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.

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.

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.

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.

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.

Bipartite Ca2+-binding motif in C2 domains of synaptotagmin and protein kinase C.

C2 domains are found in many proteins involved in membrane traffic or signal transduction. Although C2 domains are thought to bind calcium ions, the structural basis for calcium binding is unclear. Analysis of calcium binding to C2 domains of synaptotagmin I and protein kinase C-beta by nuclear magnetic resonance spectroscopy revealed a bipartite calcium-binding motif that involves the coordination of two calcium ions by five aspartate residues located on two separate loops. Sequence comparisons indicated that this may be a widely used calcium-binding motif, designated here as the C2 motif.

Distinct Ca2+ and Sr2+ binding properties of synaptotagmins. Definition of candidate Ca2+ sensors for the fast and slow components of neurotransmitter release.

Ca(2+)-dependent neurotransmitter release consists of at least two components: a major fast component that is insensitive to Sr2+ and a minor slow component that is potentiated by Sr2+ (Goda, Y., and Stevens, C. F. (1994) Proc. Natl. Acad. U. S. A. 91, 12942-12946). These results suggest that at least two Ca2+ sensors act in synaptic vesicle fusion with distinct Ca2+ and Sr2+ binding properties. We have now investigated the relative Ca2+ and Sr2+ binding activities of synaptotagmins to evaluate their potential roles as Ca2+ sensors for the fast and slow components. Our results demonstrate that the first C2 domains of synaptotagmins I, II, III, V, and VII have very similar Ca2+ requirements for phospholipid binding (range of EC50 = 2.6 microM to 5.0 microM), but distinct Sr2+ requirements (EC50 range = 23 microM to 133 microM); synaptotagmins I and II had the lowest Sr2+ affinity, and synaptotagmin III the highest Sr2+ affinity. Purified synaptotagmin I from bovine brain exhibited similar properties as its recombinant first C2 domain, suggesting that the first C2 domain fully accounts for its Ca(2+)-dependent phospholipid binding properties. Sr2+ was unable to trigger syntaxin binding by synaptotagmin I at all concentrations tested, whereas it was effective for synaptotagmin III. These results suggest that different C2 domains have distinct Sr2+ binding properties. They support the hypothesis that synaptotagmins localized on the same vesicle perform distinct functions, with synaptotagmins I and II serving as candidate Ca2+ sensors for the fast component in release and synaptotagmin III for the slow component.

High affinity binding of alpha-latrotoxin to recombinant neurexin I alpha.

alpha-Latrotoxin is a potent neurotoxin from black widow spider venom that stimulates neurotransmitter release. alpha-Latrotoxin is thought to act by binding to a high affinity receptor on presynaptic nerve terminals. In previous studies, high affinity alpha-latrotoxin binding proteins were isolated and demonstrated to contain neurexin I alpha as a major component. Neurexin I alpha is a cell surface protein that exists in multiple differentially spliced isoforms and belongs to a large family of neuron-specific proteins. Using a series of neurexin I-IgG fusion proteins, we now show that recombinant neurexin I alpha binds alpha-latrotoxin directly with high affinity (Kd approximately 4 nM). Binding of alpha-latrotoxin to recombinant neurexin I alpha is dependent on Ca2+ (EC50 approximately 30 microM). Our data suggest that neurexin I alpha is a Ca(2+)-dependent high affinity receptor for alpha-latrotoxin.

Structure of the first C2 domain of synaptotagmin I: a novel Ca2+/phospholipid-binding fold.

C2 domains are regulatory sequence motifs that occur widely in nature. Synaptotagmin I, a synaptic vesicle protein involved in the Ca2+ regulation of exocytosis, contains two C2 domains, the first of which acts as a Ca2+ sensor. We now describe the three-dimensional structure of this C2 domain at 1.9 A resolution in both the Ca(2+)-bound and Ca(2+)-free forms. The C2 polypeptide forms an eight-stranded beta sandwich constructed around a conserved four-stranded motif designated as a C2 key. Ca2+ binds in a cup-shaped depression between two polypeptide loops located at the N- and C-termini of the C2-key motif.

Ca(2+)-dependent conformational change in synaptotagmin I.

Synaptotagmin I is a Ca2+/phospholipid binding protein of synaptic vesicles with a proposed function as a Ca2+ sensor in synaptic vesicle exocytosis. Using controlled partial proteolysis as an assay, we now show that synaptotagmin I undergoes a conformational change as a function of Ca2+ binding. As observed for phospholipid binding, Ba2+ and Sr2+ but not Mg2+, substitute for Ca2+ in effecting this conformational change. The first C2 domain from synaptotagmin I that represents the Ca(2+)-dependent phospholipid binding domain of synaptotagmin also undergoes a Ca(2+)-dependent change in controlled partial proteolysis. In contrast, no effect of Ca2+ was observed with mutant C2 domains containing point mutations that abolish Ca2+ binding. The Ca2+ concentration dependence of the effect of Ca2+ on proteolysis mirrors the Ca2+ dependence of phospholipid binding. The conformational shift in synaptotagmin I caused by Ca2+/phospholipid binding could be the basis for its Ca(2+)-regulated function in triggering neurotransmitter release.

