Biomedical subjects

Y A Ushkaryov

Publications and source records attributed to Y A Ushkaryov.

At least 19 recordsLinked to original sources

PubMed2006

Insecticidal toxins from black widow spider venom.

The biological effects of Latrodectus spider venom are similar in animals from different phyla, but these symptoms are caused by distinct phylum-specific neurotoxins (collectively called latrotoxins) with molecular masses ranging from 110 to 140 kDa. To date, the venom has been found to contain five insecticidal toxins, termed alpha, beta, gamma, delta and epsilon-latroinsectotoxins (LITs). There is also a vertebrate-specific neurotoxin, alpha-latrotoxin (alpha-LTX), and one toxin affecting crustaceans, alpha-latrocrustatoxin (alpha-LCT). These toxins stimulate massive release of neurotransmitters from nerve terminals and act (1) by binding to specific receptors, some of which mediate an exocytotic signal, and (2) by inserting themselves into the membrane and forming ion-permeable pores. Specific receptors for LITs have yet to be identified, but all three classes of vertebrate receptors known to bind alpha-LTX are also present in insects. All LTXs whose structures have been elucidated (alpha-LIT, delta-LIT, alpha-LTX and alpha-LCT) are highly homologous and have a similar domain architecture, which consists of a unique N-terminal sequence and a large domain composed of 13-22 ankyrin repeats. Three-dimensional (3D) structure analysis, so far done for alpha-LTX only, has revealed its dimeric nature and an ability to form symmetrical tetramers, a feature probably common to all LTXs. Only tetramers have been observed to insert into membranes and form pores. A preliminary 3D reconstruction of a delta-LIT monomer demonstrates the spatial similarity of this toxin to the monomer of alpha-LTX.

Animals↗

PubMed2004

The multiple actions of black widow spider toxins and their selective use in neurosecretion studies.

The black widow spider venom contains several large protein toxins--latrotoxins--that are selectively targeted against different classes of animals: vertebrates, insects, and crustaceans. These toxins are synthesised as large precursors that undergo proteolytic processing and activation in the lumen of the venom gland. The mature latrotoxins demonstrate strong functional structure conservation and contain multiple ankyrin repeats, which mediate toxin oligomerisation. The three-dimensional structure has been determined for alpha-latrotoxin (alphaLTX), a representative venom component toxic to vertebrates. This reconstruction explains the mechanism of alphaLTX pore formation by showing that it forms tetrameric complexes, harbouring a central channel, and that it is able to insert into lipid membranes. All latrotoxins cause massive release of neurotransmitters from nerve terminals of respective animals after binding to specific neuronal receptors. A G protein-coupled receptor latrophilin and a single-transmembrane receptor neurexin have been identified as major high-affinity receptors for alphaLTX. Latrotoxins act by several Ca(2+)-dependent and -independent mechanisms based on pore formation and activation of receptors. Mutant recombinant alphaLTX that does not form pores has been used to dissect the multiple actions of this toxin. As a result, important insights have been gained into the receptor signalling and the role of intracellular Ca(2+) stores in the effect of alphaLTX.

Animals↗

PubMed2001

alpha-Latrotoxin, acting via two Ca2+-dependent pathways, triggers exocytosis of two pools of synaptic vesicles.

alpha-Latrotoxin stimulates three types of [(3)H]gamma-aminobutyric acid and [(14)C]glutamate release from synaptosomes. The Ca(2+)-independent component (i) is insensitive to SNAP-25 cleavage or depletion of vesicle contents by bafilomycin A1 and represents transmitter efflux mediated by alpha-latrotoxin pores. Two other components of release are Ca(2+)-dependent and vesicular but rely on distinct mechanisms. The fast receptor-mediated pathway (ii) involves intracellular Ca(2+) stores and acts upon sucrose-sensitive readily releasable vesicles; this mechanism is insensitive to inhibition of phosphatidylinositol 4-kinase (PI 4-kinase). The delayed pore-dependent exocytotic component (iii) is stimulated by Ca(2+) entering through alpha-latrotoxin pores; it requires PI 4-kinase and occurs mainly from depot vesicles. Lanthanum perturbs alpha-latrotoxin pores and blocks the two pore-mediated components (i, iii) but not the receptor-mediated release (ii). alpha-Latrotoxin mutant (LTX(N4C)) cannot form pores and stimulates only the Ca(2+)-dependent receptor-mediated amino acid exocytosis (ii) (detectable biochemically and electrophysiologically). These findings explain experimental data obtained by different laboratories and implicate the toxin receptors in the regulation of the readily releasable pool of synaptic vesicles. Our results also suggest that, similar to noradrenergic vesicles, amino acid-containing vesicles at some point in their cycle require PI 4-kinase.

