Phosphatidylcholine Biosynthesis in the Neuroblastoma-Glioma Hybrid Cell Line NG 108-15: Stimulation by Phorbol Esters

Studies were conducted on curaremimetic neurotoxin binding to the nicotinic acetylcholine receptor present on membrane fractions derived from the human medulloblastoma clonal line, TE671. High-affinity binding sites (KD = 2 nM for 1-h incubation at 20 degrees C) and low-affinity binding sites (KD = 40 nM) for 125I-labeled alpha-bungarotoxin are present in equal quantities (60 fmol/mg membrane protein). The kinetically determined dissociation constant for high-affinity binding of toxin is 0.56 nM (k1 = 6.3 X 10(-3) min-1 nM-1; k-1 = 3.5 X 10(-3) min-1) at 20 degrees C. Nicotine, d-tubocurarine, and acetylcholine are among the most effective inhibitors of high-affinity toxin binding. The quantity of toxin binding sites and their affinity for cholinergic agonists is sensitive to reduction, alkylation, and/or oxidation of membrane sulfhydryl residues. High-affinity toxin binding sites that have been subjected to reaction with the sulfhydryl reagent dithiothreitol are irreversibly blocked by the nicotinic receptor affinity reagent bromoacetylcholine. High-affinity toxin binding is inhibited in the presence of either of two polyclonal antisera or a monoclonal antibody raised against nicotinic acetylcholine receptors from fish electric tissue. Taken together, these results indicate that curaremimetic neurotoxin binding sites on membrane fractions of the TE671 cell line share some properties with nicotinic acetylcholine receptors of peripheral origin and with toxin binding sites on other neuronal tissues.     

Nicotinic Agonists Regulate α-Bungarotoxin Binding Sites of TE671 Human Medulloblastoma Cells

The TE671 human medulloblastoma cell line expresses a variety of characteristics of human neurons. Among these characteristics is the expression of membrane-bound high-affinity binding sites for alpha-bungarotoxin, which is a potent antagonist of functional nicotinic acetylcholine receptors on these cells. These toxin binding sites represent a class of nicotinic receptor isotypes present in mammalian brain. Treatment of TE671 cells during proliferative growth phase with nicotine or carbamylcholine, but not with muscarine or d-tubocurarine, induced up to a five-fold increase in the density of radiolabeled toxin binding sites in crude membrane fractions. This effect was blocked by co-incubation with the nicotinic antagonists d-tubocurarine and decamethonium, but not by mecamylamine or by muscarinic antagonists. Following a 10-13 h lag phase upon removal of agonist, recovery of the up-regulated sites to control values occurred within an additional 10-20 h. These studies indicate that the expression of functional nicotinic acetylcholine receptors on TE671 cells is subject to regulation by nicotinic agonists. Studies of the murine CNS have consistently indicated nicotine-induced up-regulation of nicotinic acetylcholine receptors, thereby supporting the identification of the toxin binding site on these cells as the functional nicotinic receptor. Although a mechanism for this effect is not apparent, nicotine-induced receptor blockade does not appear to be involved.     

A comparison of binding properties and structure of NGF receptor on PC12 pheochromocytoma and A875 melanoma cells

Rat PC12 pheochromocytoma and human A875 melanoma cells express nerve growth factor (NGF) receptors on their surfaces. Covalent crosslinking of bound 125I-NGF to PC12 or A875 intact cells or plasma membrane-enriched fractions resulted in labelling of a peptide doublet at Mr = 110,000 and a single labelled peptide at Mr = 200,000 for each of the cell and membrane preparations. However, a difference between equilibrium binding properties of NGF-receptor on PC12 and A875 cells was observed. PC12 cells exhibited biphasic binding properties with two apparent binding sites: KD = 5.2 nM sites and KD = 0.3 nM sites. The high-affinity PC12 binding sites were trypsin resistant, and 125I-NGF dissociated slowly from them. A875 cells exhibited sites with homogeneous properties (KD = 1.0 nM), all binding sites were trypsin sensitive, and 125I-NGF dissociated rapidly in the presence of unlabelled NGF. Membrane-enriched fractions from either cell type contained binding sites with a uniform low affinity (KD = 3 nM) that were trypsin sensitive, and 125I-NGF rapidly dissociated from them. Sixty to 80 percent of binding sites in membranes could be converted to the high-affinity, trypsin-resistant state by addition of wheat germ agglutinin (WGA). The loss of high-affinity, trypsin-resistant sites from PC12 cells during preparation of plasma membrane fractions does not appear to be the result of selective isolation of low-affinity sites or proteolytic degradation since there is a loss of 125I-NGF binding immediately after cell lysis which is not blocked by protease inhibitors. Also, high-affinity, trypsin-resistant binding sites are not found associated with other cell fractions. The differences between receptor properties on PC12 cells and on A875 cells apparently are the result of differences in the respective intracellular environments. Thus, significant structural homology exists between receptors on A875 and PC12 cells. Cell components other than the binding unit of the NGF receptor may be responsible for the different properties of receptor.     

