The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Yuri A. Ushkaryov - One of the best experts on this subject based on the ideXlab platform.
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α Latrotoxin and its receptors
Handbook of experimental pharmacology, 2008Co-Authors: Yuri A. Ushkaryov, Alexis Rohou, Shuzo SugitaAbstract:Alpha-Latrotoxin (Alpha-LTX) from black widow spider venom induces exhaustive release of neurotransmitters from vertebrate nerve terminals and endocrine cells. This 130-kDa protein has been employed for many years as a molecular tool to study exocytosis. However, its action is complex: in neurons, Alpha-LTX induces massive secretion both in the presence of extracellular Ca(2+) (Ca(2+) (e)) and in its absence; in endocrine cells, it usually requires Ca(2+) (e). To use this toxin for further dissection of secretory mechanisms, one needs an in-depth understanding of its functions. One such function that explains some Alpha-LTX effects is its ability to form cation-permeable channels in artificial lipid bilayers. The mechanism of Alpha-LTX pore formation, revealed by cryo-electron microscopy, involves toxin assembly into homotetrameric complexes which harbor a central channel and can insert into lipid membranes. However, in biological membranes, Alpha-LTX cannot exert its actions without binding to specific receptors of the plasma membrane. Three proteins with distinct structures have been found to bind Alpha-LTX: neurexin IAlpha, latrophilin 1, and receptor-like protein tyrosine phosphatase sigma. Upon binding a receptor, Alpha-LTX forms channels permeable to cations and small molecules; the toxin may also activate the receptor. To distinguish between the pore- and receptor-mediated effects, and to study structure-function relationships in the toxin, Alpha-LTX mutants have been used.
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mutant α Latrotoxin ltxn4c does not form pores and causes secretion by receptor stimulation this action does not require neurexins
Journal of Biological Chemistry, 2003Co-Authors: Kirill E. Volynski, C Manser, Elena V. Orlova, A. C. Ashton, Marco Capogna, Derek Thomson, Richard R Ribchester, Yuri A. UshkaryovAbstract: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.
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the Alpha Latrotoxin mutant ltxn4c enhances spontaneous and evoked transmitter release in ca3 pyramidal neurons
The Journal of Neuroscience, 2003Co-Authors: Marco Capogna, Kirill E. Volynski, Nigel J Emptage, Yuri A. UshkaryovAbstract: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.
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the α Latrotoxin mutant ltxn4c enhances spontaneous and evoked transmitter release in ca3 pyramidal neurons
The Journal of Neuroscience, 2003Co-Authors: Marco Capogna, Kirill E. Volynski, Nigel J Emptage, Yuri A. UshkaryovAbstract: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.
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Alpha-Latrotoxin, acting via two Ca2+-dependent pathways, triggers exocytosis of two pools of synaptic vesicles.
The Journal of biological chemistry, 2001Co-Authors: A. C. Ashton, Kirill E. Volynski, Vera G. Lelianova, Elena V. Orlova, Catherine Van Renterghem, Michael Seagar, Marco Canepari, Yuri A. UshkaryovAbstract: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.
Shuzo Sugita - One of the best experts on this subject based on the ideXlab platform.
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α Latrotoxin stimulates a novel pathway of ca2 dependent synaptic exocytosis independent of the classical synaptic fusion machinery
The Journal of Neuroscience, 2009Co-Authors: Mikhail Khvotchev, Shuzo Sugita, Ferenc Deak, Xinran Liu, Ege T KavalaliAbstract:Alpha-Latrotoxin induces neurotransmitter release by stimulating synaptic vesicle exocytosis via two mechanisms: (1) A Ca(2+)-dependent mechanism with neurexins as receptors, in which Alpha-Latrotoxin acts like a Ca(2+) ionophore, and (2) a Ca(2+)-independent mechanism with CIRL/latrophilins as receptors, in which Alpha-Latrotoxin directly stimulates the transmitter release machinery. Here, we show that the Ca(2+)-independent release mechanism by Alpha-Latrotoxin requires the synaptic SNARE-proteins synaptobrevin/VAMP and SNAP-25, and, at least partly, the synaptic active-zone protein Munc13-1. In contrast, the Ca(2+)-dependent release mechanism induced by Alpha-Latrotoxin does not require any of these components of the classical synaptic release machinery. Nevertheless, this type of exocytotic neurotransmitter release appears to fully operate at synapses, and to stimulate exocytosis of the same synaptic vesicles that participate in physiological action potential-triggered release. Thus, synapses contain two parallel and independent pathways of Ca(2+)-triggered exocytosis, a classical, physiological pathway that operates at the active zone, and a novel reserve pathway that is recruited only when Ca(2+) floods the synaptic terminal.
