The Experts below are selected from a list of 285 Experts worldwide ranked by ideXlab platform
Thomas C Sudhof - One of the best experts on this subject based on the ideXlab platform.
-
High Affinity Neurexin Binding to Cell Adhesion G-protein-coupled Receptor CIRL1/Latrophilin-1 Produces an Intercellular Adhesion Complex *
2013Co-Authors: Antony A. Boucard, Thomas C SudhofAbstract:Background: Neurexins and CIRL/latrophilin-1 (CL1) are independent synaptic receptors for �-Latrotoxin. Results: Neurexins and CL1 form a high affinity complex that mediates intercellular adhesion and is regulated by neurexin alternative splicing. Conclusion: Thus, two independent �-Latrotoxin receptors interact trans-cellularly to form a connection between neurons. Significance: The neurexin-CL1 complex may be involved in trans-synaptic cell adhesion and mediate �-Latrotoxin toxicity. The G-protein-coupled receptor CIRL1/latrophilin-1 (CL1) and the type-1 membrane proteins neurexins represent distinct neuronal cell adhesion molecules that exhibit no similarities except for one common function: both proteins are receptors for �-Latrotoxin, a component of black widow spider venom that induces massive neurotransmitter release at synapses. Unexpectedly, we have now identified a direct binding interaction between the extracellular domains of CL1 and neurexins tha
-
α Latrotoxin stimulates a novel pathway of ca2 dependent synaptic exocytosis independent of the classical synaptic fusion machinery
The Journal of Neuroscience, 2009Co-Authors: Thomas C Sudhof, 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.
-
genetic analysis of α Latrotoxin receptors reveals functional interdependence of cirl latrophilin 1 and neurexin 1α
Journal of Biological Chemistry, 2002Co-Authors: Sönke Tobaben, Thomas C Sudhof, Bernd StahlAbstract:α-Latrotoxin triggers massive neurotransmitter release from nerve terminals by binding to at least two distinct presynaptic receptors, neurexin 1α and CIRL1/latrophilin1 (CL1). We have now generated knockout (KO) mice that lack CL1 and analyzed them alone or in combination with neurexin 1α KO mice. Mice lacking only CL1, or both CL1 and neurexin 1α, were viable and fertile. Ca2+-independent binding of α-Latrotoxin to brain membranes was impaired similarly in CL1 single and in CL1/neurexin 1α double KO mice (∼75% decrease) but not in neurexin 1α single KO mice. In contrast, Ca2+-dependent binding (∼2 times above Ca2+-independent binding) was altered in both CL1 (∼50% decrease) and neurexin 1α single KO mice (∼25% decrease) and was decreased further in double KO mice (∼75% decrease). Synaptosomes lacking CL1 exhibited the same decrease in α-Latrotoxin-stimulated glutamate release in the presence and absence of Ca2+(∼75%). In contrast, synaptosomes lacking neurexin 1α exhibited only a small decrease in α-Latrotoxin-triggered release in the absence of Ca2+ (∼20%) but a major decrease in the presence of Ca2+ (∼75%). Surprisingly, synaptosomes lacking both CL1 and neurexin 1α displayed a relatively smaller decrease in α-Latrotoxin-stimulated glutamate release than synaptosomes lacking only CL1 in the absence of Ca2+ (∼50versus ∼75%), but the same decrease in the presence of Ca2+ (∼75%). Our data suggest the following two major conclusions. 1) CL1 and neurexin 1α together account for the majority (75%) of α-Latrotoxin receptors in brain, with the remaining receptor activity possibly due to other CL and neurexin isoforms, and 2) the two receptors act additively in binding α-Latrotoxin but not in triggering release. Together these data suggest that the two receptors act autonomously in binding of α-Latrotoxin but cooperatively in transducing the stimulation of neurotransmitter release by α-Latrotoxin.
