Acetylcholine receptor degradation measured by density labeling: effects of cholinergic ligands and evidence against recycling.

The methodology of density labeling of proteins by biosynthetic incorporation of 2H, 13C, 15N-amino acids into newly synthesized polypeptide chains allows the direct measurement of the turnover rate of the acetylcholine receptor in cultured chick skeletal muscle. In this study, receptors synthesized in medium containing 2H, 13C, 15N-amino acids were resolved from 1H, 12C, 14N-receptors by velocity sedimentation in sucrose-deuterium oxide gradients, and their proportions were determined by computer analysis of the gradient profiles. The kinetics of turnover of acetylcholine receptors are identical for developing chick muscle fibers grown in medium containing 2H, 13C, 15N-amino acids or 1H, 12C, 14N-amino acids, and the high degree of substitution of normal aminoacyl residues by 2H, 13C, 15N-residues does not affect the turnover rate of the denser receptor. Comparison of the turnover rates in continuous and pulse-labeling experiments gave independent confirmation of these results. The application of a potent, essentially irreversible blocking agent, alpha-bungarotoxin, increases the median lifetime of receptors from 17 hr for the native unbound receptor to 22 hr for the alpha-bungarotoxin-receptor complex. As predicted, the total number of alpha-bungarotoxin binding sites increased in the continued presence of alpha-bungarotoxin due to extension of receptor lifetime. To determine whether other cholinergic agents affect the turnover rate of the receptor, measurements were performed on cultures grown in the presence of 10(-4) M d-tubocurare or 10(-4) M carbachol, a reversible antagonist and a reversible agonist, respectively, of the nicotinic acetylcholine receptor. The receptor degradation rates of the drug-treated cells were identical to control values. The total number of alpha-bungarotoxin binding sites was reduced by 30% in the presence of carbachol, indicating that this agent affects the rate of synthesis of the acetylcholine receptor. Data formerly interpreted as suggesting a cycling of receptor-containing plasma membrane out of and back into the sarcolemma are now understood to reflect the alteration in receptor lifetime upon complexing with alpha-bungarotoxin. The intracellular "hidden" receptor sites were found to remain inside the myotubes and thus do not signify an intracellular pool of recycling plasma membrane.     

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.     

The effects of inhibiting oligosaccharide trimming by 1-deoxynojirimycin on the nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor has a subunit stoichiometry of alpha 2 beta gamma delta; all 5 subunits contain N-linked oligosaccharides. We investigated what role trimming of the oligosaccharides played in the post-translational processing of the subunits and assembly of the receptor by examining the receptor synthesized in the presence of an inhibitor of oligosaccharide trimming, 1-deoxynojirimycin. BC3H-1 cells express one-third fewer receptors when grown in the presence of 1-deoxynojirimycin. The receptor subunits that are expressed have decreased mobility by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating an inhibition of oligosaccharide trimming. In control cells, 40% of the translated alpha subunit acquires the capacity to bind alpha-bungarotoxin with a half-time of 40 min before assembly with the other subunits; the rest is rapidly degraded. In 1-deoxynojirimycin-treated cells approximately the same amount of alpha subunit is translated as in control cells, but that alpha subunit is degraded more rapidly, and only 25% acquires the capacity to bind alpha-bungarotoxin. From these results, we conclude that oligosaccharide processing either may aid in protecting the alpha subunit primary translation product from degradation or may be required for the conformational change or other post-translational modification(s) necessary for formation of the alpha-bungarotoxin binding form of the alpha subunit, which is then protected from proteolytic degradation. The cell surface receptor that is expressed in the presence of 1-deoxynojirimycin, however, is not altered in its affinity for cholinergic ligands. Thus, we conclude that differential N-linked oligosaccharide trimming of the 2 alpha subunits does not appear to play a part in the differences in affinities of the 2 alpha subunits for cholinergic ligands.     

A novel pharmatope tag inserted into the beta4 subunit confers allosteric modulation to neuronal nicotinic receptors

alpha-Bungarotoxin, the classic nicotinic antagonist, has high specificity for muscle type alpha1 subunits in nicotinic acetylcholine receptors. In this study, we show that an 11-amino-acid pharmatope sequence, containing residues important for alpha-bungarotoxin binding to alpha1, confers functional alpha-bungarotoxin sensitivity when strategically placed into a neuronal non-alpha subunit, normally insensitive to this toxin. Remarkably, the mechanism of toxin inhibition is allosteric, not competitive as with neuromuscular nicotinic receptors. Our findings argue that alpha-bungarotoxin binding to the pharmatope, inserted at a subunit-subunit interface diametrically distinct from the agonist binding site, interferes with subunit interface movements critical for receptor activation. Our results, taken together with the structural similarities between nicotinic and GABAA receptors, suggest that this allosteric mechanism is conserved in the Cys-loop ion channel family. Furthermore, as a general strategy, the engineering of allosteric inhibitory sites through pharmatope tagging offers a powerful new tool for the study of membrane proteins.     