Sulfhydryl Modification of Two Nicotinic Binding Sites in Mouse Brain

Two distinct binding sites with properties corresponding to those expected for nicotinic cholinergic receptors can be identified in brain by the specific binding of nicotine (or acetylcholine) and alpha-bungarotoxin. The effects of modification of these binding sites by treatment with the disulfide-reducing agent dithiothreitol were examined in tissue prepared from DBA mouse brains. Treatment with dithiothreitol reduced the binding measured with either ligand, and reoxidization of the disulfides fully restored binding. The effects of dithiothreitol treatment appeared to be due to a reduction in the maximal binding of nicotine and to a decrease in the binding affinity for alpha-bungarotoxin. Agonist affinity for the alpha-bungarotoxin binding site was reduced by treatment with low concentrations of dithiothreitol. The nicotine binding sites remaining after disulfide treatment displayed rates of ligand association and dissociation similar to those of unmodified tissue, but treatment of previously unmodified tissue with dithiothreitol accelerated the rate of nicotine dissociation. After reduction, both binding sites could be selectively alkylated with bromoacetylcholine. The results suggest that both putative nicotinic receptors in brain respond similarly to disulfide reduction and that their responses resemble those known for the nicotinic receptor of electric tissue.     

Reconstitution of active acetylcholine receptor by hybridisation of binding site-blocked with ion channel-blocked acetylcholine receptor protein

The nicotinic acetylcholine receptor regulates the ion permeability of the postsynaptic membrane. This report presents evidence that the transmitter binding site and the ion channel may be located on distinct subunits. By hybridisation of receptor complexes, in which the transmitter binding site was blocked with complexes in which the ion channel was irreversibly inhibited, we reconstituted active acetylcholine receptor complexes. The reconstituted system was similar to the native receptor in its ability to regulate the ion permeability of lipid vesicles in response to nicotinic cholinergic effectors.     

Biochemical characterization of two nicotinic receptors from the optic lobe of the chick

We have studied putative nicotinic acetylcholine receptors in the optic lobe of the newborn chick, using 125I-labeled alpha-bungarotoxin, a specific blocker of acetylcholine receptors in the neuromuscular junction, and [3H]acetylcholine, a ligand which in the presence of atropine selectively labels binding sites of nicotinic character in rat brain cortex (Schwartz et al., 1982). [3H]Acetylcholine binds reversibly to a single class of high affinity binding sites (KD = 2.2 X 10(-8) M) which occur at a tissue concentration of 5.7 pmol/g. A large fraction (approximately 60%) of these binding sites is solubilized by Triton X-100, sodium cholate, or the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Solubilization increases the affinity for acetylcholine and several nicotinic drugs from 1.5- to 7-fold. The acetylcholine-binding macromolecule resembles the receptor for alpha-bungarotoxin present in the same tissue with respect to subcellular distribution, hydrodynamic properties, lectin binding, and agonist affinity rank order. It differs from the toxin receptor in affinity for nicotinic antagonists, sensitivity to thermal inactivation, and regional distribution. The solubilized [3H]acetylcholine binding activity is separated from the toxin receptor by incubation with agarose-linked acetylcholine, by affinity chromatography on immobilized Naja naja siamensis alpha-toxin, and by precipitation with a monoclonal antibody to chick optic lobe toxin receptor.     

