3)H]-cytisine to Torpedo receptor ACh binding sites. Inhibition of the alpha7 nAChR with pre-equilibrated precoccinelline was insurmountable with respect to ACh concentration. We conclude that the coccinellines bind to one or more allosteric sites rather than to the ACh binding sites, and inhibit nAChR responses to ACh through a non-competitive mechanism. Future chemical and pharmacological investigations of other ladybird beetle alkaloids are likely to reveal other interesting alkaloids affecting ligand-gated receptors.

Structural characterization and agonist binding to human α4β2 nicotinic receptors

The Cys-loop receptor super-family of neurotransmitter-gated ion channels mediates fast synaptic transmission throughout the human nervous system. These receptors exhibit widely varying pharmacologies, yet their structural characterization has relied heavily on their homology with the naturally abundant muscle-type Torpedo nicotinic acetylcholine receptor. Here we examine for the first time the structure of a human α4β2 neuronal nicotinic acetylcholine receptor. We show that human α4β2 nicotinic receptors adopt a secondary/tertiary fold similar to that of the Torpedo nicotinic receptor with a large proportion of both α-helix and β-sheet, but exhibit a substantially increased thermal stability. Both receptors bind agonist, but with different patterns of agonist recognition – particularly in the nature of the interactions between aromatic residues and the agonist quaternary amine functional group. By comparing α4β2 and Torpedo receptors, we begin to delineate their structural similarities and differences.

Probing Structure on Well-defined Functional States of the Nicotinic Receptor Using Systematically-engineered Ionizable Residues and Proton-transfer Events

The conformational changes that underlie the closed-open transition in members of the nicotinic-receptor superfamily remain elusive and controversial. To gain insight into the structural properties of the pore-domain of the muscle-nicotinic acetylcholine-receptor channel (AChR) in the open state, while retaining the advantages of studies on intact cells and in real time, we engineered basic residues along the M1, M2, and M3 transmembrane segments of all four types of subunit and recorded the individual proton-transfer events using single-channel patch-clamp electrophysiology. Proton binding-unbinding reactions to and from individual side chains were manifest as blocking-unblocking events of the passing cation current. Two observables, namely, the extent to which the current is attenuated upon side-chain protonation, and the pKa-shifts of the engineered ionizable groups relative to bulk water, were analyzed to reveal the electrostatic properties of the local microenvironment around the transmembrane segments in the open-channel conformation. In turn, these data were interpreted in terms of secondary and tertiary structure, and compared with existing structural models of the closed state in order to elucidate the change in conformation that opens the AChR. Our open-channel data suggests that the orientation of the M1, M2, and M3 transmembrane segments of the AChR with respect to the pore and each other is very similar to that in the closed-channel structural model developed on the basis of the cryo-EM images or Torpedo's receptor at 4-A resolution. To the extent that this structural model corresponds to the actual closed-channel conformation, our results indicate that the expansion of the pore that underlies channel opening involves only a limited rearrangement of these three helices. Such a modest change seems optimal to ensure rapid closed-open interconversion rates, and hence, a fast postsynaptic response upon neurotransmitter-binding.

Acetylcholine Receptors

a-Bungarotoxin is one of a class of proteins, isolated from snake venoms, which antagonize the action of acetylcholine at vertebrate neuromuscular junctions and ‘electroplaques' of electric fish. a-Bungarotoxin blocks acetylcholine action irreversibly and may be labelled with either 125 I or 3 H. This irreversible binding is used as the basis of an in vitro assay for acetylcholine receptors, whether in intact tissue, membrane fragments or solubilized preparations. Acetylcholine receptors from Torpedo and denervated skeletal muscle have been solubilized and substantially purified using affinity chromatography. The distribution of acetylcholine receptors in several tissues has been determined, and an auto-immune response, induced by injection of purified Torpedo receptors, has been studied.