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α Latrotoxin and its receptors
Handbook of experimental pharmacology, 2008Co-Authors: Yuri A. Ushkaryov, Alexis Rohou, Shuzo SugitaAbstract:Alpha-Latrotoxin (Alpha-LTX) from black widow spider venom induces exhaustive release of neurotransmitters from vertebrate nerve terminals and endocrine cells. This 130-kDa protein has been employed for many years as a molecular tool to study exocytosis. However, its action is complex: in neurons, Alpha-LTX induces massive secretion both in the presence of extracellular Ca(2+) (Ca(2+) (e)) and in its absence; in endocrine cells, it usually requires Ca(2+) (e). To use this toxin for further dissection of secretory mechanisms, one needs an in-depth understanding of its functions. One such function that explains some Alpha-LTX effects is its ability to form cation-permeable channels in artificial lipid bilayers. The mechanism of Alpha-LTX pore formation, revealed by cryo-electron microscopy, involves toxin assembly into homotetrameric complexes which harbor a central channel and can insert into lipid membranes. However, in biological membranes, Alpha-LTX cannot exert its actions without binding to specific receptors of the plasma membrane. Three proteins with distinct structures have been found to bind Alpha-LTX: neurexin IAlpha, latrophilin 1, and receptor-like protein tyrosine phosphatase sigma. Upon binding a receptor, Alpha-LTX forms channels permeable to cations and small molecules; the toxin may also activate the receptor. To distinguish between the pore- and receptor-mediated effects, and to study structure-function relationships in the toxin, Alpha-LTX mutants have been used.
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n terminal insertion and c terminal ankyrin like repeats of Alpha Latrotoxin are critical for ca2 dependent exocytosis
The Journal of Neuroscience, 2005Co-Authors: David J Lee, Mikhail Khvotchev, Li Wang, Soon Kwang Chiew, Lakshmanan Arunachalam, Tony J Collins, Zhongping Feng, Shuzo SugitaAbstract:Alpha-Latrotoxin, a potent stimulator of exocytosis from neurons and neuroendocrine cells, has been studied intensively, but the mechanisms of its actions are poorly understood. Here, we developed a new method to generate active recombinant Alpha-Latrotoxin and conducted a structure/function analysis of the toxin in stimulating Ca2+-dependent exocytosis. Alpha-Latrotoxin consists of a conserved N-terminal domain and C-terminal ankyrin-like repeats. After cleavage of an N-terminally fused purification tag of glutathione S-transferase (GST), the recombinant toxin strongly stimulated exocytosis, whereas the GST-fused toxin was much less potent. The GST-fused toxin bound to the receptors [neurexin 1Alpha; CL1 (CIRL/latrophilin 1)] as efficiently as did the GST-cleaved toxin but was much less effective in inserting into the plasma membrane and inducing cation conductance. The toxin with deletion of the last two ankyrin-like repeats still bound the receptors but could neither stimulate exocytosis nor induce cation conductance efficiently. The abilities of the mutated toxins to stimulate exocytosis correlated well with their abilities to induce cation conductance, but not their binding to the receptors. Our results indicate that (1) C-terminal ankyrin-like repeats and a free (unfused) N terminus are both required for the toxin to form pores, which is essential for Ca2+-dependent exocytosis, and (2) receptor binding alone is not sufficient to stimulate Ca2+-dependent exocytosis.
