Purification and characterization of a nicotinic acetylcholine receptor from rat brain.

We previously reported the immunoaffinity purification of an acetylcholine receptor from chicken brain that did not bind alpha-bungarotoxin but did bind nicotine and other cholinergic agonists. Antisera and monoclonal antibodies raised against this receptor crossreacted with a receptor from rat brain that had similar pharmacological properties, and also bound to functional acetylcholine receptors in chicken ciliary ganglion cells and rat PC12 cells. Here we report purification of the receptor from rat brain using monoclonal antibody (mAb) 270 raised against receptor from chicken brain. This receptor, similar in size to monomers of receptor from Torpedo electric organ, contained two subunits--apparent Mr, 51,000 and 79,000. The Mr 51,000 subunit was bound by antisera to alpha subunits of receptor from Torpedo electric organ and by mAb 270, which is specific for the Mr 49,000 subunit analogue of receptor from chicken brain. Both subunits were bound by mAb 286, which also binds both subunits of receptors from chicken brain. The alpha-bungarotoxin binding component was purified from the same extracts. It consisted of four subunits of apparent Mr 44,700, 52,300, 56,600, and 65,200. The basic structure of receptors from muscle had evolved to an (alpha)2 beta gamma delta subunit stoichiometry by the time of primitive elasmobranches and is now little changed in mammals. The apparent (alpha)2(beta)2 or (alpha)3(beta)2 structure of the neuronal acetylcholine receptors that we have purified may derive from an early gene duplication event in the evolution of the extended gene family, which now also includes receptors from ganglia and muscle as well as neuronal alpha-bungarotoxin binding sites.     

Binding of alpha-bungarotoxin to isolated alpha subunit of the acetylcholine receptor of Torpedo californica: quantitative analysis with protein blots.

The direct binding of alpha-bungarotoxin to the alpha subunit of the acetylcholine receptor from Torpedo electric organ immobilized onto protein blots was demonstrated. Protein blots were prepared by electrophoretically transferring resolved acetylcholine receptor subunits from 10% polyacrylamide gels onto Zetabind, positively charged nylon membrane filters. Such blots, when incubated with 125I-labeled alpha-bungarotoxin, washed, and autoradiographed, gave rise to a single labeled band corresponding to the alpha subunit of the receptor. The labeling with alpha-bungarotoxin could be inhibited by pretreating the receptor-containing membranes with the affinity ligand 4-(N-maleimido)-alpha-benzyltrimethylammonium iodide. In addition, the association of toxin with the alpha subunit could be inhibited by d-tubocurarine (IC50 = 0.9 mM). Furthermore, removal of high-mannose oligosaccharide chains from the alpha subunit by treatment with endoglycosidase H did not interfere with the observed toxin binding. Thus it is demonstrated that isolated, immobilized alpha subunit of the Torpedo acetylcholine receptor can bind alpha-bungarotoxin. However, the observed binding of alpha-bungarotoxin to immobilized alpha subunit is reduced in affinity to 1/1,000 to 1/10,000 of that obtained with native receptor. The endoglycosidase H-susceptible oligosaccharide side chain(s) is not required for this interaction. Binding of alpha-bungarotoxin is to the physiologically relevant acetylcholine binding site as defined by affinity ligand alkylation.     

Binding of alpha-bungarotoxin to proteolytic fragments of the alpha subunit of Torpedo acetylcholine receptor analyzed by protein transfer on positively charged membrane filters.