1-Phosphatidylinositol 4-Kinase↗

PubMed2000

Latrophilin, neurexin, and their signaling-deficient mutants facilitate alpha -latrotoxin insertion into membranes but are not involved in pore formation.

Pure alpha-latrotoxin is very inefficient at forming channels/pores in artificial lipid bilayers or in the plasma membrane of non-secretory cells. However, the toxin induces pores efficiently in COS-7 cells transfected with the heptahelical receptor latrophilin or the monotopic receptor neurexin. Signaling-deficient (truncated) mutants of latrophilin and latrophilin-neurexin hybrids also facilitate pore induction, which correlates with toxin binding irrespective of receptor structure. This rules out the involvement of signaling in pore formation. With any receptor, the alpha-latrotoxin pores are permeable to Ca(2+) and small molecules including fluorescein isothiocyanate and norepinephrine. Bound alpha-latrotoxin remains on the cell surface without penetrating completely into the cytosol. Higher temperatures facilitate insertion of the toxin into the plasma membrane, where it co-localizes with latrophilin (under all conditions) and with neurexin (in the presence of Ca(2+)). Interestingly, on subsequent removal of Ca(2+), alpha-latrotoxin dissociates from neurexin but remains in the membrane and continues to form pores. These receptor-independent pores are inhibited by anti-alpha-latrotoxin antibodies. Our results indicate that (i) alpha-latrotoxin is a pore-forming toxin, (ii) receptors that bind alpha-latrotoxin facilitate its insertion into the membrane, (iii) the receptors are not physically involved in the pore structure, (iv) alpha-latrotoxin pores may be independent of the receptors, and (v) pore formation does not require alpha-latrotoxin interaction with other neuronal proteins.

Animals↗

PubMed2000

Tetramerisation of alpha-latrotoxin by divalent cations is responsible for toxin-induced non-vesicular release and contributes to the Ca(2+)-dependent vesicular exocytosis from synaptosomes.

A novel procedure of alpha-latrotoxin (alpha LTX) purification has been developed. Pure alpha LTX has been demonstrated to exist as a very stable homodimer. Such dimers further assemble into tetramers, and Ca(2+), Mg(2+) or higher toxin concentrations facilitate this process. However, when the venom is treated with EDTA, purified alpha LTX loses the ability to tetramerise spontaneously; the addition of Mg(2+) or Ca(2+) restores this ability. This suggests that alphaLTX has some intrinsically bound divalent cation(s) that normally support its tetramerisation. Single-particle cryoelectron microscopy and statistical image analysis have shown that: 1) the toxin has a non-compact, branching structure; 2) the alpha LTX dimers are asymmetric; and 3) the tetramers are symmetric and have a 25 A-diameter channel in the centre. Both alpha LTX oligomers bind to the same receptors in synaptosomes and rat brain sections. To study the effects of the dimers and tetramers on norepinephrine release from rat cerebrocortical synaptosomes, we used the EDTA-treated and untreated toxin preparations. The number of tetramers present in a preparation correlates with alpha LTX pore formation, suggesting that the tetramers are the pore-forming species of alpha LTX. The toxin actions mediated by the pore include: 1) Ca(2+) entry from the extracellular milieu; and 2) passive efflux of neurotransmitters via the pore that occurs independently of Ca(2+). The Ca(2+)-dependent alpha LTX-stimulated secretion conforms to all criteria of vesicular exocytosis but also depends upon intact intracellular Ca(2+) stores and functional phospholipase C (PLC). The Ca(2+)-dependent effect of the toxin is stronger when dimeric alpha LTX is used, indicating that higher receptor occupancy leads to its stronger activation, which contributes to stimulation of neuroexocytosis. In contrast, the Ca(2+)-independent release measured biochemically represents leakage of neurotransmitters through the toxin pore. These results are discussed in relation to the previously published observations.

Animals↗

PubMed2000

Structure of alpha-latrotoxin oligomers reveals that divalent cation-dependent tetramers form membrane pores.