Reconstitution of pure acetylcholine receptor in phospholipid vesicles and comparison with receptor-rich membranes by the use of a potentiometric dye

Acetylcholine receptor, isolated in Triton X-100 on a cobra alpha-neurotoxin affinity column was incorporated into unilamellar phospholipid vesicles by a detergent depletion method using Amberlite XAD-2. Vesicles of an average diameter of 25 nm were formed, as verified by freeze-fracture electron microscopy and gel filtration. 85 to 95% of the alpha-bungarotoxin binding sites of the reconstituted acetylcholine receptor were oriented towards the outside of the vesicles. In the reconstituted receptor one molecule of residual Triton X-100 per 2.5 alpha-bungarotoxin binding sites on the receptor molecule could be assessed. The reconstituted protein was not accessible to papain digestion, whereas the pure acetylcholine receptor, solubilized by Triton X-100 was split into smaller polypeptides under the same condition. Reconstituted acetylcholine receptor and receptor-rich membranes did not exhibit the same behavior as measured by use of a potentiometric dye. This is interpreted as an irreversible alteration of at least 95% of the receptors purified in the presence of Triton X-100. Furthermore, it could be shown that the fluorescence intensity changes induced by carbamylcholine in receptor-rich membranes did not reflect ion fluxes, but conformational changes of the protein or a displacement of the dye from the protein.     

Ligand-induced changes in membrane-bound acetylcholine receptor observed by ethidium fluorescence. 1. Equilibrium studies.

The interactions between the fluorescent probe ethidium and acetylcholine receptor enriched membranes from Torpedo californica are described. One class of saturable ethidium sites was blocked by alpha-bungarotoxin and therefore reflects direct binding to the receptor (Kd approximately 3 micrometers; stoichiometry--one ethidium site per two alpha-bungarotoxin sites). The second class of sites was nonsaturable and unaffected by alpha-toxin and was therefore considered nonspecific in nature. The increase in fluorescence intensity observed upon addition of cholinergic agonists and antagonists accurately reflects the dissociation constant and stoichiometry of the high-affinity receptor sites for these ligands. The effects of local anaesthetics are complex in nature and depend on the structure of the ligand. For carbamylcholine, the increase in flourescence intensity was due to an increase in the quantum yield of the dye bound to the membrane rather than a dye uptake. In general, ethidium appears not to strongly alter the properties of the membrane-bound acetylcholine receptor and can therefore be profitably used as a spectroscopic probe.     

Alpha-bungarotoxin binding to acetylcholine receptor membranes studied by low angle X-ray diffraction

The nicotinic acetylcholine receptor (nAChR) carries two binding sites for snake venom neurotoxins. alpha-Bungarotoxin from the Southeast Asian banded krait, Bungarus multicinctus, is a long neurotoxin which competitively blocks the nAChR at the acetylcholine binding sites in a relatively irreversible manner. Low angle x-ray diffraction was used to generate electron density profile structures at 14-A resolution for Torpedo californica nAChR membranes in the absence and presence of alpha-bungarotoxin. Analysis of the lamellar diffraction data indicated a 452-A lattice spacing between stacked nAChR membrane pairs. In the presence of alpha-bungarotoxin, the quality of the diffraction data and the lamellar lattice spacing were unchanged. In the plane of the membrane, the nAChRs packed together with a nearest neighbor distance of 80 A, and this distance increased to 85 A in the presence of toxin. Electron density profile structures were calculated in the absence and presence of alpha-bungarotoxin, revealing a location for the toxin binding sites. In native, fully-hydrated nAChR membranes, alpha-bungarotoxin binds to the nAChR outer vestibule and contacts the surface of the membrane bilayer.     