Agonist-induced affinity alterations of a central nervous system alpha-bungarotoxin receptor

Abstract— The ability of cholinergic agonists to block the specific interaction of α-bungarotoxin (α-Bgt) with membrane-bound sites derived from rat brain is enhanced when membranes are preincubated with agonist. Thus, pretreatment of α-Bgt receptors with agonist (but not antagonist) causes transformation of sites to a high-affinity form toward agonist. This change in receptor state occurs with a half-time on the order of minutes, and is fully reversible on dilution of agonist. The results are consistent with the identity of α-Bgt binding sites as true central nicotinic acetylcholine receptors. Furthermore, this agonist-induced alteration in receptor state may represent an in vitro correlate of physiological desensitization. As determined from the effects of agonist on toxin binding isotherms, and on the rate of toxin binding to specific sites, agonist inhibition of toxin binding to the high-affinity state is non-competitive. This result suggests that there may exist discrete toxin-binding and agonist-binding sites on central toxin receptors.     

Acetylcholine receptors of rat lymphocytes

The conditions of the binding of acetylcholine have been studied in lymphocytes isolated from rat peripheral lymph nodes. Acetylcholine appeared to penetrate the lymphocyte membrane. We have confirmed the presence of muscarinic receptors, which, however, are not involved in transport of acetylcholine through the membrane. The receptors of the nicotine type on lymphocytes are demonstrated by the decrease of acetylcholine binding in the presence of a specific antagonist, tubocurarine. These nicotinic receptors may be involved in acetylcholine transport into the cells.     

Effects of Chronic Nicotinic Ligand Exposure on Functional Activity of Nicotinic Acetylcholine Receptors Expressed by Cells of the PC12 Rat Pheochromocytoma or the TE671/RD Human Clonal Line

Studies were conducted to ascertain the temporal and dose-dependent effects of nicotinic ligand exposure on functional activity of different nicotinic acetylcholine receptor (nAChR) subtypes, as expressed by cells of the PC12 rat pheochromocytoma (ganglia-type nAChR) or the TE671/RD human (muscle-type nAChR) clonal line. Chronic (3-72-h) agonist (nicotine or carbamylcholine) treatment of cells led to a complete (TE671) or nearly complete (PC12) loss of functional nAChR responses, which is referred to as "functional inactivation." Some inactivation of nAChR function was also observed for the nicotinic ligands d-tubocurarine (d-TC), mecamylamine, and decamethonium. Half-maximal inactivation of nAChR function was observed within 3 min for TE671 cells and within 10 min for PC12 cells treated with inactivating ligands. Functional inactivation occurred with dose dependencies that could not always be reconciled with those obtained for acute agonist activation of nAChR function or for acute inhibition of those responses by d-TC, decamethonium, or mecamylamine. Treatment of TE671 or PC12 cells with the nicotinic antagonist pancuronium or alcuronium alone had no effect on levels of expression of functional nAChRs. However, evidence was obtained that either of these antagonists protected TE671 cell muscle-type nAChRs or PC12 cell ganglia-type nAChRs from functional inactivation on long-term treatment with agonists. Recovery of TE671 cell nAChR function following treatment with carbamylcholine, nicotine, or d-TC occurred with half-times of 1-3 days whether cells were maintained in situ or harvested and replated after removal of ligand. By contrast, 50% recovery of functional nAChRs on PC12 cells occurred within 2-6 h after drug removal. In either case the time course for recovery from nAChR functional inactivation is much slower than recovery from nAChR "functional desensitization," which is a reversible process that occurs on shorter-term (0-5-min) agonist exposure of cells. These results indicate that ganglia-type and muscle-type nAChRs are similar in their sensitivities to functional inactivation by nicotinic ligands but differ in their rates of recovery from and onset of those effects. The ability of drugs such as the agonists d-TC, decamethonium, and mecamylamine to induce functional inactivation may relate to their activities as partial/full agonists, channel blockers, and/or allosteric regulators. Effects of drugs such as pancuronium and alcuronium are likely to reflect simple competitive inhibition of primary ligand binding at functional activation sites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Acetylcholine receptor binding properties and ion permeability response after covalent attachment of the local anaesthetic quinacrine

Membrane vesicles rich in nicotinic acetylcholine receptor prepared from Torpedo californica electric tissue have been irreversibly modified with quinacrine mustard, an alkylating derivative of the local anaesthetic quinacrine. The reaction blocked the ion channel regulated by the acetylcholine receptor. Acetylcholine still bound to the modified membrane vesicles with K_D approx. 10^?8. The number of binding sites was reduced by up to 50%. Stopped-flow experiments showed that in contrast to what had been found with reversibly binding quinacrine no fluorescence changes caused by energy transfer from the irradiated protein to the fluorescent local anaesthetic occurred after addition of agonist. This indicates that the conformational changes associated with the activation of the ion channel are blocked by the covalent reaction with quinacrine mustard. Analysis of the membrane vesicles by SDS-polyacrylamide gel electrophoresis showed that all polypeptide chains assumed to be part of the receptor complex had reacted with the mustard. Even small components, probably lipids, migrating with the dye front, showed fluorescence.     