We report here the first three-dimensional structure of alpha-latrotoxin, a black widow spider neurotoxin, which forms membrane pores and stimulates secretion in the presence of divalent cations. We discovered that alpha-latrotoxin exists in two oligomeric forms: it is dimeric in EDTA but forms tetramers in the presence of Ca2+ or Mg2+. The dimer and tetramer structures were determined independently at 18 A and 14 A resolution, respectively, using cryo-electron microscopy and angular reconstitution. The alpha-latrotoxin monomer consists of three domains. The N- and C-terminal domains have been identified using antibodies and atomic fitting. The C4-symmetric tetramers represent the active form of alpha-latrotoxin; they have an axial channel and can insert into lipid bilayers with their hydrophobic base, providing the first model of alpha-latrotoxin pore formation.

Amino Acid Sequence↗

PubMed1999

Norepinephrine exocytosis stimulated by alpha-latrotoxin requires both external and stored Ca2+ and is mediated by latrophilin, G proteins and phospholipase C.

alpha-latrotoxin (LTX) stimulates massive release of neurotransmitters by binding to a heptahelical transmembrane protein, latrophilin. Our experiments demonstrate that latrophilin is a G-protein-coupled receptor that specifically associates with heterotrimeric G proteins. The latrophilin-G protein complex is very stable in the presence of GDP but dissociates when incubated with GTP, suggesting a functional interaction. As revealed by immunostaining, latrophilin interacts with G alpha q/11 and G alpha o but not with G alpha s, G alpha i or G alpha z, indicating that this receptor may couple to several G proteins but it is not promiscuous. The mechanisms underlying LTX-evoked norepinephrine secretion from rat brain nerve terminals were also studied. In the presence of extracellular Ca2+, LTX triggers vesicular exocytosis because botulinum neurotoxins E, Cl or tetanus toxin inhibit the Ca(2+)-dependent component of the toxin-evoked release. Based on (i) the known involvement of G alpha q in the regulation of inositol-1,4,5-triphosphate generation and (ii) the requirement for Ca2+ in LTX action, we tested the effect of inhibitors of Ca2+ mobilization on the toxin-evoked norepinephrine release. It was found that aminosteroid U73122, which inhibits the coupling of G proteins to phospholipase C, blocks the Ca(2+)-dependent toxin's action. Thapsigargin, which depletes intracellular Ca2+ stores, also potently decreases the effect of LTX in the presence of extracellular Ca2+. On the other hand, clostridial neurotoxins or drugs interfering with Ca2+ metabolism do not inhibit the Ca2(+)-independent component of LTX-stimulated release. In the absence of Ca2+, the toxin induces in the presynaptic membrane non-selective pores permeable to small fluorescent dyes; these pores may allow efflux of neurotransmitters from the cytoplasm. Our results suggest that LTX stimulates norepinephrine exocytosis only in the presence of external Ca2+ provided intracellular Ca2+ stores are unperturbed and that latrophilin, G proteins and phospholipase C may mediate the mobilization of stored Ca2+, which then triggers secretion.

Animals↗

PubMed1999

The latrophilin family: multiply spliced G protein-coupled receptors with differential tissue distribution.

Latrophilin is a brain-specific Ca2+-independent receptor of alpha-latrotoxin, a potent presynaptic neurotoxin. We now report the finding of two novel latrophilin homologues. All three latrophilins are unusual G protein-coupled receptors. They exhibit strong similarities within their lectin, olfactomedin and transmembrane domains but possess variable C-termini. Latrophilins have up to seven sites of alternative splicing; some splice variants contain an altered third cytoplasmic loop or a truncated cytoplasmic tail. Only latrophilin-1 binds alpha-latrotoxin; it is abundant in brain and is present in endocrine cells. Latrophilin-3 is also brain-specific, whereas latrophilin-2 is ubiquitous. Together, latrophilins form a novel family of heterogeneous G protein-coupled receptors with distinct tissue distribution and functions.

Alternative Splicing↗

PubMed1999

Functional expression of alpha-latrotoxin in baculovirus system.

To facilitate the study of the mechanism of alpha-latrotoxin action, it is necessary to create a biologically active recombinant toxin. Mature alpha-latrotoxin is naturally produced by post-translational cleavage, probably at two furin sites located at the N- and C-termini of the precursor. A recombinant baculovirus has now been constructed, which encodes the melittin signal peptide fused to the 130-kDa mature toxin between the furin sites. Insect cells, infected with this baculovirus, secreted recombinant alpha-latrotoxin. This was partially purified and proved indistinguishable from the natural toxin with respect to its molecular mass, immunostaining, toxicity to mice, binding to alpha-latrotoxin receptors (latrophilin or neurexin Ialpha) and electrophysiological recording in the mouse diaphragm. The successful expression of recombinant alpha-latrotoxin permits mutational analysis of the toxin.