Tryptophan 86 of the α Subunit in the Torpedo Nicotinic Acetylcholine Receptor Is Important for Channel Activation by the Bisquaternary Ligand Suberyldicholine

Suberyldicholine, a bisquaternary compound, is a potent nicotinic acetylcholine receptor agonist. Previously, we suggested that at least some of the unusual binding properties of this ligand may be a consequence of its ability to cross-link two binding "subsites" within each of the high-affinity agonist binding domains [Dunn, S. M. J., and Raftery, M. A. (1997) Biochemistry 36, 3846-3853]. Tryptophan 86 of the alpha subunit has previously been implicated in the binding of agonist to this receptor. However, on the basis of the crystal structure of a homologous acetylcholine binding protein, this residue is predicted to lie 15-20 A from the high-affinity site, i.e., a distance that approximates the interonium distance of suberyldicholine. Tryptophan 86 was mutated to either an alanine or a phenylalanine, and the mutated subunit was coexpressed with wild-type beta, gamma, and delta subunits in Xenopus oocytes. Although the alanine mutation resulted in a loss of receptor expression, the alphaW86F mutant receptor was expressed on the oocyte surface, albeit with a much reduced efficiency. Acetylcholine-evoked currents of the alphaW86F receptor were not significantly different from those of the wild type with respect to the concentration dependence of channel activation, receptor desensitization, or d-tubocurarine inhibition. In contrast, the EC(50) for suberyldicholine-mediated activation of the alphaW86F receptor was increased by approximately 500-fold. Furthermore, suberyldicholine-evoked currents in the mutant receptor did not desensitize and were insensitive to block by d-tubocurarine. Thus, tryptophan 86 of the Torpedo receptor alpha subunit may be part of a subsite for recognition of suberyldicholine and other bisquaternary ligands.

Physostigmine modulation of acetylcholine currents in COS cells transfected with mouse muscle nicotinic receptor

Physostigmine (Phy), a reversible inhibitor of acetylcholine (ACh) esterase (AChE), may also act as a low potency agonist and a modulator of the nicotinic receptor. The actions of Phy on mouse muscle nicotinic receptors in the COS-7 cell line were studied by the patch-clamp technique. Currents were recorded in the whole-cell mode 3–7 days after cell transfection by plasmids coding αβγδ combination of receptor subunits. The application of ACh to cells clamped at −10 mV produced inward currents which displayed desensitization. The application of Phy in concentrations up to 1 × 10 −3 M did not give reliable specific whole-cell membrane responses. The application of Phy in concentrations of 10 −6–10 −4 M together with ACh modulated the amplitude; accelerated desensitization of currents induced by ACh and increased the final extent of desensitization in a concentration-dependent manner. This finding is in contrast to the suppression and slowing down of desensitization by Phy and 1-methyl-galanthamine observed in Torpedo receptors.

Activation of the Torpedo nicotinic acetylcholine receptor

The Torpedo nicotinic acetylcholine receptor is a heteropentamer (alpha2betagammadelta) in which structurally homologous subunits assemble to form a central ion pore. Viewed from the synaptic cleft, the likely arrangement of these subunits is alpha-gamma-alpha-delta-beta lying in an anticlockwise orientation. High affinity binding sites for agonists and competitive antagonists have been localized to the alpha-gamma and alpha-delta subunit interfaces. We investigated the involvement of amino acids lying at an adjacent interface (gamma-alpha) in receptor properties. Recombinant Torpedo receptors, expressed in Xenopus oocytes, were used to investigate the consequences of mutating alphaArg55 and gammaGlu93, residues that are conserved in most species of the peripheral nicotinic receptors. Based on homology modeling, these residues are predicted to lie in close proximity to one another and it has been suggested that they may form a salt bridge in the receptor's three-dimensional structure (Sine et al. 2002 J Biol Chem277, 29 210-29 223). Although substitution of alphaR55 by phenylalanine or tryptophan resulted in approximately a six-fold increase in the EC50 value for acetylcholine activation, the charge reversal mutation (alphaR55E) had no significant effect. In contrast, the replacement of gammaE93 by an arginine conferred an eight-fold increase in the potency for acetylcholine-induced receptor activation. In the receptor carrying the double mutations, alphaR55E-gammaE93R or alphaR55F-gammaE93R, the potency for acetylcholine activation was partially restored to that of the wild-type. The results suggest that, although individually these residues influence receptor activation, direct interactions between them are unlikely to play a major role in the stabilization of different conformational states of the receptor.

Interaction of a semirigid agonist with Torpedo acetylcholine receptor.

