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 α-latrotoxin (αLTX), a representative venom component toxic to vertebrates. This reconstruction explains the mechanism of αLTX 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 αLTX. Latrotoxins act by several Ca
2+-dependent and -independent mechanisms based on pore formation and activation of receptors. Mutant recombinant αLTX 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 αLTX.
Heptahelical, or G‐protein‐coupled, receptors control many cellular functions and normally consist of one polypeptide chain. In contrast, heptahelical receptors that belong to the long N‐terminus, ...group B (LNB) family are cleaved constitutively into two fragments. The N‐terminal fragments (NTFs) resemble cell‐adhesion proteins and the C‐terminal fragments (CTFs) are typical G‐protein‐coupled receptors (GPCRs) with seven transmembrane regions. However, the functional roles of this cleavage and of any subsequent NTF–CTF interactions remain to be identified. Using latrophilin, a well‐studied member of the LNB family, we now demonstrate that cleavage is critical for delivery of this receptor to the cell surface. On the plasma membrane, NTF and CTF behave as separate membrane proteins involved, respectively, in cell‐surface reception and signalling. The two fragments can also internalise independently. However, separated NTF and CTF can re‐associate on solubilisation. Agonist binding to NTF on the cell surface also induces re‐association of fragments and provokes signal transduction via CTF. These findings define a novel principle of structural and functional organisation of the cleaved, two‐subunit GPCRs.
Alpha-latrotoxin (LTX) stimulates vesicular exocytosis by at least two mechanisms that include (1) receptor binding-stimulation and (2) membrane pore formation. Here, we use the toxin mutant LTX(N4C) ...to selectively study the receptor-mediated actions of LTX. LTX(N4C) binds to both LTX receptors (latrophilin and neurexin) and greatly enhances the frequency of spontaneous and miniature EPSCs recorded from CA3 pyramidal neurons in hippocampal slice cultures. The effect of LTX(N4C) is reversible and is not attenuated by La3+ that is known to block LTX pores. On the other hand, LTX(N4C) action, which requires extracellular Ca2+, is inhibited by thapsigargin, a drug depleting intracellular Ca2+ stores, by 2-aminoethoxydiphenyl borate, a blocker of inositol(1,4,5)-trisphosphate-induced Ca2+ release, and by U73122, a phospholipase C inhibitor. Furthermore, measurements using a fluorescent Ca2+ indicator directly demonstrate that LTX(N4C) increases presynaptic, but not dendritic, free Ca2+ concentration; this Ca2+ rise is blocked by thapsigargin, suggesting, together with electrophysiological data, that the receptor-mediated action of LTX(N4C) involves mobilization of Ca2+ from intracellular stores. Finally, in contrast to wild-type LTX, which inhibits evoked synaptic transmission probably attributable to pore formation, LTX(N4C) actually potentiates synaptic currents elicited by electrical stimulation of afferent fibers. We suggest that the mutant LTX(N4C), lacking the ionophore-like activity of wild-type LTX, activates a presynaptic receptor and stimulates Ca2+ release from intracellular stores, leading to the enhancement of synaptic vesicle exocytosis.
alpha-Latrotoxin stimulates three types of (3)Hgamma-aminobutyric acid and (14)Cglutamate 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.
Alpha-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 (LTXN4C) 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 LTXN4C triggers exocytosis. An interesting feature of this receptor-mediated transmitter release is its dependence on extracellular Ca2+. Because Ca2+ is also strictly required for LTX interaction with neurexin, the latter might be the only receptor mediating the LTXN4C action. To test this hypothesis, we used conditions (substitution of Ca2+ in the medium with Sr2+) under which LTXN4C does not bind to any member of the neurexin family but still interacts with latrophilin. We show that, in all the systems tested, Sr2+ fully replaces Ca2+ in supporting the stimulatory effect of LTXN4C. These results indicate that LTXN4C can cause neurotransmitter release just by stimulating a receptor and that neurexins are not critical for this receptor-mediated action.
Pure α-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 α-latrotoxin pores are permeable to Ca2+ and small molecules including fluorescein isothiocyanate and norepinephrine. Bound α-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 Ca2+). Interestingly, on subsequent removal of Ca2+, α-latrotoxin dissociates from neurexin but remains in the membrane and continues to form pores. These receptor-independent pores are inhibited by anti-α-latrotoxin antibodies. Our results indicate that (i) α-latrotoxin is a pore-forming toxin, (ii) receptors that bind α-latrotoxin facilitate its insertion into the membrane, (iii) the receptors are not physically involved in the pore structure, (iv) α-latrotoxin pores may be independent of the receptors, and (v) pore formation does not require α-latrotoxin interaction with other neuronal proteins.
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.
alpha -Latrotoxin (LTX) stimulates vesicular exocytosis by at least two mechanisms that include (1) receptor binding-stimulation and (2) membrane pore formation. Here, we use the toxin mutant LTX ...super(N4C) to selectively study the receptor-mediated actions of LTX. LTX super(N4C) binds to both LTX receptors (latrophilin and neurexin) and greatly enhances the frequency of spontaneous and miniature EPSCs recorded from CA3 pyramidal neurons in hippocampal slice cultures. The effect of LTX super(N4C) is reversible and is not attenuated by La super(3+) that is known to block LTX pores. On the other hand, LTX super(N4C) action, which requires extracellular Ca super(2+), is inhibited by thapsigargin, a drug depleting intracellular Ca super(2+) stores, by 2-aminoethoxydiphenyl borate, a blocker of inositol(1,4,5)-trisphosphate-induced Ca super(2+) release, and by U73122, a phospholipase C inhibitor. Furthermore, measurements using a fluorescent Ca super(2+) indicator directly demonstrate that LTX super(N4C) increases presynaptic, but not dendritic, free Ca super(2+) concentration; this Ca super(2+) rise is blocked by thapsigargin, suggesting, together with electrophysiological data, that the receptor-mediated action of LTX super(N4C) involves mobilization of Ca super(2+) from intracellular stores. Finally, in contrast to wild-type LTX, which inhibits evoked synaptic transmission probably attributable to pore formation, LTX super(N4C) actually potentiates synaptic currents elicited by electrical stimulation of afferent fibers. We suggest that the mutant LTX super(N4C), lacking the ionophore-like activity of wild-type LTX, activates a presynaptic receptor and stimulates Ca super(2+) release from intracellular stores, leading to the enhancement of synaptic vesicle exocytosis.
To facilitate the study of the mechanism of α-latrotoxin action, it is necessary to create a biologically active recombinant toxin. Mature α-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 α-latrotoxin. This was partially purified and proved indistinguishable from the natural toxin with respect to its molecular mass, immunostaining, toxicity to mice, binding to α-latrotoxin receptors (latrophilin or neurexin Iα) and electrophysiological recording in the mouse diaphragm. The successful expression of recombinant α-latrotoxin permits mutational analysis of the toxin.
alpha -Latrotoxin stimulates three types of super(3)H gamma -aminobutyric acid and super(14)Cglutamate release from synaptosomes. The Ca super(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 super(2+)-dependent and vesicular but rely on distinct mechanisms. The fast receptor-mediated pathway (ii) involves intracellular Ca super(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 super(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 super(N4C)) cannot form pores and stimulates only the Ca super(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.
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