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α Latrotoxin action probed with recombinant toxin receptors recruit α Latrotoxin but do not transduce an exocytotic signal
The EMBO Journal, 1998Co-Authors: Mikhail Khvotchev, Konstantin Ichtchenko, Nikita Kiyatkin, Lance Simpson, Shuzo SugitaAbstract:Alpha-Latrotoxin stimulates neurotransmitter release probably by binding to two receptors, CIRL/latrophilin 1 (CL1) and neurexin IAlpha. We have now produced recombinant Alpha-Latrotoxin (LtxWT) that is as active as native Alpha-Latrotoxin in triggering synaptic release of glutamate, GABA and norepinephrine. We have also generated three Alpha-Latrotoxin mutants with substitutions in conserved cysteine residues, and a fourth mutant with a four-residue insertion. All four Alpha-Latrotoxin mutants were found to be unable to trigger release. Interestingly, the insertion mutant LtxN4C exhibited receptor-binding affinities identical to wild-type LtxWT, bound to CL1 and neurexin IAlpha as well as LtxWT, and similarly stimulated synaptic hydrolysis of phosphatidylinositolphosphates. Therefore, receptor binding by Alpha-Latrotoxin and stimulation of phospholipase C are insufficient to trigger exocytosis. This conclusion was confirmed in experiments with La3+ and Cd2+. La3+ blocked release triggered by LtxWT, whereas Cd2+ enhanced it. Both cations, however, had no effect on the stimulation by LtxWT of phosphatidylinositolphosphate hydrolysis. Our data show that receptor binding by Alpha-Latrotoxin and activation of phospholipase C do not by themselves trigger exocytosis. Thus receptors recruit Alpha-Latrotoxin to its point of action without activating exocytosis. Exocytosis probably requires an additional receptor-independent activity of Alpha-Latrotoxin that is selectively inhibited by the LtxN4C mutation and by La3+.
Alfonso Grasso - One of the best experts on this subject based on the ideXlab platform.
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monoclonal antibody fragment from combinatorial phage display library neutralizes Alpha Latrotoxin activity and abolishes black widow spider venom lethality in mice
Toxicon, 2008Co-Authors: F Bugli, Alfonso Grasso, Rosalia Graffeo, Francesco Paroni Sterbini, Riccardo Torelli, Luca Masucci, Michela Sali, Stefano Rufini, Enzo Ricci, Giovanni FaddaAbstract:Alpha-Latrotoxin (Alpha-ltx), a component of the venom of black widow spiders (BWSV), binds to higher vertebrates presynaptic nerve terminals, stimulating massive neurotransmitter release. This neurotoxic protein is responsible for most of the symptoms elicited in men by the bite of black widow spider (BWS), i.e. a neurological syndrome named latrodectism. By reasoning that targeting this single component would abrogate most of the effect of BWS envenomation, we took advantage of the antibody phage display technology to generate monoclonal Fab fragments able to bind and neutralize the Alpha-ltx. To this aim, we immunized Balb/c mice with purified toxin and cloned their antibody repertoire in the pCombIII phage display vector. By combining a high-stringency affinity selection with a sensitive 45Ca(2+) uptake assay, we isolated a Fab fragment (FM1) able to bind the Alpha-ltx in the low nM range and neutralize its ionophore activity, in vitro and in vivo. After the onset of overt symptomatology, administration of FM1 to experimentally envenomed mice induced remission of symptoms and prevented lethality. Since Alpha-ltx is the only molecule responsible for the great toxicity of BWS bites in mammals, the FM1 Fab, highly effective in neutralizing the toxin in vivo, represents a promising immunotherapy reagent for treating latrodectic patients.