Characterization of Curaremimetic Neurotoxin Binding Sites on Cellular Membrane Fragments Derived from the Rat Pheochromocytoma PC 12

Studies were conducted on the properties of 125I-labeled alpha-bungarotoxin binding sites on cellular membrane fragments derived from the PC12 rat pheochromocytoma. Two classes of specific toxin binding sites are present at approximately equal densities (50 fmol/mg of membrane protein) and are characterized by apparent dissociation constants of 3 and 60 nM. Nicotine and d-tubocurarine are among the most potent inhibitors of high-affinity toxin binding. The affinity of high-affinity toxin binding sites for nicotinic cholinergic agonists is reversibly or irreversibly decreased, respectively, on treatment with dithiothreitol or dithiothreitol and N-ethylmaleimide. The nicotinic receptor affinity reagent bromoacetylcholine irreversibly blocks high-affinity toxin binding to PC12 cell membranes that have been treated with dithiothreitol. Two polyclonal antisera raised against the nicotinic acetylcholine receptor from Electrophorus electricus inhibit high-affinity toxin binding. These detailed studies confirm that curaremimetic neurotoxin binding sites on the PC12 cell line are comparable to toxin binding sites from neural tissues and to nicotinic acetylcholine receptors from the periphery. Because toxin binding sites are recognized by anti-nicotinic receptor antibodies, the possibility remains that they are functionally analogous to nicotinic receptors.     

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.     

Preparation of right-side-out, acetylcholine receptor enriched intact vesicles from Torpedo californica electroplaque membranes.

Intact vesicles enriched in acetylcholine receptor from Torpedo californica electroplaque membranes can be separated from collapsed or leaky vesicles and membrane sheets on sucrose density gradients. alpha-Bungarotoxin binding in intact vesicles reveals that approximately 95% of the acetylcholine receptor containing vesicles are formed outside-out (with the synaptic membrane face exposed on the vesicle exterior). The binding data also indicated that only 5% or less of the sites for alpha-bungarotoxin binding to synaptic membranes are located on the interior, cytoplasmic face. Intact vesicles are stable to gentle pelleting and resuspension but are easily osmotically shocked. The vesicles are impermeable to sucrose and Ficoll, but glycerol readily transverses to membrane barrier. Intact vesicles provide a sealed, oriented membrane preparation for studies of vectorial acetylcholine receptor mediated processes.     

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.     

Newly synthesized acetylcholine receptors are located in the Golgi apparatus

Chick skeletal muscle cells in tissue culture were fixed and treated with saponin to allow [125I]alpha-bungarotoxin access into the cells while preserving ultrastructure. The kinetics of binding of iodinated alpha-bungarotoxin to intracellular acetylcholine (ACh) receptors and to surface A Ch receptors were comparable. About half of the intracellular ACh receptors are newly synthesized and in the pathway leading to incorporation into the plasma membrane. Correlated electron microscope autoradiographic and kinetic studies of this receptor population suggest that a substantial fraction of the newly synthesized ACh receptors are located in the Golgi apparatus, where they reside for approx. 2 h.     

Thymopoietin,a thymic polypeptide,potently interacts at muscle and neuronal nicotinic α-bungarotoxin receptors

Current studies suggest that several distinct populations of nicotinic acetylcholine (ACh) receptors exist. One of these is the muscle-type nicotinic receptors with which neuromuscular nicotinic receptor ligands and the snake toxin alpha-bungarotoxin interact. alpha-Bungarotoxin potently binds to these nicotinic receptors and blocks their function, two characteristics that have made the alpha-toxin a very useful probe for the characterization of these sites. In neuronal tissues, several populations of nicotinic receptors have been identified which, although they share a nicotinic pharmacology, have unique characteristics. The alpha-bungarotoxin-insensitive neuronal nicotinic receptors, which may be involved in mediating neuronal excitability, bind nicotinic agonists with high affinity but do not interact with alpha-bungarotoxin. Subtypes of these alpha-toxin-insensitive receptors appear to exist, as evidenced by findings that some are inhibited by neuronal bungarotoxin whereas others are not. In addition to the alpha-bungarotoxin-insensitive sites, alpha-bungarotoxin-sensitive neuronal nicotinic receptors are also present in neuronal tissues. These latter receptors bind alpha-bungarotoxin with high affinity and nicotinic agonists with an affinity in the microM range. The function of the nicotinic alpha-bungarotoxin receptors are as yet uncertain. Thymopoietin, a polypeptide linked to immune function, appears to interact specifically with nicotinic receptor populations that bind alpha-bungarotoxin. Thus, in muscle tissue where alpha-bungarotoxin both binds to the receptor and blocks activity, thymopoietin also potently binds to the receptor and inhibits nicotinic receptors-mediated function. In neuronal tissues, thymopoietin interacts only with the nicotinic alpha-bungarotoxin site and not the alpha-bungarotoxin-insensitive neuronal nicotinic receptor population. These observations that thymopoietin potently and specifically interacts with nicotinic alpha-bungarotoxin-sensitive receptors in neuronal and muscle tissue, together with findings that thymopoietin is an endogenously occurring agent, could suggest that this immune-related polypeptide represents a ligand for the alpha-bungarotoxin receptors. The function of thymopoietin at the alpha-bungarotoxin receptor is as yet uncertain; however, a potential trophic, as well as other roles are suggested.     

