α-Latrotoxin (LTX) stimulates massive neurotransmitter release by two mechanisms: Ca 2⍣ -dependent and -independent. Our studies on norepinephrine secretion from nerve terminals now reveal the different molecular basis of these two actions. The Ca 2⍣ -dependent LTX-evoked vesicle exocytosis (abolished by botulinum neurotoxins) is 10-fold more sensitive to external Ca 2⍣ than secretion triggered by depolarization or A23187; it does not, however, depend on the cation entry into terminals but requires intracellular Ca 2⍣ and is blocked by drugs depleting Ca 2⍣ 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 Ca 2⍣ -independent LTX-stimulated release is not inhibited by botulinum neurotoxins or drugs interfering with Ca 2⍣ 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 LTXstimulated exocytosis depends upon the co-operative action of external and intracellular Ca 2⍣ involving G proteins and PLC, whereas the Ca 2⍣ -independent release is largely non-vesicular.

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␣-Latrotoxin (LTX) causes massive release of neurotransmitters via a complex mechanism involving (i) activation of receptor(s) and (ii) toxin insertion into the plasma membrane with (iii) subsequent pore formation. Using cryo-electron microscopy, electrophysiological and biochemical methods, we demonstrate here that the recently described toxin mutant (LTX N4C ) is unable to insert into membranes and form pores due to its inability to assemble into tetramers. However, this mutant still binds to major LTX receptors (latrophilin and neurexin) and causes strong transmitter exocytosis in synaptosomes, hippocampal slice cultures, neuromuscular junctions, and chromaffin cells. In the absence of mutant incorporation into the membrane, receptor activation must be the only mechanism by which LTX N4C triggers exocytosis. An interesting feature of this receptormediated transmitter release is its dependence on extracellular Ca 2؉ . Because Ca 2؉ is also strictly required for LTX interaction with neurexin, the latter might be the only receptor mediating the LTX N4C action. To test this hypothesis, we used conditions (substitution of Ca 2؉ in the medium with Sr 2؉ ) under which LTX N4C does not bind to any member of the neurexin family but still interacts with latrophilin. We show that, in all the systems tested, Sr 2؉ fully replaces Ca 2؉ in supporting the stimulatory effect of LTX N4C . These results indicate that LTX N4C can cause neurotransmitter release just by stimulating a receptor and that neurexins are not critical for this receptor-mediated action.␣-Latrotoxin (LTX) 1 stimulates exhaustive release of neurotransmitters in vertebrates. This toxin has been extensively used to probe molecular mechanisms that control exocytosis of both synaptic vesicles (SVs) and large dense-core vesicles (LDCVs) in such diverse models as brain, neuromuscular junctions, and endocrine cells (for reviews see Refs. 1-3).LTX acts only after binding to presynaptic receptors (4). Once receptor-bound, the toxin can trigger exocytosis by several mechanisms: (i) activation of the receptors (5-8), (ii) formation of non-selective pores in the membrane (9 -11), and (iii) hypothetical intracellular interaction with the exocytotic machinery (12).Because toxin pores damage cell membranes and produce strong cytotoxic effects (e.g. 13-15), only the receptor-transduced LTX action is likely to reveal intact, physiologically important exocytotic mechanisms. Unfortunately, this action is difficult to study using the wild-type LTX (LTX WT ) because it easily inserts into membranes and forms ionic pores (9 -11, 16). This problem could be overcome by designing LTX mutants that would lack the propensity of membrane insertion, and a promising toxin variant has been described recently (LTX N4C ) (17). This mutant had the same affinity for the receptors as LTX WT (17) but failed to form pores in synaptosomes or receptor-transfected BHK cells (8). Lacking the major (ionophore) activity, LTX N4C was originally thought to be altogether inactive (12, 17). However, we h...

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.

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.

Under optimised conditions for intoxication, botulinum neurotoxin type A was shown to inhibit approximately 90% of Ca2+-dependent K+-evoked release of [3H]acetylcholine, [3H]noradrenaline, and [3H]dopamine from rat cerebrocortical synaptosomes; cholinergic terminals were most susceptible. In each case, the dose-response curve for the neurotoxin was extended, with about 50% of evoked release being inhibited at approximately 10 nM whereas 200 nM was required for the maximal blockade. This may suggest some heterogeneity in the release process. The action of the toxin was time and temperature dependent and appeared to involve binding and sequestration steps prior to blockade of release. The neurotoxin failed to exert any effect on synaptosomal integrity or on Ca2+-independent release of the transmitters tested; it produced only minimal changes in neurotransmitter uptake although small secondary effects were detected with cholinergic terminals. Blockade by the neurotoxin of Ca2+-dependent resting release of transmitter was apparent; Sr2+, Ba2+, or high concentrations of Ca2+ restored the resting release of 3H-catecholamine but not [3H]acetylcholine. Interestingly, none of the latter conditions or 4-aminopyridine could reverse the toxin-induced blockade of evoked release. This lack of specificity in its action on synaptosomes, and other published findings, lead to the conclusion that toxin-sensitive component(s) exist in all nerve terminals that are concerned with transmitter release.