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Victor I. Tsetlin – One of the best experts on this subject based on the ideXlab platform.
Weak toxin WTX from Naja kaouthia cobra venom interacts with both nicotinic and muscarinic acetylcholine receptorsFEBS Journal, 2009Co-Authors: Dmitry Y. Mordvintsev, Yakov L. Polyak, Dmitry I. Rodionov, Jan Jakubík, Vladimir Dolezal, Evert Karlsson, Victor I. Tsetlin, Yuri N. UtkinAbstract:
Iodinated [125I] weak toxin from Naja kaouthia (WTX) cobra venom was injected into mice, and organ-specific binding was monitored. Relatively high levels of [125I]WTX were detected in the adrenal glands. Rat adrenal membranes were therefore used for analysis of [125I]WTX-binding sites. Specific [125I]WTX binding was partially inhibited by both alpha-cobratoxin, a blocker of the alpha7 and muscle-type nicotinic acetylcholine receptors (nAChRs), and by atropine, an antagonist of the muscarinic acetylcholine receptor (mAChR). Binding to rat adrenal nAChR had a Kd of 2.0+/-0.8 microM and was inhibited by alpha-cobratoxin but not by a short-chain Alpha-Neurotoxin antagonist of the muscle-type nAChR, suggesting a specific interaction with the alpha7-type nAChR. WTX binding was reduced not only by atropine but also by other muscarinic agents (oxotremorine and muscarinic toxins from Dendroaspis angusticeps), indicating an interaction with mAChR. This interaction was further characterized using individual subtypes of human mAChRs expressed in Chinese hamster ovary cells. WTX concentrations up to 30 microM did not inhibit binding of [3H]acetylcholine to any subtype of mAChR by more than 50%. Depending on receptor subtype, WTX either increased or had no effect on the binding of the muscarinic antagonist [3H]N-methylscopolamine, which binds to the orthosteric site, a finding indicative of an allosteric interaction. Furthermore, WTX alone activated G-protein coupling with all mAChR subtypes and reduced the efficacy of acetylcholine in activating G-proteins with the M1, M4, and M5 subtypes. Our data demonstrate an orthosteric WTX interaction with nAChR and an allosteric interaction with mAChRs.
Naturally occurring and synthetic peptides acting on nicotinic acetylcholine receptors.Current pharmaceutical design, 2009Co-Authors: Igor E Kasheverov, Yuri N. Utkin, Victor I. TsetlinAbstract:
Nicotinic acetylcholine receptors (nAChRs) are pentameric membrane-bound proteins belonging to the large family of ligand-gated ion channels. nAChRs possess various binding sites which interact with compounds of different chemical nature, including peptides. Historically first peptides found to act on nAChR were synthetic fragments of snake Alpha-Neurotoxins, competitive receptor antagonists. Later it was shown that fragments of glycoprotein from rabies virus, having homology to Alpha-Neurotoxins, and polypeptide neurotoxins waglerins from the venom of Wagler’s pit viper Trimeresurus (Tropidolaemus) wagleri bind in a similar way, waglerins being efficient blockers of muscle-type nAChRs. Neuropeptide substance P appears to interact with the channel moiety of nAChR. beta-Amyloid, a peptide forming senile plaques in Alzheimer’s disease, also can bind to nAChR, although the mode of binding is still unclear. However, the most well-studied peptides interacting with the ligand-binding sites of nAChRs are so-called alpha-conotoxins, peptide neurotoxins from marine snails of Conus genus. First alpha-conotoxins were discovered in the late 1970s, and now it is a rapidly growing family due to isolation of peptides from multiple Conus species, as well as to cloning, and chemical synthesis of new analogues. Because of their unique selectivity towards distinct nAChR subtypes, alpha-conotoxins became valuable tools in nAChR research. Recent X-ray structures of alpha-conotoxin complexes with acetylcholine–binding protprotein, a model of nAChR ligand-binding domains, revealed the details of the nAChR ligand-binding sites and provided the basis for design of novel ligands.