Proteolytic fragments of the alpha subunit of the acetylcholine receptor retain the ability to bind alpha-bungarotoxin following resolution by polyacrylamide gel electrophoresis and immobilization on protein transfers. The alpha subunit of the acetylcholine receptor of Torpedo electric organ was digested with four proteases: Staphylococcus aureus V-8 protease, papain, bromelain, and proteinase K. The proteolytic fragments resolved on 15% polyacrylamide gels were electrophoretically transferred onto positively charged nylon membrane filters. When incubated with 0.3 nM 125I-labeled alpha-bungarotoxin and autoradiographed, the transfers yielded patterns of labeled bands characteristic for each protease. The molecular masses of the fragments binding toxin ranged from 7 to 34 kDa, with major groupings in the 8-, 18-, and 28-kDa ranges. The apparent affinity of the fragments for alpha-bungarotoxin as determined from the IC50 value was 6.7 X 10(-8) M. The labeling of fragments with alpha-bungarotoxin could be inhibited by prior affinity alkylation of receptor-containing membranes with 4-(N-maleimido)-alpha-benzyltrimethylammonium iodide. These findings demonstrate that immobilized proteolytic fragments as small as 1/5 the size of the alpha subunit retain the structural characteristics necessary for binding alpha-bungarotoxin, although the toxin is bound to the fragments with lower affinity than to the native receptor. The effect of affinity ligand alkylation demonstrates that the alpha-bungarotoxin binding site detected on the proteolytic fragments is the same as the affinity-labeled acetylcholine binding site on the intact acetylcholine receptor.     

Molecular weight and structural nonequivalence of the mature alpha subunits of Torpedo californica acetylcholine receptor.

A discrepancy of about 20% exists between the molecular weight of the alpha subunit of Torpedo californica electroplax acetylcholine receptor as determined by gel electrophoresis of the mature protein (Mr 40,000 +/- 2000) and by nucleotide sequence analysis of cDNA (Mr approximately equal to 50,000). We demonstrate by amino acid sequence analysis that post-translational processing does not occur and that the mature subunit has a Mr of approximately equal to 50,000. The functional acetylcholine receptor contains two copies of this alpha subunit in addition to one each of related beta, gamma, and delta subunits. The binding sites for cholinergic ligands that are located on the alpha subunits have been shown to be nonequivalent. Amino acid sequence analysis of peptides obtained by proteolytic cleavage of the alpha subunit reveals that N-asparagine glycosylation at a single site (residue 141) occurs to a different extent in the two copies of this polypeptide in the mature protein and provides an explanation for nonequivalence of their binding sites.     

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.     

Functional expression of two neuronal nicotinic acetylcholine receptors from cDNA clones identifies a gene family.

A family of genes coding for proteins homologous to the alpha subunit of the muscle nicotinic acetylcholine receptor has been identified in the rat genome. These genes are transcribed in the central and peripheral nervous systems in areas known to contain functional nicotinic receptors. In this paper, we demonstrate that three of these genes, which we call alpha 3, alpha 4, and beta 2, encode proteins that form functional nicotinic acetylcholine receptors when expressed in Xenopus oocytes. Oocytes expressing either alpha 3 or alpha 4 protein in combination with the beta 2 protein produced a strong response to acetylcholine. Oocytes expressing only the alpha 4 protein gave a weak response to acetylcholine. These receptors are activated by acetylcholine and nicotine and are blocked by Bungarus toxin 3.1. They are not blocked by alpha-bungarotoxin, which blocks the muscle nicotinic acetylcholine receptor. Thus, the receptors formed by the alpha 3, alpha 4, and beta 2 subunits are pharmacologically similar to the ganglionic-type neuronal nicotinic acetylcholine receptor. These results indicate that the alpha 3, alpha 4, and beta 2 genes encode functional nicotinic acetylcholine receptor subunits that are expressed in the brain and peripheral nervous system.     

Analysis of ligand binding to the synthetic dodecapeptide 185-196 of the acetylcholine receptor alpha subunit.