Animals↗

PubMed1998

Enhancement of spontaneous transmitter release at neonatal mouse neuromuscular junctions by the glial cell line-derived neurotrophic factor (GDNF).

1. The acute effects of neurotrophic factors on the frequency of spontaneous transmitter release (miniature endplate potentials (MEPPs)) from motor nerve terminals has been examined in skeletal muscles of neonatal mice aged between 9 and 20 days. The following factors were tested at a concentration of 50 ng ml-1: brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), ciliary neuronotrophic factor (CNTF), leukaemia inhibitory factor (LIF), insulin-like growth factors 1 and 2 (IGF-1 and IGF-2), and glial cell line-derived neurotrophic factor (GDNF). In some experiments, the responses to 2 microM LaCl3 and 10 mM K+, or to 2-5 nM purified alpha-latrotoxin (alpha-LTX) were also measured. 2. Neither BDNF, NT-3, NT-4, LIF, IGF-1 or IGF-2 - singly or in combination - caused any significant change in MEPP frequency. GDNF, however, produced a highly significant, 2-fold increase in neurotransmitter release that was reproduced in fourteen muscles. 3. Potentiation of MEPP frequency in GDNF was of the same order as that induced by tetanic stimulation or substitution of the bathing medium with hypertonic saline; but substantially less than that induced either by lanthanum ions or alpha-latrotoxin. 4. The data suggest that concentrations of GDNF that produce maximal enhancement of motoneurone survival in vitro and in vivo also produce acute, non-saturating enhancement in transmitter release at immature mammalian neuromuscular synapses. Taken together with other reports, these findings suggest that GDNF may mediate both functional and structural plasticity of neonatal neuromuscular junctions.

Animals↗

PubMed1998

Vesicle exocytosis stimulated by alpha-latrotoxin is mediated by latrophilin and requires both external and stored Ca2+.

alpha-Latrotoxin (LTX) stimulates massive neurotransmitter release by two mechanisms: Ca2+-dependent and -independent. Our studies on norepinephrine secretion from nerve terminals now reveal the different molecular basis of these two actions. The Ca2+-dependent LTX-evoked vesicle exocytosis (abolished by botulinum neurotoxins) is 10-fold more sensitive to external Ca2+ than secretion triggered by depolarization or A23187; it does not, however, depend on the cation entry into terminals but requires intracellular Ca2+ and is blocked by drugs depleting Ca2+ stores and by inhibitors of phospholipase C (PLC). These data, together with binding studies, prove that latrophilin, which is linked to G proteins and inositol polyphosphate production, is the major functional LTX receptor. The Ca2+-independent LTX-stimulated release is not inhibited by botulinum neurotoxins or drugs interfering with Ca2+ metabolism and occurs via pores in the presynaptic membrane, large enough to allow efflux of neurotransmitters and other small molecules from the cytoplasm. Our results unite previously contradictory data about the toxin's effects and suggest that LTX-stimulated exocytosis depends upon the co-operative action of external and intracellular Ca2+ involving G proteins and PLC, whereas the Ca2+-independent release is largely non-vesicular.

Animals↗

PubMed1997

Alpha-latrotoxin receptor, latrophilin, is a novel member of the secretin family of G protein-coupled receptors.

alpha-Latrotoxin (LTX) stimulates massive exocytosis of synaptic vesicles and may help to elucidate the mechanism of regulation of neurosecretion. We have recently isolated latrophilin, the synaptic Ca2+-independent LTX receptor. Now we demonstrate that latrophilin is a novel member of the secretin family of G protein-coupled receptors that are involved in secretion. Northern blot analysis shows that latrophilin message is present only in neuronal tissue. Upon expression in COS cells, the cloned protein is indistinguishable from brain latrophilin and binds LTX with high affinity. Latrophilin physically interacts with a Galphao subunit of heterotrimeric G proteins, because the two proteins co-purify in a two-step affinity chromatography. Interestingly, extracellular domain of latrophilin is homologous to olfactomedin, a soluble neuronal protein thought to participate in odorant binding. Our findings suggest that latrophilin may bind unidentified endogenous ligands and transduce signals into nerve terminals, thus implicating G proteins in the control of synaptic vesicle exocytosis.