High affinity binding of alpha-bungarotoxin to the purified alpha-subunit and to its 27,000-dalton proteolytic peptide from Torpedo marmorata acetylcholine receptor. Requirement for sodium dodecyl sulfate

Intact nicotinic acetylcholine receptor (AChR) tightly binds alpha-bungarotoxin. The two toxin-binding sites are presumed to be on the two alpha-subunits, either on or near the ACh-binding sites. Isolated alpha-subunits have been found to maintain weak binding to alpha-bungarotoxin (KD approximately 0.2 microM). We describe here conditions under which the alpha-subunit and a 27,000-dalton proteolytic peptide bound alpha-bungarotoxin with high affinity. The four subunits of Torpedo marmorata AChR, as well as several proteolytic peptides of the alpha-subunit, were first purified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. We found that the purified alpha-subunit (but not the beta-, gamma- or delta-subunits) and its 27,000-dalton peptide specifically bound 125I-labeled alpha-bungarotoxin with KD approximately 3 and 6 nM, i.e., about two orders of magnitude lower than the intact AChR. Nearly 100% of the sites were recovered. The recovery of this high affinity binding required the presence of SDS (approximately 0.02%) but non-denaturing detergents had a strongly inhibitory effect. Unlabeled alpha-toxins competed with labeled alpha-bungarotoxin, alpha-bungarotoxin being more effective than all the other toxins tested. Decamethonium and hexamethonium competed efficiently with alpha-bungarotoxin binding but carbamylcholine had only a weak effect. The main immunogenic region of the AChR was only partially preserved since conformation-dependent monoclonal antibodies to this region bound the alpha subunit-toxin complexes, but much less efficiently than the intact AChR. We conclude that SDS can be advantageous to the recovery of high toxin binding to the alpha subunit which still has not completely recovered its native conformation.     

Synthesis, insertion into the plasma membrane, and turnover of alpha- bungarotoxin receptors in chick sympathetic neurons

alpha-Bungarotoxin was used to identify an integral membrane protein in the plasma membrane of chick sympathetic neurons. The synthesis, insertion into the plasma membrane, and turnover of the alpha-bungarotoxin receptor were studied using isotopically labeled amino acids (2H, 13C, 15N) to directly label receptor molecules. Neurons incubated in medium containing dense amino acids continued to insert unlabeled receptors from a pool of previously synthesized molecules for 2 h. Density-labeled receptors began to appear in the plasma membrane after this 2-h period. Synthesis of receptors, but not insertion into the surface, was blocked by cycloheximide (100 microgram/ml). Neither colchicine (0.05 microgram/ml) of actinomycin D (5 microgram/ml) has any effect on alpha-bungarotoxin receptor synthesis or insertion. Autoradiographic studied revealed that receptors occur on growth cones, axons, and cell bodies of single neurons and explanted ganglia. The rate of insertion of newly synthesized receptors into the plasma membrane of axons extending from explanted sympathetic ganglia was approximately the same as that into the cell body portion of the ganglion. Cytochalasin B (2 microgram/ml) rapidly distrupted growth cones but had no effect on receptor insertion. These experiments suggested that the growth cone is not the sole or even the primary site for insertion of this membrane protein. The kinetics of turnover of the alpha-bungarotoxin receptor were a first-order exponential with t 1/2 = 11 h. Neurons that had their surface receptors labeled with 125I-alpha-bungarotoxin produced [125I]iodotyrosine. This process was inhibited by low temperature (23 degrees C) and also by a metabolic inhibitor. This is interpreted as evidence that receptors turn over by a mechanism in which they are internalized and then proteolytically degraded.     

Is agonist self-inhibition at the nicotinic acetylcholine receptor a nonspecific action?