A synthetic dodecapeptide corresponding to residues 185-196 of the Torpedo acetylcholine receptor alpha subunit, which contains the adjacent cysteine residues at positions 192 and 193, was recently shown by us to contain the essential elements for alpha-bungarotoxin binding. In the present study, we have used Sepharose-linked peptides for quantitative analysis of the cholinergic binding properties of this and other synthetic peptides. Sepharose-linked peptides corresponding to residues 1-20, 126-143, 143-158, 169-181, 185-196, 193-210, and 394-409 of the alpha subunit of Torpedo acetylcholine receptor, as well as a peptide corresponding to residues 185-196 of the alpha subunit of human acetylcholine receptor, were tested for their toxin-binding capacity. Of these immobilized peptides, only peptide 185-196 of the Torpedo acetylcholine receptor bound toxin significantly, thus verifying that this synthetic peptide contains essential components of the receptor toxin-binding site. Analysis of toxin binding to the peptide yielded a dissociation constant of 3.5 X 10(-5) M. This binding was inhibited by various cholinergic ligands. The inhibition potency obtained was alpha-bungarotoxin greater than Naja naja siamensis toxin greater than d-tubocurarine greater than decamethonium greater than acetylcholine greater than carbamoylcholine. This pharmacological profile resembles that of the nicotinic acetylcholine receptor and therefore suggests that the synthetic dodecapeptide also includes the neurotransmitter binding site. Reduction and carboxymethylation of the cysteine residues on peptide 185-196 inhibit its capacity to bind toxin, demonstrating that an intact disulfide is required for toxin binding. A decrease in toxin binding was also obtained following chemical modification of the tryptophan residue at position 187, thus implying its possible involvement in toxin binding. The failure to detect binding of toxin to the corresponding human sequence 185-196, in which the tryptophan residue is replaced by serine, supports this hypothesis.     

Mapping of the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor.

Synthetic peptides and their respective antibodies have been used in order to map the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor. By using antibodies to a synthetic peptide corresponding to residues 169-181 of the alpha subunit, we demonstrate that this sequence is included within the 18-kDa toxin binding fragment previously reported. Furthermore, the 18-kDa fragment was also found to bind a monoclonal antibody (5.5) directed against the cholinergic binding site. Sequential proteolysis of the acetylcholine receptor with trypsin, prior to Staphylococcus aureus V8 protease digestion, resulted in a 15-kDa toxin binding fragment that is included within the 18-kDa fragment but is shorter than it only at its carboxyl terminus. This 15-kDa fragment therefore initiates beyond Asp-152 and terminates in the region of Arg-313/Lys-314. In addition, experiments are reported that indicate that in the intact acetylcholine receptor, Cys-128 and/or Cys-142 are not crosslinked by disulfide bridges with any of the cysteines (at positions 192, 193, and 222) that reside in the 15-kDa toxin binding fragment. Finally, the synthetic dodecapeptide Lys-His-Trp-Val-Tyr-Tyr-Thr-Cys-Cys-Pro-Asp-Thr, which is present in the 15-kDa fragment (corresponding to residues 185-196 of the alpha subunit) was shown to bind alpha-bungarotoxin directly. This binding was completely inhibited by competition with d-tubocurarine.     

d-Tubocurarine binding sites are located at alpha-gamma and alpha-delta subunit interfaces of the nicotinic acetylcholine receptor.

The competitive nicotinic antagonist d-[3H]tubocurarine was used as a photoaffinity label for the acetylcholine binding sites on the nicotinic acetylcholine receptor (AcChoR) from Torpedo. Irradiation with 254-nm UV light of AcChoR-rich membranes equilibrated with d-[3H]tubocurarine resulted in covalent incorporation into the alpha, gamma, and delta subunits that could be blocked by alpha-bungarotoxin or by carbamoylcholine. The concentrations of d-[3H]tubocurarine required for half-maximal specific incorporation into the gamma and delta subunits were 40 nM and 0.9 microM, respectively, consistent with the dissociation constants for the high- and low-affinity binding sites (Kd = 35 nM and 1.2 microM). The concentration dependence of incorporation into alpha subunit was biphasic and consistent with labeling of both the high- and low-affinity d-tubocurarine binding sites. The specific photolabeling of each AcChoR subunit was inhibited by carbamoylcholine with appropriate dose dependence. These results establish that, in addition to the alpha subunits, the gamma and delta subunits also contribute directly to the acetylcholine binding sites and that each binding site is at an interface of subunits. Because the AcChoR subunits are homologous and are arranged pseudosymmetrically about a central axis, the photolabeling results are inconsistent with an arrangement of subunits in the AcChoR rosette of alpha beta alpha gamma delta and indicate that either the gamma or delta subunit resides between the alpha subunits.     