Synaptotagmin I is a high affinity receptor for clathrin AP-2: implications for membrane recycling.

In nerve terminals, Ca(2+)-stimulated synaptic vesicle exocytosis is rapidly followed by endocytosis. Synaptic vesicle endocytosis requires clathrin-coated pits similar to receptor-mediated endocytosis in fibroblasts. Binding of clathrin AP-2 (adaptor complex) to an unidentified high affinity membrane receptor appears to be necessary for coated pit assembly in fibroblasts. We now show that synaptic vesicles have a high affinity AP-2 site (KD, approximately 1 x 10(-10) M) similar to the one observed in fibroblasts. Using a combination of competition and direct binding assays, we demonstrate that synaptotagmin I, an intrinsic membrane protein of synaptic vesicles, has all of the properties of the AP-2 receptor and that AP-2 binds to the second C2 domain in the molecule. Thus, synaptotagmin I may be a multifunctional protein with a function in endocytosis in addition to the previously proposed role in exocytosis.

A single C2 domain from synaptotagmin I is sufficient for high affinity Ca2+/phospholipid binding.

Synaptotagmin I is a Ca(2+)- and phospholipid-binding protein of synaptic vesicles with an essential function in neurotransmission. Ca2+/phospholipid binding by synaptotagmin I may be mediated by its C2 domains, sequence motifs that have been implicated in the Ca2+ regulation of a variety of proteins. However, it is currently unknown if C2 domains are sufficient for Ca2+/phospholipid binding or if they even directly participate in Ca2+/phospholipid binding. In order to address this question, we have studied the Ca2+/phospholipid-binding properties of the first C2 domain of synaptotagmin I. Our results show that this C2 domain by itself binds Ca2+ and phospholipids with high affinity (half-maximal binding at 4-6 microM free Ca2+) and exhibits strong positive cooperativity. The C2 domain is specific for negatively charged phospholipids and for those divalent cations that are known to stimulate synaptic vesicle exocytosis (Ca2+ > Sr2+, Ba2+ >>> Mg2+). These studies establish that C2 domains can serve as independently folding Ca2+/phospholipid-binding domains. Furthermore, the cation specificity and the cooperativity of Ca2+ binding by the C2 domain from synaptotagmin I support a role for this protein in mediating the Ca2+ signal in neurotransmitter release.

Binding of synaptotagmin to the alpha-latrotoxin receptor implicates both in synaptic vesicle exocytosis.

A vertebrate neurotoxin, alpha-latrotoxin, from black widow spider venom causes synaptic vesicle exocytosis and neurotransmitter release from presynaptic nerve terminals. Although the mechanism of action of alpha-latrotoxin is not known, it does require binding of alpha-latrotoxin to a high-affinity receptor on the presynaptic plasma membrane. The alpha-latrotoxin receptor seems to be exclusively at the presynaptic plasmamembrane. Here we report that the alpha-latrotoxin receptor specifically binds to a synaptic vesicle protein, synaptotagmin, and modulates its phosphorylation. Synaptotagmin is a synaptic vesicle-specific membrane protein that binds negatively charged phospholipids and contains two copies of a putative Ca(2+)-binding domain from protein kinase C (the C2-domain), suggesting a regulatory role in synaptic vesicle fusion. Our findings suggest that a physiological role of the alpha-latrotoxin receptor may be the docking of synaptic vesicles at the active zone. The direct interaction of the alpha-latrotoxin receptor with a synaptic vesicle protein also suggests a mechanism of action for this toxin in causing neurotransmitter release.

Human adrenoceptor system response to the development of high altitude pulmonary arterial hypertension.

High-altitude pulmonary arterial hypertension affects people with a long-term residence at altitudes over 2500 m above the sea level and is characterized by elevated pulmonary artery pressure (over 35/15 mmHg). In order to assess the role of beta-adrenoreceptors in the development of high-altitude pulmonary arterial hypertension, we studied the beta-adrenoreceptor density on mononuclear leukocytes in Kirghiz male natives of Eastern Pamir (3600-4200 m above sea level) with the diagnosis of high-altitude pulmonary arterial hypertension, and in healthy men. It was shown that patients with signs of right ventricular hypertrophy (RVH) of the second and third grade have beta-adrenoreceptor density 4.5 times lower than control (2.27 +/- 0.22 vs. 9.85 +/- 1.28 fmol/10(6) cells). Values of Kd also proved to be lower, by 2.5 times (0.57 +/- 0.14 vs. 1.44 +/- 0.18 nM). Stimulation of adenylate cyclase by isoproterenol and other beta-agonists was lower in patients than in controls (+33% and +120%, respectively). These results demonstrate that the desensitization of beta-adrenoreceptors is present in patients with high-altitude pulmonary arterial hypertension associated with severe right ventricular hypertrophy. Patients with pulmonary arterial hypertension due to mitral stenosis do not have any signs of beta-adrenoreceptor desensitization associated with high plasma levels of catecholamines.