The binding of the semirigid agonist [(3)H]arecolone methiodide to the Torpedo nicotinic acetylcholine receptor has been correlated with its functional properties measured both in flux studies with Torpedo membrane vesicles and by single-channel analysis after reconstitution in giant liposomes. Under both equilibrium and preequilibrium conditions, the binding of arecolone methiodide is similar to that of other agonists such as acetylcholine. At equilibrium, it binds to two sites per receptor with high affinity (K(d) = 99 +/- 12 nM), and studies of its dissociation kinetics suggest that each of these sites is made up of two subsites that are mutually exclusive at equilibrium. The kinetics of arecolone methiodide binding were monitored by the changes in the receptor intrinsic fluorescence, and the data are consistent with a model in which the initial binding event is followed by sequential conformational transitions of the receptor-ligand complex. In flux studies, arecolone methiodide was approximately 3-fold more potent (EC(50) = 31 +/- 5 microM) than acetylcholine but its maximum flux rate was 4-10-fold lower. This phenomenon has been studied further by single-channel analysis of Torpedo receptors reconstituted in giant liposomes. Whereas the flexible agonist carbamylcholine (5 microM) was shown to induce channels with conductances of 56 and 34 pS with approximately equal frequency, arecolone methiodide (2 microM) preferentially induced the channel of lower conductance. These results are interpreted in terms of a simple model in which the rigidity of arecolone methiodide restrains the conformation that the receptor-ligand complex can adopt, thus favoring the lower conductance state.

Variability among the Sites by Which Curaremimetic Toxins Bind to Torpedo Acetylcholine Receptor, as Revealed by Identification of the Functional Residues of α-Cobratoxin

alpha-Cobratoxin, a long chain curaremimetic toxin from Naja kaouthia venom, was produced recombinantly (ralpha-Cbtx) from Escherichia coli. It was indistinguishable from the snake toxin. Mutations at 8 of the 29 explored toxin positions resulted in affinity decreases for Torpedo receptor with DeltaDeltaG higher than 1.1 kcal/mol. These are R33E > K49E > D27R > K23E > F29A >/= W25A > R36A >/= F65A. These positions cover a homogeneous surface of approximately 880 A(2) and mostly belong to the second toxin loop, except Lys-49 and Phe-65 which are, respectively, on the third loop and C-terminal tail. The mutations K23E and K49E, and perhaps R33E, induced discriminative interactions at the two toxin-binding sites. When compared with the short toxin erabutoxin a (Ea), a number of structurally equivalent residues are commonly implicated in binding to muscular-type nicotinic acetylcholine receptor. These are Lys-23/Lys-27, Asp-27/Asp-31, Arg-33/Arg-33, Lys-49/Lys-47, and to a lesser and variable extent Trp-25/Trp-29 and Phe-29/Phe-32. In addition, however, the short and long toxins display three major differences. First, Asp-38 is important in Ea in contrast to the homologous Glu-38 in alpha-Cbtx. Second, all of the first loop is insensitive to mutation in alpha-Cbtx, whereas its tip is functionally critical in Ea. Third, the C-terminal tail may be specifically critical in alpha-Cbtx. Therefore, the functional sites of long and short curaremimetic toxins are not identical, but they share common features and marked differences that might reflect an evolutionary pressure associated with a great diversity of prey receptors.

How do snake curaremimetic toxins discriminate between nicotinic acetylcholine receptor subtypes

Curaremimetic toxins from snake venoms form a large family of small proteins that adopt a similar fold and which bind to Torpedo nicotinic acetylcholine receptors with high affinity. Notwithstanding its apparent homogeneity, the toxin family is subdivided into short-chain (60–62 residues and four disulfide bonds) and long-chain toxins (66–74 residues and five disulfide bonds). In agreement with this structurally-based distinction we recently showed that only long-chain toxins bind with high affinity to the neuronal nicotinic acetylcholine α7 receptor. We suggested that a small loop cyclized by a disulfide bond and uniquely present in long-chain toxins may act as a major discriminative element. To assess the validity of this proposal we prepared various derivatives of a long-chain toxin, using stepwise solid-phase synthesis. We found that replacement of both half cystines of the small loop by a serine caused a 35-fold affinity decrease for the neuronal receptor and only a 6-fold affinity decrease for Torpedo receptor. In addition, insertion of this loop at a homologous position of a short-chain toxin caused a 20-fold affinity increase for the neuronal receptor whereas it did not modify its affinity for the Torpedo receptor. Our findings, therefore, reveal that a small structural deviation from a toxin fold can generate exquisite discriminative recognition for some receptor subtypes.

Determinants Involved in the Affinity of α-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.