Critical residues influence the affinity and selectivity of alpha-conotoxin MI for nicotinic acetylcholine receptors.

The mammalian skeletal muscle acetylcholine receptor contains two nonequivalent acetylcholine binding sites, one each at the alpha/delta and alpha/gamma subunit interfaces. Alpha-Conotoxin MI, a 14-amino acid competitive antagonist, binds at both interfaces but has approximately 10(4) higher affinity for the alpha/delta site. We performed an "alanine walk" to identify the residues in alpha-MI that contribute to this selective interaction with the alpha/delta site. Electrophysiological measurements with Xenopus oocytes expressing normal receptors or receptors lacking either the gamma or delta subunit were made to assay toxin-receptor interaction. Alanine substitutions in most amino acid positions had only modest effects on toxin potency at either binding site. However, substitutions in two positions, proline-6 and tyrosine-12, dramatically reduced toxin potency at the high-affinity alpha/delta site while having comparatively little effect on low-affinity alpha/gamma binding. When tyrosine-12 was replaced by alanine, the toxin's selectivity for the high-affinity site (relative to that for the low-affinity site) was reduced from 45,000- to 30-fold. A series of additional amino acid substitutions in this position showed that increasing side chain size/hydrophobicity increases toxin potency at the alpha/delta site without affecting alpha/gamma binding. In contrast, when tyrosine-12 is diiodinated, toxin binding is nearly irreversible at the alpha/delta site but also increases by approximately 500-fold at the alpha/gamma site. The effects of position 12 substitutions are accounted for almost entirely by changes in the rate of toxin dissociation from the high-affinity alpha/delta binding site.     

Intracellular recording in avian brain of a nicotinic response that is insensitive to K-bungarotoxin

We examined nicotinic acetylcholine receptors in the avian brain using a combination of autoradiographic and intracellular electrophysiological techniques. We found that the lateral spiriform nucleus (SPL) in the mesencephalon has a very high density of 3H-nicotine binding sites but no detectable 125I-K-bungarotoxin (125I-K-BuTx) or 125I-alpha-bungarotoxin (125I-alpha-BuTx) bindings sites. Intracellular recordings in brain slices revealed that SPL neurons depolarize in response to nicotine and carbachol (in the presence of atropine). These depolarizations were blocked by the classic nicotinic antagonists d-tubocurarine and dihydro-beta-erythroidine. As predicted for nicotinic receptors with a high affinity for nicotine, neither K-BuTx nor alpha-BuTx blocked these nicotinic responses. Thus, although the existence of high-affinity 3H-nicotine binding sites has been known for some time, we now report the in situ detection of a functional nicotinic receptor that has a high affinity for nicotine and is K-BuTx-insensitive.     

Interactions of antinicotinic acetylcholine receptor antibodies with rat brain and muscle antigenic determinants

Studies were performed to determine whether antibodies prepared against nicotinic acetylcholine receptors (nAcChoR) from electric tissue are reactive toward nAcChoR-like antigenic determinants in rat brain. Reference experiments involved the use of Torpedo electroplax and rat innervated muscle as tissue controls and an anti-alpha-bungarotoxin antiserum as a probe for curaremimetic neurotoxin binding sites. As evinced by their ability to inhibit immunoprecipitation of Torpedo nAcChoR, brain or muscle membranes specifically interact with polyclonal antisera raised against Electrophorus electroplax nAcChoR. When the extent of polyclonal anti-nAcChoR antibody binding to muscle membranes is measured by protein A binding protocols, receptor-like antigenic determinants and toxin binding sites are found to be present in approximately equal quantities. In contrast, nAcChoR-like antigenic determinants on rat brain membranes are present at concentrations in excess of those of toxin binding sites. The results are consistent with the earlier observation that some antibodies prepared against nAcChoR from peripheral tissues recognize rat brain high-affinity alpha-bungarotoxin binding sites. The results also suggest the existence of nAcChoR-like entities in brain that do not bind toxin with a high affinity.     