A model for short α-neurotoxin bound to nicotinic acetylcholine receptor from Torpedo californica: Comparison with long-chain α-neurotoxins and α-conotoxinsComputational biology and chemistry, 2005Co-Authors: Dmitry Y. Mordvintsev, Igor E Kasheverov, Ya. L. Polyak, O. V. Levtsova, Ye. V. Tourleigh, Konstantin V. Shaitan, Yu. N. Utkin, Victor I. TsetlinAbstract:
Short-chain Alpha-Neurotoxins from snakes are highly selective antagonists of the muscle-type nicotinic acetylcholine receptors (nAChR). Although their spatial structures are known and abundant information on topology of binding to nAChR is obtained by labeling and mutagenesis studies, the accurate structure of the complex is not yet known. Here, we present a model for a short Alpha-Neurotoxin, neurotoxin II from Naja oxiana (NTII), bound to Torpedo californica nAChR. It was built by comparative modeling, docking and molecular dynamics using 1H NMR structure of NTII, cross-linking and mutagenesis data, cryoelectron microscopy structure of Torpedo marmorata nAChR [Unwin, N., 2005. Refined structure of the nicotinic acetacetylcholineeptor at 4A resolution. J. Mol. Biol. 346, 967-989] and X-ray structures of acetylcholine–binding protprotein (AChBP) with agonists [Celie, P.H., van Rossum-Fikkert, S.E., van Dijk, W.J., Brejc, K., Smit, A.B., Sixma, T.K., 2004. Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures. Neuron 41 (6), 907-914] and antagonists: alpha-cobratoxin, a long-chain Alpha-Neurotoxin [Bourne, Y., Talley, T.T., Hansen, S.B., Taylor, P., Marchot, P., 2005. Crystal structure of Cbtx-AChBP complex reveals essential interactions between snake Alpha-Neurotoxins and nicotinic receptors. EMBO J. 24 (8), 1512-1522] and alpha-conotoxin [Celie, P.H., Kasheverov, I.E., Mordvintsev, D.Y., Hogg, R.C., van Nierop, P., van Elk, R., van Rossum-Fikkert, S.E., Zhmak, M.N., Bertrand, D., Tsetlin, V., Sixma, T.K., Smit, A.B., 2005. Crystal structure of nicotinic acetacetylcholineeptor homolog AChBP in complex with an alpha-conotoxin PnIA variant. Nat. Struct. Mol. Biol. 12 (7), 582-588]. In complex with the receptor, NTII was located at about 30 A from the membrane surface, the tip of its loop II plunges into the ligand-binding pocket between the alpha/gamma or alpha/delta nAChR subunits, while the loops I and III contact nAChR by their tips only in a ‘surface-touch’ manner. The toxin structure undergoes some changes during the final complex formation (for 1.45 rmsd in 15-25 ps according to AMBER’99 molecular dynamics simulation), which correlates with NMR data. The data on the mobility and accessibility of spin- and fluorescence labels in free and bound NTII were used in MD simulations. The binding process is dependent on spontaneous outward movement of the C-loop earlier found in the AChBP complexes with alpha-cobratoxin and alpha-conotoxin. Among common features in binding of short- and long Alpha-Neurotoxins is the rearrangement of aromatic residues in the binding pocket not observed for alpha-conotoxin binding. Being in general very similar, the binding modes of short- and long Alpha-Neurotoxins differ in the ways of loop II entry into nAChR.
Palmer Taylor – One of the best experts on this subject based on the ideXlab platform.
Structural Dynamics of the α-Neurotoxin−Acetylcholine-Binding Protein Complex: Hydrodynamic and Fluorescence Anisotropy Decay Analyses†Biochemistry, 2005Co-Authors: Ryan E. Hibbs, David A. Johnson, Jianxin Shi, Scott B. Hansen, Palmer TaylorAbstract:
The three-fingered Alpha-Neurotoxins have played a pivotal role in elucidating the structure and function of the muscle-type and neuronal alpha7 nicotinic acetylcholine receptors (nAChRs). To advance our understanding of the Alpha-Neurotoxin-nAChR interaction, we examined the flexibility of Alpha-Neurotoxin bound to the acetylcholine–binding protprotein (AChBP), which shares structural similarity and sequence identities with the extracellular domain of nAChRs. Because the crystal structure of five alpha-cobratoxin molecules bound to AChBP shows the toxins projecting radially like propeller “blades” from the perimeter of the donut-shaped AChBP, the toxin molecules should increase the frictional resistance and thereby alter the hydrodynamic properties of the complex. alpha-Bungarotoxin binding had little effect on the frictional coefficients of AChBP measured by analytical ultracentrifugation, suggesting that the bound toxins are flexible. To support this conclusion, we measured the anisotropy decay of four site-specifically labeled alpha-cobratoxins (conjugated at positions Lys(23), Lys(35), Lys(49), and Lys(69)) bound to AChBP and free in solution and compared their anisotropy decay properties with fluorescently labeled cysteine mutants of AChBP. The results indicated that the core of the toxin molecule is relatively flexible when bound to AChBP. When hydrodynamic and anisotropy decay analyses are taken together, they establish that only one face of the second loop of the Alpha-Neurotoxin is immobilized significantly by its binding. The results indicate that bound Alpha-Neurotoxin is not rigidly oriented on the surface of AChBP but rather exhibits segmental motion by virtue of flexibility in its fingerlike structure.