-
Genetic analysis of alpha-Latrotoxin receptors reveals functional interdependence of CIRL/latrophilin 1 and neurexin 1 alpha
The Journal of biological chemistry, 2001Co-Authors: Sönke Tobaben, Thomas C Sudhof, Bernd StahlAbstract:Abstract α-Latrotoxin triggers massive neurotransmitter release from nerve terminals by binding to at least two distinct presynaptic receptors, neurexin 1α and CIRL1/latrophilin1 (CL1). We have now generated knockout (KO) mice that lack CL1 and analyzed them alone or in combination with neurexin 1α KO mice. Mice lacking only CL1, or both CL1 and neurexin 1α, were viable and fertile. Ca2+-independent binding of α-Latrotoxin to brain membranes was impaired similarly in CL1 single and in CL1/neurexin 1α double KO mice (∼75% decrease) but not in neurexin 1α single KO mice. In contrast, Ca2+-dependent binding (∼2 times above Ca2+-independent binding) was altered in both CL1 (∼50% decrease) and neurexin 1α single KO mice (∼25% decrease) and was decreased further in double KO mice (∼75% decrease). Synaptosomes lacking CL1 exhibited the same decrease in α-Latrotoxin-stimulated glutamate release in the presence and absence of Ca2+(∼75%). In contrast, synaptosomes lacking neurexin 1α exhibited only a small decrease in α-Latrotoxin-triggered release in the absence of Ca2+ (∼20%) but a major decrease in the presence of Ca2+ (∼75%). Surprisingly, synaptosomes lacking both CL1 and neurexin 1α displayed a relatively smaller decrease in α-Latrotoxin-stimulated glutamate release than synaptosomes lacking only CL1 in the absence of Ca2+ (∼50versus ∼75%), but the same decrease in the presence of Ca2+ (∼75%). Our data suggest the following two major conclusions. 1) CL1 and neurexin 1α together account for the majority (75%) of α-Latrotoxin receptors in brain, with the remaining receptor activity possibly due to other CL and neurexin isoforms, and 2) the two receptors act additively in binding α-Latrotoxin but not in triggering release. Together these data suggest that the two receptors act autonomously in binding of α-Latrotoxin but cooperatively in transducing the stimulation of neurotransmitter release by α-Latrotoxin.
-
α-Latrotoxin and Its Receptors: Neurexins and CIRL/Latrophilins
Annual review of neuroscience, 2001Co-Authors: Thomas C SudhofAbstract:▪ Abstract α-Latrotoxin, a potent neurotoxin from black widow spider venom, triggers synaptic vesicle exocytosis from presynaptic nerve terminals. α-Latrotoxin is a large protein toxin (120 kDa) that contains 22 ankyrin repeats. In stimulating exocytosis, α-Latrotoxin binds to two distinct families of neuronal cell-surface receptors, neurexins and CLs (Cirl/latrophilins), which probably have a physiological function in synaptic cell adhesion. Binding of α-Latrotoxin to these receptors does not in itself trigger exocytosis but serves to recruit the toxin to the synapse. Receptor-bound α-Latrotoxin then inserts into the presynaptic plasma membrane to stimulate exocytosis by two distinct transmitter-specific mechanisms. Exocytosis of classical neurotransmitters (glutamate, GABA, acetylcholine) is induced in a calcium-independent manner by a direct intracellular action of α-Latrotoxin, while exocytosis of catecholamines requires extracellular calcium. Elucidation of precisely how α-Latrotoxin works is likely ...
Alexander G. Petrenko - One of the best experts on this subject based on the ideXlab platform.