A large intracellular pool of inactive Na channel alpha subunits in developing rat brain.

An intracellular pool of Na channel alpha subunits has been detected in developing brain cells in vivo and in vitro by phosphorylation with cAMP-dependent protein kinase, immunoprecipitation with specific antiserum, and NaDodSO4 gel electrophoresis or by radioimmunoassay. These alpha subunits are membrane-bound, contain complex carbohydrate chains, and have an apparent molecular weight of 260,000 like mature alpha subunits. In contrast to mature alpha subunits, the intracellular subunits are not covalently attached to a beta 2 subunit, and they do not bind saxitoxin with high affinity. They comprise 67-77% of the total immunoreactive alpha subunit in developing rat brain cells but are not a prominent component in the adult brain. It is proposed that this intracellular pool of alpha subunits forms a ready reserve of preformed subunits for incorporation into the surface membrane during periods of active membrane biogenesis. The results suggest that disulfide linkage of the alpha and beta 2 subunits, insertion into the cell surface membrane, and attainment of a functional conformation are closely related late events in the biogenesis of the Na channel. These processes may regulate the number of functional Na channels in the developing brain.     

Preferential masking by the receptor of immunoreactive sites on the alpha subunit of human choriogonadotropin.

125I-Labeled human choriogonadotropin (125I-hCG) bound to rat ovarian receptor was solubilized in Triton X-100. By using increasing concentrations of nine different antisera specific for the individual subunits of human choriogonadotropin (hCG), free 125I-hCG or 125I-hCG-receptor complex was precipitated by double-antibody technique. The ability of any antiserum to bind to the hormone-specific beta subunit was not affected by hCG binding to receptor, suggesting that this subunit is not directly involved with the receptor in the final state of the hormone-receptor complex. In contrast, every antiserum specific for the alpha subunit was dramatically inhibited in binding to the solubilized 125I-hCG-receptor complex. These results suggest that the alpha subunit directly interacts with the receptor, thereby masking immunoreactive sites normally available on the free hormone. Because a number of reports describe binding activity of high concentrations of immunopurified beta subunits of hCG, we propose a two-step model for the binding of hCG to receptor and postulate separate and distinct roles for the subunits. We propose that the binding of hCG to the receptor involves a specific low-affinity initial interaction of the beta subunit with the receptor that activates a second site for the high-affinity binding of alpha subunit and stabilization of the hormone-receptor complex.     

Nicotinic cholinergic signaling in hippocampal astrocytes involves calcium-induced calcium release from intracellular stores

In this report we provide evidence that neuronal nicotinic acetylcholine receptors (nAChRs) are present on hippocampal astrocytes and their activation produces rapid currents and calcium transients. Our data indicate that these responses obtained from astrocytes are primarily mediated by an AChR subtype that is functionally blocked by alpha-bungarotoxin (alpha Bgt) and contains the alpha7 subunit (alpha Bgt-AChRs). Furthermore, their action is unusual in that they effectively increase intracellular free calcium concentrations by activating calcium-induced calcium release from intracellular stores, triggered by influx through the receptor channels. These results reveal a mechanism by which alpha Bgt-AChRs on astrocytes can efficiently modulate calcium signaling in the central nervous system in a manner distinct from that observed with these receptors on neurons.     