Orientation of α-Neurotoxin at the Subunit Interfaces of the Nicotinic Acetylcholine Receptor†Biochemistry, 2000Co-Authors: Siobhan Malany, Hitoshi Osaka, Steven M. Sine, Palmer TaylorAbstract:
The Alpha-Neurotoxins are three-fingered peptide toxins that bind selectively at interfaces formed by the alpha subunit and its associating subunit partner, gamma, delta, or epsilon of the nicotinic acetacetylcholineeptor. Because the Alpha-Neurotoxin from Naja mossambica mossambica I shows an unusual selectivity for the alpha gamma and alpha delta over the alpha epsilon subunit interface, residue replacement and mutant cycle analysis of paired residues enabled us to identify the determinants in the gamma and delta sequences governing alpha-toxin recognition. To complement this approach, we have similarly analyzed residues on the alpha subunit face of the binding site dictating specificity for alpha-toxin. Analysis of the alpha gamma interface shows unique pairwise interactions between the charged residues on the alpha-toxin and three regions on the alpha subunit located around residue Asp(99), between residues Trp(149) and Val(153), and between residues Trp(187) and Asp(200). Substitutions of cationic residues at positions between Trp(149) and Val(153) markedly reduce the rate of alpha-toxin binding, and these cationic residues appear to be determinants in preventing alpha-toxin binding to alpha 2, alpha 3, and alpha 4 subunit containing receptors. Replacement of selected residues in the alpha-toxin shows that Ser(8) on loop I and Arg(33) and Arg(36) on the face of loop II, in apposition to loop I, are critical to the alpha-toxin for association with the alpha subunit. Pairwise mutant cycle analysis has enabled us to position residues on the concave face of the three alpha-toxin loops with respect to alpha and gamma subunit residues in the alpha-toxin binding site. Binding of NmmI alpha-toxin to the alpha gamma interface appears to have dominant electrostatic interactions not seen at the alpha delta interface.
Pairwise electrostatic interactions between α-neurotoxins and γ, δ, and ε subunits of the nicotinic acetylcholine receptorThe Journal of biological chemistry, 2000Co-Authors: Hitoshi Osaka, Siobhan Malany, Steven M. Sine, Brian E. Molles, Palmer TaylorAbstract:
Alpha-Neurotoxins bind with high affinity to alpha-gamma and alpha-delta subunit interfaces of the nicotinic acetacetylcholineeptor. Since this high affinity complex likely involves a van der Waals surface area of approximately 1200 A(2) and 25-35 residues on the receptor surface, analysis of side chains should delineate major interactions and the orientation of bound Alpha-Neurotoxin. Three distinct regions on the gamma subunit, defined by Trp(55), Leu(119), Asp(174), and Glu(176), contribute to alpha-toxin affinity. Of six charge reversal mutations on the three loops of Naja mossambica mossambica alpha-toxin, Lys(27) –> Glu, Arg(33) –> Glu, and Arg(36) –> Glu in loop II reduce binding energy substantially, while mutations in loops I and III have little effect. Paired residues were analyzed by thermodynamic mutant cycles to delineate electrostatic linkages between the six alpha-toxin charge reversal mutations and three key residues on the gamma subunit. Large coupling energies were found between Arg(33) at the tip of loop II and gammaLeu(119) (-5.7 kcal/mol) and between Lys(27) and gammaGlu(176) (-5.9 kcal/mol). gammaTrp(55) couples strongly to both Arg(33) and Lys(27), whereas gammaAsp(174) couples minimally to charged alpha-toxin residues. Arg(36), despite strong energetic contributions, does not partner with any gamma subunit residues, perhaps indicating its proximity to the alpha subunit. By analyzing cationic, neutral and anionic residues in the mutant cycles, interactions at gamma176 and gamma119 can be distinguished from those at gamma55.