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ca2 independent insulin exocytosis induced by Alpha Latrotoxin requires latrophilin a g protein coupled receptor
The EMBO Journal, 1998Co-Authors: Jochen Lang, Alfonso Grasso, Yuri A. Ushkaryov, Claes B WollheimAbstract:Alpha-Latrotoxin (Alpha-LTX) induces exocytosis of small synaptic vesicles (SSVs) in neuronal cells both by a calcium-independent mechanism and by opening cation-permeable pores. Since the basic molecular events regulating exocytosis in neurons and endocrine cells may be similar, we have used the exocytosis of insulin-containing large dense core vesicles (LDCVs) as a model system. In primary pancreatic beta-cells and in the derived cell lines INS-1 and MIN6, Alpha-LTX increased insulin release in the absence of extracellular calcium, but the insulin-secreting cell lines HIT-T15 and RINm5F were unresponsive. Alpha-LTX did not alter membrane potential or cytosolic calcium, and its stimulatory effect on exocytosis was still observed in pre-permeabilized INS-1 cells kept at 0.1 microM Ca2+. Consequently, pore formation or ion fluxes induced by Alpha-LTX could be excluded. The Ca2+-independent Alpha-LTX-binding protein, latrophilin, is a novel member of the secretin family of G protein-coupled receptors (GPCR). Sensitivity to Alpha-LTX correlated with expression of latrophilin, but not with synaptotagmin I or neurexin IAlpha expression. Moreover, transient expression of latrophilin in HIT-T15 cells conferred Alpha-LTX-induced exocytosis. Our results indicate that direct stimulation of exocytosis by a GPCR mediates the Ca2+-independent effects of Alpha-LTX in the absence of altered ion fluxes. Therefore, direct regulation by receptor-activated heterotrimeric G proteins constitutes an important feature of the endocrine exocytosis of insulin-containing LDCVs and may also apply to SSV exocytosis in neurons.
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The cloning of a cDNA encoding a protein (latrodectin) which co-purifies with the Alpha-Latrotoxin from the black widow spider Latrodectus tredecimguttatus (Theridiidae).
European journal of biochemistry, 1995Co-Authors: Mario Pescatori, Alessandro Mastrogiacomo, Andrew Bradbury, Françoise Bouet, Nicola Gargano, Alfonso GrassoAbstract:A cDNA encoding a polypeptide of 88 amino acids was cloned following the rapid amplification of cDNA ends (RACE) procedure using mRNA isolated from the venom glands of the Mediterranean black widow spider (Latrodectus tredecimguttatus) and oligonucleotides based on the sequence of a tryptic fragment putatively from Alpha-Latrotoxin. Apart from a potential signal peptide, the rest of this small protein, named latrodectin, was highly hydrophilic, having a calculated molecular mass of 7945 Da and a pI of 4.3. Northern-blot analysis showed that the mRNA was specifically expressed in the venom gland of L. tredecimguttatus and that it was well conserved between two geographically remote species (L. geometricus and L. indistinctus). A polyclonal serum raised in rabbits against the C-terminal sequence of latrodectin detected cross-reactive proteins in the venom fluid, venom gland extracts, and in purified Alpha-Latrotoxin, suggesting that latrodectin is intimately associated with Alpha-Latrotoxin. Finally, we produced a recombinant protein in a cell system infected with baculovirus and developed an immunoaffinity purification procedure for latrodectin to facilitate further structural and functional analyses of the molecule.
Kirill E. Volynski - One of the best experts on this subject based on the ideXlab platform.
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mutant α Latrotoxin ltxn4c does not form pores and causes secretion by receptor stimulation this action does not require neurexins
Journal of Biological Chemistry, 2003Co-Authors: Kirill E. Volynski, C Manser, Elena V. Orlova, A. C. Ashton, Marco Capogna, Derek Thomson, Richard R Ribchester, Yuri A. UshkaryovAbstract: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.
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the α Latrotoxin mutant ltxn4c enhances spontaneous and evoked transmitter release in ca3 pyramidal neurons
The Journal of Neuroscience, 2003Co-Authors: Marco Capogna, Kirill E. Volynski, Nigel J Emptage, Yuri A. UshkaryovAbstract: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.
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the Alpha Latrotoxin mutant ltxn4c enhances spontaneous and evoked transmitter release in ca3 pyramidal neurons
The Journal of Neuroscience, 2003Co-Authors: Marco Capogna, Kirill E. Volynski, Nigel J Emptage, Yuri A. UshkaryovAbstract: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.