Multiple Ras Signals Pattern theDrosophilaOvarian Follicle Cells

DuringDrosophilaoogenesis, spatially restricted activity of the TORPEDO receptor tyrosine kinase first recruits follicle cells adjacent to the oocyte to a posterior cell fate and then specifies dorsal follicle cells. Another receptor tyrosine kinase, BREATHLESS, stimulates migration of the anterior follicle cells known as border cells. Since Ras is known to mediate many receptor tyrosine kinase effects, we have investigated the role of Ras in follicle cell fate determination, differentiation, and migration throughout oogenesis. Early ectopic Ras activity induced transient expression of posterior follicle cell markers in anterior follicle cells, but did not inhibit anterior differentiation. Later ectopic Ras activity inhibited anterior follicle cell differentiation but did not induce posterior marker expression. Complete transformation of anterior follicle cells to posterior follicle cells required early ectopic Ras activity in egg chambers where terminal differentiation of anterior cells was inhibited. These results suggest that,in vivoasin vitro,Ras can have diverse effects on different cells, but, in addition, Ras activity can have different effects on the same cells at different stages in their development.

A single site on the ϵ subunit is responsible for the change in ACh receptor channel conductance during skeletal muscle development

Four critically positioned amino acids on each of the αβ, δ and γ sunits of the Torpedo nicotinic acetylcholine receptor are determinants of channel conductance. Our results show that the γ and ϵ subunits of Xenopus muscle receptors are identical at all four positions, despite the fact that α 2 βδϵ receptors have a 50% greater conductance than α 2 βδγ receptors. Instead, the functional difference is conferred by a single charged residue that lies extracellular to all four positions, corresponding to a location in the Torpedo receptor previously shown to have no influence on conductance. Substitution of a positively charged lysine residue in γ by the neutral methionine in ϵ at this extracellular position is responsible for the increased conductance during maturation of the amphibian neuromuscular junction.

Aromatic trivalent arsenicals: covalent yet reversible reagents for the agonist binding site of nicotinic receptors

The agonist binding site of nicotinic acetylcholine receptors (AChRs) includes a disulfide bond that is easily reduced with dithiothreitol to a pair of thiols, and can be then either reoxidized with dithiobis(nitrobenzoic acid) (DTNB) or irreversibly alkylated with bromoacetylcholine (BAC). Aromatic trivalent arsenicals form stable complexes with pairs of appropriately-spaced thiols, but not single thiols. Furthermore, once complexed in proteins, trivalent arsenicals can be removed with dimercaptans, such as 2,3-dimercaptopropanesulfonic acid (DMPS). In an effort to develop reagents that will covalently, yet reversibly label AChRs, we investigated the effects of two model arsenicals, p-aminophenyldichloroarsine (APA) and 4-bromoacetyl-aminophenylarsenoxide (BAPA) on two types of nicotinic receptors: AChRs from Torpedo electroplax and neuronal receptors from chick retina. APA and BAPA significantly decrease the number of 125I-α-bungarotoxin binding sites in reduced Torpedo AChRs. Furthermore, arsenylation of neuronal and Torpedo receptors with APA or BAPA (1) prevents reoxidation with DTNB, (2) is reversible with DMPS, and (3) protects against irreversible alkylation by BAC. In Torpedo receptors, the EC 50 of protection against BAC alkylation with APA or BAPA is approximately 30 nM. APA arsenylation of Torpedo receptors persists up to 20 h, but can be reversed at any time with DMPS. These results suggest that heterobifunctional arsenicals could anchor labeling groups in the agonist binding site in order to map the agonist binding site, quantitate receptors, or purify and reconstitute functional receptors.

Modulation of anti-acetylcholine receptor antibody specificities and of experimental autoimmune myasthenia gravis by synthetic peptides.

Synthetic peptides corresponding to selected sequences from the nicotinic acetylcholine receptor (AChR) were employed to identify possible antigenic determinants within the receptor which can modulate the anti-AChR response and experimental autoimmune myasthenia gravis (EAMG). Immunization of rabbits with peptides T alpha 73-89, T alpha 351-368, T delta 354-367 and H alpha 351-368, prior to AChR inoculation, affected the course of EAMG in six out of eight rabbits. These six protected rabbits survived three inoculations of AChR and survived for at least five months after the third injection with AChR, whereas control rabbits died following one or two injections of AChR. The survival of peptide-preimmunized rabbits injected with AChR seemed to correlate with the antibody specificities in immunoblots. Following AChR inoculation there was a shift in reactivity, from a subunit-restricted response, to reactivity with all subunits of the receptor. This shift was delayed in protected rabbits. This may indicate that the reactivity with the entire Torpedo receptor molecule represents a loss of tolerance to AChR which culminates in the autoimmune disease, EAMG.