-
Protein-tyrosine Phosphatase-ς Is a Novel Member of the Functional Family of α-Latrotoxin Receptors
The Journal of biological chemistry, 2002Co-Authors: Valery Krasnoperov, Konstantin Ichtchenko, Mary A. Bittner, Ronald W. Holz, Leonid Buryanovsky, Thomas A. Neubert, Alexander G. PetrenkoAbstract:Abstract Receptor-like protein-tyrosine phosphatase sigma (PTPς) is essential for neuronal development and function. Here we report that PTPς is a target of α-Latrotoxin, a strong stimulator of neuronal exocytosis. α-Latrotoxin binds to the cell adhesion-like extracellular region of PTPς. This binding results in the stimulation of exocytosis. The toxin-binding site is located in the C-terminal part of the PTPς ectodomain and includes two fibronectin type III repeats. The intracellular catalytic domains of PTPς are not required for the α-Latrotoxin binding and secretory response triggered by the toxin in chromaffin cells. These features of PTPς resemble two other previously described α-Latrotoxin receptors, neurexin and CIRL. Thus, α-Latrotoxin represents an unusual example of the neurotoxin that has three independent, equally potent, and yet structurally distinct targets. The known structural and functional characteristics of PTPς, neurexin, and CIRL suggest that they define a functional family of neuronal membrane receptors with complementary or converging roles in presynaptic function via a mechanism that involves cell-to-cell and cell-to-matrix interaction.
-
Calcium-Independent Receptor for α-Latrotoxin and Neurexin 1α Facilitate Toxin-Induced Channel Formation: Evidence That Channel Formation Results from Tethering of Toxin to Membrane
Molecular pharmacology, 2000Co-Authors: Michael D. Hlubek, Valery Krasnoperov, Alexander G. Petrenko, Edward L. Stuenkel, Ronald W. HolzAbstract:α-Latrotoxin binding to the calcium-independent receptor for α-Latrotoxin (CIRL-1), a putative G-protein-coupled receptor, stimulates secretion from chromaffin and PC12 cells. Using patch clamp techniques and microspectrofluorimetry, we demonstrate that the interaction of α-Latrotoxin with CIRL-1 produces a high conductance channel that permits increases in cytosolic Ca2+. α-Latrotoxin interaction with CIRL-1 transiently expressed in bovine chromaffin cells produced a 400-pS channel, which rarely closed under Ca2+-free conditions. The major effect of overexpressing CIRL-1 was to greatly increase the sensitivity of chromaffin cells to channel formation by α-Latrotoxin. α-Latrotoxin interaction with CIRL-1 transiently overexpressed in non-neuronal human embryonic kidney 293 (HEK293) cells produced channels that were nearly identical with those observed in chromaffin cells. Channel currents were reduced by millimolar Ca2+. At α-Latrotoxin concentrations below 500 pM, channel formation occurred many seconds after binding of toxin to CIRL-1 indicating distinct steps in channel formation. In all cases there was a rapid, sequential addition of channels once the first channel appeared. An analysis of CIRL-1 mutants indicated that channel formation in HEK293 cells is unlikely to be transduced by a G-protein-dependent mechanism. α-Latrotoxin interaction with a fusion construct composed of the extracellular domain of CIRL-1 anchored to the membrane by the transmembrane domain of vesicular stomatitis virus glycoprotein, and with neurexin 1α, an α-Latrotoxin receptor structurally unrelated to CIRL-1, produced channels virtually identical with those observed with wild-type CIRL-1. We propose that α-Latrotoxin receptors recruit toxin to facilitate its insertion across the membrane and that α-Latrotoxin itself controls the conductance properties of the channels it produces.