Amino Acid Sequence↗

PubMed1996

Isolation and biochemical characterization of a Ca2+-independent alpha-latrotoxin-binding protein.

alpha-Latrotoxin, a black widow spider neurotoxin, can bind to high affinity receptors on the presynaptic plasma membrane and stimulate massive neurotransmitter release in the absence of Ca2+. Neurexins, previously isolated as alpha-latrotoxin receptors, require Ca2+ for their interaction with the toxin and, thus, may not participate in the Ca2+-independent alpha-latrotoxin activity. We now report the isolation of a novel protein that binds alpha-latrotoxin with high affinity in the presence of various divalent cations (Ca2+, Mg2+, Ba2+, and Sr2+) as well as in EDTA. This protein, termed here latrophilin, has been purified from detergent-solubilized bovine brain membranes by affinity chromatography on immobilized alpha-latrotoxin and concentrated on a wheat germ agglutinin affinity column. The single polypeptide chain of latrophilin is N-glycosylated and has an apparent molecular weight of 120,000. Sucrose gradient centrifugations demonstrated that latrophilin and alpha-latrotoxin form a stable equimolar complex. In the presence of the toxin, anti-alpha-latrotoxin antibodies precipitated iodinated latrophilin, whose binding to immobilized toxin was characterized by a dissociation constant of 0.5-0.7 nM. This presumably membrane-bound protein is localized to and differentially distributed among neuronal tissues, with about four times more latrophilin expressed in the cerebral cortex than in the cerebellum; subcellular fractionation showed that the protein is highly enriched in synaptosomal plasma membranes. Our data suggest that latrophilin may represent the Ca2+-independent receptor and/or molecular target for alpha-latrotoxin.

Amino Acid Sequence↗

PubMed1995

Cartography of neurexins: more than 1000 isoforms generated by alternative splicing and expressed in distinct subsets of neurons.

Neurexins, a family of cell surface proteins specific to brain, are transcribed from two promoters in three genes, resulting in three alpha- and three beta-neurexins. In situ hybridization revealed differential but overlapping distributions of neurexin isoforms in different classes of neurons. PCRs demonstrated that alpha-neurexins are alternatively spliced at five canonical positions, and beta-neurexins at two. Characterization of many independent bovine neurexin I alpha cDNAs suggests that different splice sites are used independently. This creates the potential to express more than 1000 distinct neurexin proteins in brain. The splicing pattern is conserved in rat and cow. Thus, in addition to somatic gene rearrangement (immunoglobulins and T cell receptors) and large gene families (odorant receptors), alternative splicing potentially represents a third mechanism for creating a large number of cell surface receptors that are expressed by specific subsets of cells.

Alternative Splicing↗

PubMed1994

Conserved domain structure of beta-neurexins. Unusual cleaved signal sequences in receptor-like neuronal cell-surface proteins.

Neurexins, a family of neuronal cell-surface proteins, consist of the longer alpha-neurexins (I alpha, II alpha, and III alpha) and the shorter beta-neurexins (I beta and II beta) with identical C termini but distinct N termini. alpha-Neurexins have the structure of cell surface receptors, but the membrane topology and conservation of beta-neurexins is unknown. We have now characterized cDNA clones encoding bovine neurexins I beta and III beta, thereby demonstrating the presence of a beta-form for neurexin III and the evolutionary conservation of beta-neurexins in mammals. Similar to alpha-neurexins, beta-neurexins were found to be highly O-glycosylated after expression by transfection in COS cells, suggesting that alpha- and beta-neurexins utilize the same O-glycosylation cassette and have similar transmembrane orientations. To determine if beta-neurexins contain a cleaved or uncleaved signal sequence for membrane translocation, beta-neurexin-IgG fusion proteins were expressed in COS cells, and their N termini were directly sequenced. This revealed that the N terminus of all three beta-neurexins contains an unusual cleaved signal sequence. Together our data show that all known neurexin genes generate alpha and beta forms with similar transmembrane organizations and receptor-like structures. Due to the presence of a long atypical cleaved signal peptide, beta-neurexins contain only a short unique sequence before splicing into the alpha-neurexin sequence. Thus, beta-neurexins are essentially N terminally truncated alpha-neurexins.

Amino Acid Sequence↗

PubMed1993

Cellubrevin is a ubiquitous tetanus-toxin substrate homologous to a putative synaptic vesicle fusion protein.