Models of the extracellular domain of the nicotinic receptors and of agonist- and Ca2+-binding sites

We constructed a three-dimensional model of the amino-terminal extracellular domain of three major types of nicotinic acetylcholine receptor, (alpha7)5, (alpha4)2(beta2)3, and (alpha1)2beta1gammadelta, on the basis of the recent x-ray structure determination of the molluscan acetylcholine-binding protein. Comparative analysis of the three models reveals that the agonist-binding pocket is much more conserved than the overall structure. Differences exist, however, in the side chains of several residues. In particular, a phenylalanine residue, present in beta2 but not in alpha7, is proposed to contribute to the high affinity for agonists in receptors containing the beta2 subunit. The semiautomatic docking of agonists in the ligand-binding pocket of (alpha7)5 led to positions consistent with labeling and mutagenesis experiments. Accordingly, the quaternary ammonium head group of nicotine makes a pi-cation interaction with W148 (alpha7 numbering), whereas the pyridine ring is close to both the cysteine pair 189-190 and the complementary component of the binding site. The intrinsic affinities inferred from docking give a rank order epibatidine > nicotine > acetylcholine, in agreement with experimental values. Finally, our models offer a structural basis for potentiation by external Ca2+.     

Monoclonal antibodies used to probe acetylcholine receptor structure: localization of the main immunogenic region and detection of similarities between subunits.

Seventeen cell lines producing monoclonal antibodies against Torpedo californica (torpedo) acetylcholine receptor (AcChoR) and its subunits were established. By using these antibodies as probes, we identified: (i) a similar antigenic determinant on alpha and beta torpedo subunits, (ii) a similar antigenic determinant on gamma and delta subunits, (iii) antigenic determinants unique for alpha or beta torpedo AcChoR subunits, (iv) a small region on the alpha subunit that dominates the immunogenicity of native torpedo AcChoR in rats (a monoclonal antibody directed at this region could bind to rat AcChoR in vivo and cause passive experimental autoimmune myasthenia gravis), and (v) antigenic determinants on torpedo subunits recognized in AcChoR from other species. The unexpected similarities between alpha and beta and between gamma and delta subunits raise the possibility that the complex four-subunit structure of AcChoR was derived from a simpler precursor and suggests that these antigenic similarities might reflect some structural and functional homologies.     

Purification and characterization of an alpha-bungarotoxin receptor that forms a functional nicotinic channel.

Neither the structure nor the function of alpha-bungarotoxin (alpha Bgtx) binding molecules in the nervous system have yet been completely defined, although it is known that some of these molecules are related to cation channels and some are not. Using an improved method of affinity chromatography, we have isolated a toxin binding molecule from chicken optic lobe that contains at least three subunits with apparent Mr values of 52,000, 57,000, and 67,000. The Mr 57,000 subunit binds alpha Bgtx and seems to be present in two copies per receptor. The receptor is recognized by antibodies raised against the alpha Bgtx receptors of human neuroblastoma cells, fetal calf muscle, and chicken optic lobe but not by antibodies raised against Torpedo acetylcholine receptor, the serum of myasthenic patients, or monoclonal antibody, 35. 125I-labeled alpha Bgtx binding to the isolated receptor is blocked, with the same potency, by nicotinic agonists and antagonists, such as nicotine, neuronal bungarotoxin and, d-tubocurarine. When reconstituted in a planar lipid bilayer, the purified alpha Bgtx receptor forms cationic channels with a conductance of 50 pS. These channels are activated in a dose-dependent manner by carbamylcholine and blocked by d-tubocurarine.     

In vitro synthesis, glycosylation, and membrane insertion of the four subunits of Torpedo acetylcholine receptor.

We have characterized the early biosynthetic forms of the Torpedo electroplax acetylcholine receptor by using a cell-free protein synthesizing system. We obtained primary translation products of approximately 38, 50, 49, and 60 kilodaltons for the alpha, beta, gamma, and delta polypeptides, respectively, by using immunoprecipitation with subunit-specific antisera. These chains could each be labeled by the formylated initiator [35S]Met-tRNA. On cotranslational incubation with pancreatic rough microsomes, glycosylated forms of each subunit were obtained that had molecular weights close to those of their mature authentic counterparts. Extensive trypsinization reduced the glycosylated forms of the receptor subunits to glycosylated membrane-protected fragments of approximately 35 (alpha), 37 (beta), 45 (gamma), and 44 (delta) kilodaltons. In this system, then, each receptor chain spans the membrane at least once. This in vitro-synthesized material apparently exhibited neither oligomeric assembly nor alpha-bungarotoxin binding.     