-
A novel ubiquitously expressed α-Latrotoxin receptor is a member of the CIRL family of G-protein-coupled receptors
The Journal of biological chemistry, 1999Co-Authors: Konstantin Ichtchenko, Valery Krasnoperov, Mary A. Bittner, Ronald W. Holz, Oleg G. Chepurny, Alvin R. Little, Alexander G. PetrenkoAbstract:Abstract Poisoning with α-Latrotoxin, a neurotoxic protein from black widow spider venom, results in a robust increase of spontaneous synaptic transmission and subsequent degeneration of affected nerve terminals. The neurotoxic action of α-Latrotoxin involves extracellular binding to its high affinity receptors as a first step. One of these proteins, CIRL, is a neuronal G-protein-coupled receptor implicated in the regulation of secretion. We now demonstrate that CIRL has two close homologs with a similar domain structure and high degree of overall identity. These novel receptors, which we propose to name CIRL-2 and CIRL-3, together with CIRL (CIRL-1) belong to a recently identified subfamily of large orphan receptors with structural features typical of both G-protein-coupled receptors and cell adhesion proteins. Northern blotting experiments indicate that CIRL-2 is expressed ubiquitously with highest concentrations found in placenta, kidney, spleen, ovary, heart, and lung, whereas CIRL-3 is expressed predominantly in brain similarly to CIRL-1. It appears that CIRL-2 can also bind α-Latrotoxin, although its affinity to the toxin is about 14 times less than that of CIRL-1. When overexpressed in chromaffin cells, CIRL-2 increases their sensitivity to α-Latrotoxin stimulation but also inhibits Ca2+-regulated secretion. Thus, CIRL-2 is a functionally competent receptor of α-Latrotoxin. Our findings suggest that although the nervous system is the primary target of low doses of α-Latrotoxin, cells of other tissues are also susceptible to the toxic effects of α-Latrotoxin because of the presence of CIRL-2, a low affinity receptor of the toxin.
-
Structural Requirements for α-Latrotoxin Binding and α-Latrotoxin-stimulated Secretion A STUDY WITH CALCIUM-INDEPENDENT RECEPTOR OF α-Latrotoxin (CIRL) DELETION MUTANTS
The Journal of biological chemistry, 1999Co-Authors: Valery Krasnoperov, Mary A. Bittner, Ronald W. Holz, Oleg Chepurny, Alexander G. PetrenkoAbstract:Stimulation of neurotransmitter release by alpha-Latrotoxin requires its binding to the calcium-independent receptor of alpha-Latrotoxin (CIRL), an orphan neuronal G protein-coupled receptor. CIRL consists of two noncovalently bound subunits, p85, a heptahelical integral membrane protein, and p120, a large extracellular polypeptide with domains homologous to lectin, olfactomedin, mucin, the secretin receptor family, and a novel structural motif common for large orphan G protein-coupled receptors. The analysis of CIRL deletion mutants indicates that the high affinity alpha-Latrotoxin-binding site is located within residues 467-891, which comprise the first transmembrane segment of p85 and the C-terminal half of p120. The N-terminal lectin, olfactomedin, and mucin domains of p120 are not required for the interaction with alpha-Latrotoxin. Soluble p120 and all its fragments, which include the 467-770 residues, bind alpha-Latrotoxin with low affinity suggesting the importance of membrane-embedded p85 for the stabilization of the complex of the toxin with p120. Two COOH-terminal deletion mutants of CIRL, one with the truncated cytoplasmic domain and the other with only one transmembrane segment left of seven, supported both alpha-Latrotoxin-induced calcium uptake in HEK293 cells and alpha-Latrotoxin-stimulated secretion when expressed in chromaffin cells, although with a different dose dependence than wild-type CIRL and its N-terminal deletion mutant. Thus the signaling domains of CIRL are not critically important for the stimulation of exocytosis in intact chromaffin cells by alpha-Latrotoxin.
-
Structural Requirements for a-Latrotoxin Binding and a-Latrotoxin-stimulated Secretion
1999Co-Authors: Valery Krasnoperov, Mary A. Bittner, Ronald W. Holz, Oleg G. Chepurny, Alexander G. PetrenkoAbstract:Stimulation of neurotransmitter release by a-Latrotoxin requires its binding to the calcium-independent receptor of a-Latrotoxin (CIRL), an orphan neuronal G protein-coupled receptor. CIRL consists of two noncovalently bound subunits, p85, a heptahelical integral membrane protein, and p120, a large extracellular polypeptide with domains homologous to lectin, olfactomedin, mucin, the secretin receptor family, and a novel structural motif common for large orphan G proteincoupled receptors. The analysis of CIRL deletion mutants indicates that the high affinity a-Latrotoxin-binding site is located within residues 467‐ 891, which comprise the first transmembrane segment of p85 and the C-terminal half of p120. The N-terminal lectin, olfactomedin, and mucin domains of p120 are not required for the interaction with a-Latrotoxin. Soluble p120 and all its fragments, which include the 467‐770 residues, bind a-Latrotoxin with low affinity suggesting the importance of membrane-embedded p85 for the stabilization of the complex of the toxin with p120. Two COOH-terminal deletion mutants of CIRL, one with the truncated cytoplasmic domain and the other with only one transmembrane segment left of seven, supported both a-Latrotoxin-induced calcium uptake in HEK293 cells and a-Latrotoxin-stimulated secretion when expressed in chromaffin cells, although with a different dose dependence than wild-type CIRL and its N-terminal deletion mutant. Thus the signaling domains of CIRL are not critically important for the stimulation of exocytosis in intact chromaffin cells by a-Latrotoxin.