Chemical modification of the nicotinic cholinergic receptor of PC-12 nerve cell

The identity of the protein that mediates the nicotinic acetylcholine sensitivity in neuronal cells has been investigated by chemical modification and affinity labeling. When an ion flux assay is used, it is possible to measure specifically the activity of the ionophore associated with the nicotinic acetylcholine receptor in cultured nerve cells (PC-12 pheochromocytoma). This activity is modulated by modification of the redox state of at least one disulfide bridge located at the vicinity of the agonist binding site. The oxidizing agent 5,5'-dithiobis(nitrobenzoic acid) restores the complete receptor response which had been inhibited by reduction with dithiothreitol. N-Ethylmaleimide and the nicotinic affinity labels [4-(N-maleimido)benzyl]-alpha-trimethylammonium iodide and bromoacetylcholine react also with the reduced receptor and irreversibly block the agonist-dependent response of the ionophore. The two affinity labels show strong affinities for the receptor, and apparent IC50 values of 20 and 560 nM can be respectively evaluated. Bromoacetylcholine, being an acetylcholine analogue, blocks the receptor function by desensitization, a process in which the constant interaction with the activator causes a shift into an inactive form of the receptor. Bromoacetylcholine can also be shown to activate untreated as well as reduced cells. In this case, the bound label induces a lasting response which is terminated by the irreversible desensitization of the modified receptor. These experiments thus show that the PC-12 nicotinic ionophore shares functional and structural similarities with peripheral receptors. They suggest that nicotinic affinity labels developed for the muscle receptor can also be used as specific markers of the nicotinic neural ionophore.     

Nicotinic Modulation of [3H]Dopamine Release from Striatal Synaptosomes: Pharmacological Characterisation

Presynaptic nicotinic acetylcholine receptors on striatal nerve terminals modulate the release of dopamine. We have compared the effects of a number of nicotinic agonists and antagonists on a perfused synaptosome preparation preloaded with [3H]dopamine. (-)-Nicotine, acetylcholine, and the nicotinic agonists cytisine and 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), at micromolar concentrations, stimulated the release of [3H]dopamine from striatal nerve terminals. Carbamylcholine was a much weaker agonist. The actions of (-)-nicotine, cytisine, and DMPP were inhibited by low concentrations of the nicotinic antagonists dihydro-beta-erythroidine, mecamylamine, pempidine, and neosurugatoxin; alpha-bungarotoxin was without effect, and extending the time of exposure to this toxin resulted in only very modest inhibition. This pharmacology points to a specific nicotinic receptor mechanism that is clearly distinct from that at the neuromuscular junction. Atropine failed to antagonise the effects of acetylcholine and carbamylcholine, suggesting that no muscarinic component is involved. The nicotinic receptor ligands (-)-[3H]nicotine and 125I-alpha-bungarotoxin bound to specific sites enriched in the synaptosome preparation. Drugs tested on the perfused synaptosomes were examined for their ability to interact with these two ligand binding sites in brain membranes. The differential sensitivity to the neurotoxins alpha-bungarotoxin and neosurugatoxin of the 125I-alpha-bungarotoxin and (-)-[3H]nicotine binding sites, respectively, leads to a tentative correlation of the (-)-[3H]nicotine site with the presynaptic nicotinic receptor on striatal nerve terminals.     

Antibodies against preselected peptides to map functional sites on the acetylcholine receptor

Rabbit immune sera and mouse monoclonal antibodies were raised against the synthetic peptide Tyr-Cys-Glu-Ile-Ile-Val matching in sequence residues 127-132 of the alpha-subunit of all nicotinic acetylcholine receptors sequenced so far. Representative cholinergic ligands did not interfere with the binding of these antibodies to the receptor from Torpedo marmorata, indicating that this sequence is not part of the binding sites for cholinergic ligands. The applicability of antigenic sites analysis to the mapping of functional sites on receptor proteins is discussed.     