Agonist concentration-response relationships at nicotinic postsynaptic receptors were established by measuring 86Rb+ efflux from acetylcholine receptor rich native Torpedo membrane vesicles under three different conditions: integrated net ion efflux (in 10 s) from untreated vesicles, integrated net efflux from vesicles in which most acetylcholine sites were irreversibly blocked with alpha-bungarotoxin, and initial rates of efflux (5-100 ms) from vesicles that were partially blocked with alpha-bungarotoxin. Exposure to acetylcholine, carbamylcholine, suberyldicholine, phenyltrimethylammonium, or (-)-nicotine over 10(8)-fold concentration ranges results in bell-shaped ion flux response curves due to stimulation of acetylcholine receptor channel opening at low concentrations and inhibition of channel function at 60-2000 times higher concentrations. Concentrations of agonists that inhibit their own maximum 86Rb+ efflux by 50% (KB values) are 110, 211, 3.0, 39, and 8.9 mM, respectively, for the agonists listed above. For acetylcholine and carbamylcholine, KB values determined from both 10-s and 15-ms efflux measurements are the same, indicating that the rate of agonist-induced desensitization increases to maximum at concentrations lower than those causing self-inhibition. For all partial and full agonists studied, Hill coefficients for self-inhibition are close to 1.0. Concentrations of agonists up to 8 times KB did not change the order parameter reported by a spin-labeled fatty acid incorporated in Torpedo membranes. We conclude that agonist self-inhibition cannot be attributed to a general nonspecific membrane perturbation. Instead, these results are consistent with a saturable site of action either at the lipid-protein interface or on the acetylcholine receptor protein itself.     

Chromosomes move poleward in anaphase along stationary microtubules that coordinately disassemble from their kinetochore ends

The binding sites on the nicotinic acetylcholine receptor of labels specific for the alpha-, beta-, and delta-subunits were determined by electron image analysis, using tubular crystals of receptors grown from the postsynaptic membranes of Torpedo marmorata electric organ. The labels were alpha-bungarotoxin (which attaches to the acetylcholine binding sites on the pair of alpha-subunits), Fab35 (a monoclonal antibody Fab fragment directed against the main immunogenic region of the alpha-subunit), Fab111 (a monoclonal antibody Fab fragment directed against a cytoplasmic site on the beta-subunit), and wheat germ agglutinin (which binds to N-acetylglucosamine residues on the delta-subunit). These labels, bound to receptors in the crystals, were located by comparing labeled with native structures, averaged in each case over more than 5,000 molecules. From the assignments made, we find that the clockwise arrangement of subunits around the receptor, viewed from the synaptic face, is: alpha, beta, alpha, gamma, and delta; that the main immunogenic region is at (or close to) the side of the alpha-subunit; and that the two acetylcholine binding sites are at the synaptic end of the alpha-subunits, 27-28 A from the central axis and approximately 53 A apart. In the crystal lattice, neighboring molecules are paired so that their delta- and alpha-subunits are juxtaposed, an organization that appears to relate closely to the grouping of receptors in vivo.     

Effects of valency on thermodynamic parameters of specific membrane interactions

We have measured the equilibrium binding of dioleoylphosphatidylcholine vesicles (800-A diameter) containing various densities of incorporated palmitoyl-alpha-bungarotoxin (PBGT) to acetylcholine receptor (AchR) enriched microsac membranes. We have previously shown that these PBGT vesicles bind specifically to the microsacs mediated by direct interactions with the AchRs [Grant, S. W., Babbitt, B. P., West, L. K., & Huang, L. (1982) Biochemistry 21, 1274-1279]. The percent binding of liposomal lipid and associated PBGT to excess AchR sites, as well as the inhibition of binding by pretreatment of microsacs with excess alpha-bungarotoxin (alpha BGT), was strongly dependent upon the protein/lipid molar ratio of the vesicles. In addition, there existed a threshold level of approximately six PBGT molecules per vesicle at which the binding increased dramatically. The apparent association constant, KAapp, for lipid vesicle-microsac membrane binding increased approximately 4800-fold (from 3.95 X 10(4) to 1.90 X 10(8) M-1) due to an increase of 20-fold in the vesicle-associated PBGT surface density. Direct competition for binding to microsac membranes between vesicles with different PBGT/lipid molar ratios indicated that multivalent binders could easily replace binders of lower valency when receptor sites were limited. Measurement of the temperature dependence of the KAapp indicated that weak (low valency) and medium strength (intermediate valency) PBGT vesicle binders bound to microsacs in a fashion similar to the binding of alpha BGT and PBGT to detergent-solubilized AchRs. Strong PBGT vesicle binders (high valency) appear to bind by a somewhat different mechanism. All results are discussed in terms of the effects of ligand (PBGT) valency on the binding strength of vesicles to microsac membranes.