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Alpha-Latrotoxin, acting via two Ca2+-dependent pathways, triggers exocytosis of two pools of synaptic vesicles.
The Journal of biological chemistry, 2001Co-Authors: A. C. Ashton, Kirill E. Volynski, Vera G. Lelianova, Elena V. Orlova, Catherine Van Renterghem, Michael Seagar, Marco Canepari, Yuri A. UshkaryovAbstract: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.
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Latrophilin, Neurexin, and Their Signaling-deficient Mutants Facilitate α-Latrotoxin Insertion into Membranes but Are Not Involved in Pore Formation
The Journal of biological chemistry, 2000Co-Authors: Kirill E. Volynski, Eugene V. Grishin, Frederic A. Meunier, Vg Lelianova, Ee Dudina, T. M. Volkova, Ma Rahman, C Manser, Jo Dolly, Rh AshleyAbstract: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.
Angela Vincent - One of the best experts on this subject based on the ideXlab platform.
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miller fisher anti gq1b antibodies Alpha Latrotoxin like effects on motor end plates
Annals of Neurology, 1999Co-Authors: Jaap J Plomp, Peter C M Molenaar, Graham M Ohanlon, Bart C Jacobs, J Veitch, M R Daha, P A Van Doorn, F G A Van Der Meche, Angela Vincent, B P MorganAbstract:In the Miller Fisher syndrome (MFS) variant of the Guillain-Barre syndrome, weakness is restricted to extraocular muscles and occasionally other craniobulbar muscles. Most MFS patients have serum antibodies against ganglioside type GQ1b of which the pathophysiological relevance is unclear. We examined the in vitro effects of MFS sera, MFS IgG, and a human monoclonal anti-GQ1b IgM antibody on mouse neuromuscular junctions (NMJs). It was found that anti-GQ1b antibodies bind at NMJs where they induce massive quantal release of acetylcholine from nerve terminals and eventually block neuromuscular transmission. This effect closely resembled the effect of the paralytic neurotoxin Alpha-Latrotoxin at the mouse NMJs, implying possible involvement of Alpha-Latrotoxin receptors or associated downstream pathways. By using complement-deficient sera, the effect of anti-GQ1b antibodies on NMJs was shown to be entirely dependent on activation of complement components. However, neither classical pathway activation nor the formation of membrane attack complex was required, indicating the effects could be due to involvement of the alternative pathway and intermediate complement cascade products. Our findings strongly suggest that anti-GQ1b antibodies in conjunction with activated complement components are the principal pathophysiological mediators of motor symptoms in MFS and that the NMJ is an important site of their action.
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lack of effect of miller fisher sera plasmas on transmitter release from pc12 cells
Journal of Neuroimmunology, 1997Co-Authors: Michael Benatar, Hugh J Willison, Angela VincentAbstract:IgG antibodies to GQ1b ganglioside are found in > 90% of patients with the Miller Fisher Syndrome (MFS). MFS sera or IgG preparations have marked effects on neurotransmitter release at the neuromuscular junction, but their mode(s) of action remain unclear. To establish a cell-based system for investigating the mechanism of action of MFS serum preparations, we looked at neurotransmitter release from three cell lines. We failed to demonstrate substantial 14C-acetylcholine release from two motor-neuronal cell lines, VSC4.1 and NSC19, and therefore studied 3H-noradrenaline release from NGF-differentiated PC12 cells, a neural-crest derived catecholaminergic cell line. K(+)-induced release was inhibited by botulinum toxin and basal release was enhanced by Alpha-Latrotoxin, resembling that at the neuromuscular junction, although K(+)-induced release was dependent on L-type rather than P/Q-type calcium channels. The cells expressed polysialylated gangliosides on the cell surface. Incubation in heat-inactivated or untreated MFS preparations did not, however, affect basal or K(+)-induced release. Thus the PC12 cells do not appear to be sensitive to the effects of serum antibodies from MFS patients.