Binding of Agonists and Antagonists to Muscarinic Acetylcholine Receptors on Intact Cultured Heart Cells

The binding of agonists and antagonists to muscarinic acetylcholine receptors on intact cultured cardiac cells has been compared with the binding observed in homogenized membrane preparations. The antagonists [3H]quinuclidinyl benzilate and [3H]N-methylscopolamine bind to a single class of receptor sites on intact cells with affinities similar to those seen in membrane preparations. In contrast with the heterogeneity of agonist binding sites observed in membrane preparations, the agonist carbachol binds to a homogeneous class of low-affinity sites on intact cells with an affinity identical to that found for the low-affinity agonist site in membrane preparations in the presence of guanyl nucleotides. Kinetic studies of antagonist binding to receptors in the absence and presence of agonist did not provide evidence for the existence of a transient (greater than 30 s) high-affinity agonist site that was subsequently converted to a site of lower affinity. Nathanson N. M. Binding of agonists and antagonists to muscarinic acetylcholine receptors on intact cultured heart cells.     

昆虫中肠Bt晶体蛋白受体的研究进展

苏云金芽孢杆菌Ｂａｃｉｌｌｕｓｔｈｕｒｉｎｇｉｅｎｓｉｓ杀虫作用的主要成份是胞内产生的伴孢晶体,晶体蛋白经昆虫吞食,在肠道降解为激活的毒性肽。普遍认为毒性肽的作用机制主要有两个步骤：１）与中肠表面的受体专一结合;２）在细胞膜上形成跨膜通道。杀虫晶体蛋白的专一性与中肠细胞膜表面的受体蛋白紧密相连,晶体蛋白的杀虫作用是通过昆虫中肠细胞的专一性受体而起作用。本文通过说明受体蛋白的生物学特性、分子本质及与昆虫抗性的关系,概述了近年来中肠受体蛋白的研究进展。１　昆虫中肠受体蛋白的生物学特性１１　受体蛋白…     

Functional immobilisation of the nicotinic acetylcholine receptor in tethered lipid membranes

The nicotinic acetylcholine receptor from Torpedo was immobilised in tethered membranes. Surface plasmon resonance was used to quantify the binding of ligands and antibodies to the receptor. The orientation and structural integrity of the surface-reconstituted receptor was probed using monoclonal antibodies, demonstrating that approximately 65% of the receptors present their ligand-binding site towards the lumen of the flow cell and that at least 85% of these receptors are structurally intact. The conformation of the receptor in tethered membranes was investigated with Fourier transform infrared spectroscopy and found to be practically identical to that of receptors reconstituted in lipid vesicles. The affinity of small receptor ligands was determined in a competition assay against a monoclonal antibody directed against the ligand-binding site which yielded dissociation constants in agreement with radioligand binding assays. The presented method for the functional immobilisation of the nicotinic acetylcholine receptor in tethered membranes might be generally applicable to other membrane proteins.     

Complex ganglioside expression and tetanus toxin binding by PC12 pheochromocytoma cells

Ganglioside expression and tetanus toxin binding were studied in the rat pheochromocytoma cell line PC12. Seven ganglioside species were readily detected in extracts of PC12 cells; two were identified as tri- and tetrasialogangliosides, which are common brain constituents but unusual components of neuronal cell lines. Carbohydrate composition, acid and enzyme hydrolyses, and mass spectral analysis revealed that the major species is GT 1b, a predominant mammalian brain ganglioside previously reported to support high affinity tetanus toxin binding (Rogers, T. B., and Snyder, S. H. (1981) J. Biol. Chem. 256, 2402-2407). Direct binding of 125I-tetanus toxin to PC12 gangliosides on TLC plates revealed selective binding to the tri- and tetrasialogangliosides. Radioiodinated toxin also bound with high affinity to intact PC12 cells or their isolated membranes. The binding affinity (Kd = 1.25 nM), density of receptors (Bmax = 238 pmol/mg of membrane protein), and dependence on pH, ionic strength, and temperature were similar to those previously reported for toxin binding to rat brain synaptic membranes. Differentiation of PC12 cells caused an increase in expression of the tri- and tetrasialogangliosides and a closely matched increase in tetanus toxin binding to cell membranes. These data provide evidence that complex gangliosides may act as tetanus toxin receptors, and demonstrate the utility of the PC12 cell line for studies of tetanus toxicity and complex ganglioside expression.