Vincent A Chiappinelli – One of the best experts on this subject based on the ideXlab platform.
two novel alpha neurotoxins isolated from the taipan snake oxyuranus scutellatus exhibit reduced affinity for nicotinic acetylcholine receptors in brain and skeletal muscleBiochemistry, 1996Co-Authors: Fernando Z Zamudio, Kathleen M Wolf, Brian M Martin, Vincent A ChiappinelliAbstract:
: Three novel toxic peptides were purified to homogeneity from the venom of the Australian taipan snake, Oxyuranus scutellatus scutellatus. On the basis of complete amino acid sequence analyses, two of these toxins belong to the family of short-chain Alpha-Neurotoxins found in elapid and hydrophid snake venoms and are the first postsynaptic neurotoxins identified in taipan venom. Radioligand binding studies confirm that taipan toxins 1 and 2 inhibit the binding of [125I]-alpha-bungarotoxin to nicotinic acetylcholine receptors in skeletal muscle with IC50 values of 2.4-2.5 nM but are 5-fold less potent in this assay than alpha-bungarotoxin or the two short-chain Alpha-Neurotoxins erabutoxin a and erabutoxin b. Taipan toxins 1 and 2 do not antagonize [125I]-alpha-bungarotoxin binding to central neuronal nicotinic receptors at concentrations up to 3 microM. We find that erabutoxin a and erabutoxin b do inhibit the binding of [125I]-alpha-bungarotoxin to central neuronal nicotinic receptors but are over 350-fold less potent than long-chain Alpha-Neurotoxins at these receptors. The novel Alpha-Neurotoxins from taipan venom do not inhibit the binding of [3H]nicotine to high-affinity nicotine receptors in brain, a property they share with alpha-bungarotoxin and the erabutoxins. The results demonstrate that at least two neuromuscular junction-blocking peptides are present in taipan venom. Nonconservative substitutions at position 32 in both taipan toxin 1 and 2 may be responsible for the observed decreases in affinities of the toxins of 5-fold for muscle receptors (compared to alpha-bungarotoxin) and over 10-fold for alpha-bungarotoxin-sensitive nicotinic receptors in brain (compared to the structurally similar short-chain Alpha-Neurotoxins erabutoxin a and erabutoxin b).
affinity of native kappa bungarotoxin and site directed mutants for the muscle nicotinic acetylcholine receptorBiochemistry, 1994Co-Authors: James J. Fiordalisi, Vincent A Chiappinelli, Regina Alrabiee, Gregory A GrantAbstract:
kappa-Bungarotoxin (kappa-bgt) is a 66-residue peptide originally purified from snake venom that acts as an antagonist at certain acetylcholine receptors. It is one of four homologous kappa-neurotoxins that are distinguished from the structurally related Alpha-Neurotoxins by their ability to block the alpha 3-subunit-containing neuronal nicotinic acetacetylcholineeptor (nAChR). It has been reported that venom-purified kappa-bgt also displays some affinity for the alpha 1-subunit-containing muscle nAChR to which the Alpha-Neurotoxins bind with high affinity. Here we report the effects of particular mutations on the ability of recombinant kappa-bgt to block the binding of 125I-alpha-bgt to nAChRs found in fetal mouse muscle and chick skeletal muscle. While the replacement of a proline resiresidue found in all kappa-neurotoxins with an alanine (P-42-A) has relatively little effect, the introduction of a lysine, which is found in 90% of active Alpha-Neurotoxins at the same position (P-42-K), eliminates muscle receptor affinity at the concentrations tested. In contrast, the replacement of a glutamine in kappa-bgt with a tryptophan found in all active Alpha-Neurotoxins (Q-32-W) increases the affinity of kappa-bgt for the muscle receptor. When the arginine residue found in all active alpha- and kappa-neurotoxins is replaced by an alanine (R-40-A), the ability of kappa-bgt to block the muscle receptor is reduced to undetectable levels.(ABSTRACT TRUNCATED AT 250 WORDS)
site directed mutagenesis of kappa bungarotoxin implications for neuronal receptor specificityBiochemistry, 1994Co-Authors: James J. Fiordalisi, Gregory A Grant, Regina Alrabiee, Vincent A ChiappinelliAbstract:
Postsynaptic polypeptide neurotoxins isolated from the venoms of elapid and hydrophid snakes exhibit the ability to bind selectively to and inhibit different types of receptors that function in nerve signal transmission. On the basis of their amino acid sequences and three-dimensional structures, these neurotoxins are clearly related, but nothing is yet known about the basis for their physiological receptor specificity. In this report, site-directed mutants of kappa-bungarotoxin, produced by an Escherichia coli expression system, are tested to determine the function of selected amino acid side chains in the interaction between toxin and receptor. Highly conserved residues at the bottom of the second loop (a region that has been shown to be a major point of contact with the receptor), particularly those residues at the junction between the beta-sheet and the end of the loop, were selected. The results demonstrate that a single amino acid substitution of the invariant arginine residue (Arg-40 to Ala-40) renders the toxin unable to inhibit nerve transmission in the chick ciliary ganglion up to a concentration of 10 microM. Significantly, the results also show that conversion to alanine of the nearby proline resiresidue (Pro-42) found to be invariant in all kappa-neurotoxins, but not found in any potent Alpha-Neurotoxin, produces a toxin with full inhibitory capacity. However, the introduction of a lysine residue at this position (P-42-K), like that found in alpha-bungarotoxin, reduces activity significantly.(ABSTRACT TRUNCATED AT 250 WORDS)
Liwen Niu – One of the best experts on this subject based on the ideXlab platform.