Nicotinic acetylcholine receptor from chick optic lobe.

An alpha-bungarotoxin-sensitive nicotinic cholinergic receptor from chick optic lobe has been completely purified. Its standard sedimentation coefficient is 9.1 S. The value near 12 S reported for the related component from other brain regions can be reproduced when the initial extraction is by Triton X-100 (rather than Lubrol PX), but other protein is then complexed with it. A single subunit of apparent molecular weight 54,000 is detected, and this subunit is specifically labeled by bromo-[3H]acetylcholine, but only after disulfide reduction. The same size subunit likewise is labeled in the protein (purified similarly) from the rest of the chick brain which can also bind alpha-bungarotoxin and nicotinic ligands. Immunological crossreactivity is demonstrated between both of these proteins with an antiserum to pure acetylcholine receptor from skeletal muscle. The acetylcholine receptor from chick optic lobe and the alpha-bungarotoxin-binding protein from the rest of the brain appear similar or identical by a series of criteria and are related to (but with differences from) peripheral acetylcholine receptors.     

How the mongoose can fight the snake: the binding site of the mongoose acetylcholine receptor.

The ligand binding site of the nicotinic acetylcholine receptor (AcChoR) is within a short peptide from the alpha subunit that includes the tandem cysteine residues at positions 192 and 193. To elucidate the molecular basis of the binding properties of the AcChoR, we chose to study nonclassical muscle AcChoRs from animals that are resistant to alpha-neurotoxins. We have previously reported that the resistance of snake AcChoR to alpha-bungarotoxin (alpha-BTX) may be accounted for by several major substitutions in the ligand binding site of the receptor. In the present study, we have analyzed the binding site of AcChoR from the mongoose, which is also resistant to alpha-neurotoxins. It was shown that mongoose AcChoR does not bind alpha-BTX in vivo or in vitro. cDNA fragments of the alpha subunit of mongoose AcChoR corresponding to codons 122-205 and including the presumed ligand binding site were cloned, sequenced, and expressed in Escherichia coli. The expressed protein fragments of the mongoose, as well as of snake receptors, do not bind alpha-BTX. The mongoose fragment is highly homologous (greater than 90%) to the respective mouse fragment. Out of the seven amino acid differences between the mongoose and mouse in this region, five cluster in the presumed ligand binding site, close to cysteines 192 and 193. These changes are at positions 187 (Trp----Asn), 189 (Phe----Thr), 191 (Ser----Ala), 194 (Pro----Leu), and 197 (Pro----His). The mongoose like the snake AcChoR has a potential glycosylation site in the binding site domain. Sequence comparison between species suggests that substitutions at positions 187, 189, and 194 are important in determining the resistance of mongoose and snake AcChoR to alpha-BTX. In addition, it was shown that amino acid residues that had been reported to be necessary for acetylcholine binding are conserved in the toxin-resistant animals as well.     

Binding of thymopoietin to the acetylcholine receptor.

Thymopoietin is a polypeptide hormone of the thymus with physiological effects on the immune system and on acetylcholine-mediated transmission at the neuromuscular synapse. Elucidation of the structure and function of the nicotinic acetylcholine receptor has been facilitated by the use of the electric organs of Torpedo ray or Electrophorus eel as rich sources of the receptor and by the use of snake polypeptide toxins such as alpha-bungarotoxin as highly selective labels of the acetylcholine binding site. We now show that thymopoietin binds with high affinity (Ka approximately equal to 2.5 X 10(9) M-1) to the acetylcholine binding region of the acetylcholine receptor of Torpedo californica, as evidenced by similar and complete inhibition of the binding of radiolabeled thymopoietin or alpha-bungarotoxin by either of these polypeptides. These findings raise intriguing questions concerning the mechanisms whereby alpha-bungarotoxin and the thymopoietin affect acetylcholine receptor function, since these two polypeptides with such similar binding properties have very different functional effects.