Tetanus toxin inhibits neurotransmitter release by selectively blocking fusion of synaptic vesicles. Recently tetanus toxin was shown to proteolytically degrade synaptobrevin II (also named VAMP-2), a synaptic vesicle-specific protein, in vitro and in nerve terminals. As targets of tetanus toxin, synaptobrevins probably function in the exocytotic fusion of synaptic vesicles. Here we describe a new synaptobrevin homologue, cellubrevin, that is present in all cells and tissues tested and demonstrate that it is a membrane trafficking protein of a constitutively recycling pathway. Like synaptobrevin II, cellubrevin is proteolysed by tetanus toxin light chain in vitro and after transfection. Our results suggest that constitutive and regulated vesicular pathways use homologous proteins for membrane trafficking, probably for membrane fusion at the plasma membrane, indicating a greater mechanistic and evolutionary similarity between these pathways than previously thought.

Amino Acid Sequence↗

PubMed1993

Neurexin III alpha: extensive alternative splicing generates membrane-bound and soluble forms.

The structure of neurexin III alpha was elucidated from overlapping cDNA clones. Neurexin III alpha is highly homologous to neurexins I alpha and II alpha and shares with them a distinctive domain structure that resembles a cell surface receptor. cDNA cloning and PCR experiments revealed alternative splicing at four positions in the mRNA for neurexin III alpha. Alternative splicing was previously observed at the same positions in either neurexin I alpha or neurexin II alpha or both, suggesting that the three neurexins are subject to extensive alternative splicing. This results in hundreds of different neurexins with variations in small sequences at similar positions in the proteins. The most extensive alternative splicing of neurexin III alpha was detected at its C-terminal site, which exhibits a minimum of 12 variants. Some of the alternatively spliced sequences at this position contain in-frame stop codons, suggesting the synthesis of secreted proteins. None of the sequences of the other splice sites in this or the other two neurexins include stop codons. RNA blot analysis demonstrate that neurexin III alpha is expressed in a brain-specific pattern. Our results suggest that the neurexins constitute a large family of polymorphic cell surface proteins that includes secreted variants, indicating a possible role as signaling molecules.

Alternative Splicing↗

PubMed1993

Polypeptide composition of the alpha-latrotoxin receptor. High affinity binding protein consists of a family of related high molecular weight polypeptides complexed to a low molecular weight protein.

alpha-Latrotoxin is a vertebrate neurotoxin from black widow spider venom that causes massive neurotransmitter release. In order to gain insight into the mechanism of action of alpha-latrotoxin, we have studied alpha-latrotoxin-binding proteins from bovine and rat brain. Proteins purified by affinity chromatography on immobilized alpha-latrotoxin were investigated. Two sets of proteins were isolated: 1) three polypeptides of M(r) 79,000, 65,000, and 43,000 that were eluted from immobilized alpha-latrotoxin by increasing KCl concentrations in the presence of Ca2+, and 2) a family of related proteins ranging in molecular weight from 160,000 to 220,000 and a low molecular weight component of M(r) 29,000 that were eluted from immobilized alpha-latrotoxin only after removal of Ca2+. Amino acid sequences of these proteins demonstrated that all of these proteins represent novel proteins except for the M(r) 65,000 polypeptide, which is identical with synaptotagmin (Petrenko, A. G., Perin, M. S., Davletov, B. A., Ushkaryov, Y. A., Geppert, M., and Südhof, T. C. (1991) Nature 353, 65-68). Surprisingly, the M(r) 79,000 and 43,000 proteins were also found in tissues insensitive to alpha-latrotoxin action. Since these proteins do not bind 125I-alpha-latrotoxin with high affinity, their purification probably is not physiologically significant. On the other hand, the fractions containing the M(r) 160,000-220,000 and 29,000 polypeptides bound alpha-latrotoxin with high affinity. Sucrose gradient centrifugations and anion exchange chromatography suggested that most of the M(r) 160,000-220,000 proteins were complexed with the M(r) 29,000 protein. alpha-Latrotoxin binding correlated with the presence of the M(r) 160,000-220,000 proteins and M(r) 29,000 polypeptide, and alpha-latrotoxin formed stable complexes with the M(r) 160,000-220,000 proteins. Accordingly, the alpha-latrotoxin receptor consists of a high molecular weight protein (M(r) 160,000-220,000) that is complexed with one or several copies of an M(r) 29,000 polypeptide. In addition, the receptor is found in a less tight association with synaptotagmin but not with other polypeptides.

Amino Acid Sequence↗