Purification, N-terminal sequencing, crystallization and preliminary structural determination of atratoxin-b, a short-chain Alpha-Neurotoxin from Naja atra venom.Acta crystallographica. Section D Biological crystallography, 2003Co-Authors: Xiaohua Lou, Maikun Teng, Guoqiang Pan, Rong Fan, Wenhan Deng, Pingfan Rao, Liwen NiuAbstract:
Atratoxin-b, a short-chain Alpha-Neurotoxin purified from Naja atra (mainland Chinese cobra) venom using a three-step chromatography procedure, has an apparent molecular mass of 6950 Da with an alkaline pI value (>9.5) and consists of one single polypeptide chain as estimated by MALDI-TOF mass spectrometry and SDS-PAGE. The protein is toxic to mice, with an in vitro LD(50) of about 0.18 mg kg(-1). Its N-terminal amino-acid sequence, LECHNQQSSQTPTIT, displays a very high homology to those of other Alpha-Neurotoxins. The overall three-dimensional structure of atratoxin-b is very similar to that of the homologous erabutoxin-a, as shown by the crystallographic molecular replacement and preliminary refinement results, with an R factor and R(free) of 27 and 29%, respectively. The microcrystal slowly grew to dimensions of approximate 0.1 x 0.1 x 0.15 mm over eight months using hanging-drop vapour-diffusion method. It gave a set of diffraction data to 1.56 A resolution using X-rays of wavelength 1.1516 A generated by the X-ray Diffraction and Scattering Station of beamline U7B at the National Synchrotron Radiation Laboratory (Hefei, China); this is the first example of the use of this beamline in protein crystallography. The crystals belong to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = 49.28, c = 44.80 A, corresponding to one molecule per asymmetric unit and a volume-to-mass ratio of 1.96 A(3) Da(-1).
Purification, N-terminal sequencing, crystallization and preliminary X-ray diffraction analysis of atratoxin, a new short-chain Alpha-Neurotoxin from the venom of Naja naja atra.Acta crystallographica. Section D Biological crystallography, 2002Co-Authors: Qingqiu Huang, Xiaohua Lou, Maikun Teng, Liwen NiuAbstract:
Atratoxin, a new Alpha-Neurotoxin purified to homogeneity by a series of liquid chromatographies from the venom of Naja naja atra (mainland Chinese cobra), is a small single-polypeptide alkaline protein with a pI of about 9.5 and molecular weight of 6952 Da estimated by mass spectrometry. Although the sequencing of the N-terminal 15 residues (LECHNQQTTQQPEGG) shows that this neurotoxic protein contains most of the residues, especially at the conserved positions, of the consensus sequence of short-chain Alpha-Neurotoxins, the natural mutations in the N-terminal Loop-1 presented by the sequence alignment may have structural or functional implications for the interactions between Alpha-Neurotoxins and related receptors. Single crystals of atratoxin have been grown from drops containing the necessary Cu(2+) ions by the conventional hanging-drop vapour-diffusion method. The crystals diffract X-rays to 1.6 A resolution and belong to space group C222(1), with unit-cell parameters a = 47.36, b = 47.83, c = 91.31 A, corresponding to a volume-to-mass ratio of 1.85 A(3) Da(-1) and two molecules in each asymmetric unit.
Ferdinand Hucho – One of the best experts on this subject based on the ideXlab platform.
How do acetylcholine receptor ligands reach their binding sitesEuropean journal of biochemistry, 1999Co-Authors: Patricio Sáez-briones, Victor I. Tsetlin, Michael Krauss, Mathias Dreger, Andreas Herrmann, Ferdinand HuchoAbstract:
The access pathway to the binding sites for large competitive antagonists of the nicotinic acetacetylcholineeptor from Torpedo californica electric tissue was analyzed by binding and photolabeling experiments with Alpha-Neurotoxins. Binding assays with [125I]alpha-bungarotoxin showed an increase in the number of accessible binding sites upon stepwise solubilization of the receptor-rich membranes. Similarily, ligand binding is facilitated upon fluidization of the membrane by increasing the temperature. The access to the binding sites seems to be sterically ‘hindered’ in the densely packed membrane state. Using a novel series of large biotinylated photoactivatable derivatives of neurotoxin II, we observed that the accessibility to the alpha/gamma- but not to the alpha/delta-binding site was considerably decreased for some derivatives under native conditions. This effect was less apparent at higher temperatures and could be abolished by complete solubilization. These observations support the nonequivalence of the receptor’s binding sites. Together, our data suggest (a) that Alpha-Neurotoxins approach their binding sites from the membrane-facing periphery of the receptor’s extramembrane domain rather than through the channel mouth and (b) that different entrance pathways to each binding site exist which vary in their sensitivity to the physical state of the plasma membrane.
Benzophenone-type photoactivatable derivatives of Alpha-Neurotoxins and alpha-conotoxins in studies on Torpedo nicotinic acetylcholine receptor.Journal of receptor and signal transduction research, 1999Co-Authors: Igor E Kasheverov, Maxim N. Zhmak, E Chivilyov, P Saez-brionez, Y U Utkin, Ferdinand Hucho, Victor I. TsetlinAbstract:
By chemical modification of different lysine residues, benzoylbenzoyl (BzBz) groups were introduced into neurotoxin II Naja naja oxiana (NT-II), a short-chain snake venom Alpha-Neurotoxin, while p-benzoylphenylalanyl (Bpa) residue was incorporated in the course of peptide synthesis at position 11 of alpha-conotoxin G1, a neurotoxic peptide from marine snails. Although the crosslinking yields for iodinated BzBz derivatives of NT-II and for Bpa analogue of G1 to the membrane-bound Torpedo californica nicotinic acetacetylcholineeptor (AChR) are relatively low, the subunit labeling patterns confirm the earlier conclusions, derived from arylazide or diazirine photolabels, that Alpha-Neurotoxins and alpha-conotoxins bind at the subunit interfaces. Detecting the labeled alpha-subunit with iodinated Bpa analogue of G1 provided a direct proof for the contact between this subunit and alpha-conotoxin molecule.
The Handedness of the Subunit Arrangement of the Nicotinic Acetylcholine Receptor from Torpedo californicaEuropean journal of biochemistry, 1995Co-Authors: Jan Machold, Yuri N. Utkin, Victor I. Tsetlin, Christoph Weise, Ferdinand HuchoAbstract:
Cross-linking an Alpha-Neurotoxin with a known three-dimensional structure and with photoactivatable groups in known positions to native membrane-bound acetylcholine receptor reveals its quaternary structure, including the handedness of its circular subunit arrangement. Photolabelling with Alpha-Neurotoxin carrying the photoactivatable group at position Lys46 is inhibited by the competitive antagonist (+)-tubocurarine in a biphasic manner, indicating that it reacts with both alpha-subunits that were shown to have different affinities for this antagonist [Neubig, R. R. & Cohen, J. B. (1979) Biochemistry 18, 5464-5475]. Lys46 is located on loop III of the neurotoxin. The other information necessary for the elucidation of the handedness was provided by the recent finding that the central loop of the toxin (loop II) is oriented towards the central pore of the receptor, securing the overall orientation of the bound toxin [Machold, J., Utkin, Y. N., Kirsch, D., Kaufmann, R., Tsetlin, V. & Hucho, F. (1995b) Proc. Natl Acad. Sci. USA 92, 7282-7286]. Looking at the receptor from the synaptic side of the postsynaptic membrane, it was concluded that the clockwise subunit arrangement is alpha H-gamma-alpha L-delta-beta (alpha H and alpha L are the alpha-subunits binding (+)-tubocurarine with high and low affinity, respectively). Its mirror image alpha alpha L-gamma-alpha H-beta-delta could thus be excluded.