Yuri A. Ushkaryov - One of the best experts on this subject based on the ideXlab platform.
-
Activation of α-Latrotoxin Receptors in Neuromuscular Synapses Leads to a Prolonged Splash Acetylcholine Release
Bulletin of experimental biology and medicine, 2009Co-Authors: V. G. Lelyanova, Derek Thomson, Richard R. Ribchester, E. A. Tonevitsky, Yuri A. UshkaryovAbstract:The mechanisms of acetylcholine release in presynaptic terminals of motoneurons induced by mutant alpha-Latrotoxin (LT(N4C)) were analyzed. In contrast to wild-type alpha-Latrotoxin that causes both continuous and splash secretion of acetylcholine and necessarity block neuromuscular transmission, LT(N4C) causes only splash release lasting over many hours. Thus, activation of alpha-Latrotoxin receptors controls long-lasting enhanced secretion of acetylcholine.
-
penelope s web using α Latrotoxin to untangle the mysteries of exocytosis
Journal of Neurochemistry, 2009Co-Authors: John Paul Silva, Jason Suckling, Yuri A. UshkaryovAbstract:For more than three decades, the venom of the black widow spider and its principal active components, Latrotoxins, have been used to induce release of neurotransmitters and hormones and to study the mechanisms of exocytosis. Given the complex nature of α-Latrotoxin actions, this research has been continuously overshadowed by many enigmas, misconceptions and perpetual changes of the underlying hypotheses. Some of the toxin’s mechanisms of action are still not completely understood. Despite all these difficulties, the extensive work of several generations of neurobiologists has brought about a great deal of fascinating insights into presynaptic processes and has led to the discovery of several novel proteins and synaptic systems. For example, α-Latrotoxin studies have contributed to the widespread acceptance of the vesicular theory of transmitter release. Presynaptic receptors for α-Latrotoxin – neurexins, latrophilins and protein tyrosine phosphatase σ – and their endogenous ligands have now become centerpieces of their own areas of research, with a potential of uncovering new mechanisms of synapse formation and regulation that may have medical implications. However, any future success of α-Latrotoxin research will require a better understanding of this unusual natural tool and a more precise dissection of its multiple mechanisms.
-
Penelope’s web: using α‐Latrotoxin to untangle the mysteries of exocytosis
Journal of neurochemistry, 2009Co-Authors: John Paul Silva, Jason Suckling, Yuri A. UshkaryovAbstract:For more than three decades, the venom of the black widow spider and its principal active components, Latrotoxins, have been used to induce release of neurotransmitters and hormones and to study the mechanisms of exocytosis. Given the complex nature of α-Latrotoxin actions, this research has been continuously overshadowed by many enigmas, misconceptions and perpetual changes of the underlying hypotheses. Some of the toxin’s mechanisms of action are still not completely understood. Despite all these difficulties, the extensive work of several generations of neurobiologists has brought about a great deal of fascinating insights into presynaptic processes and has led to the discovery of several novel proteins and synaptic systems. For example, α-Latrotoxin studies have contributed to the widespread acceptance of the vesicular theory of transmitter release. Presynaptic receptors for α-Latrotoxin – neurexins, latrophilins and protein tyrosine phosphatase σ – and their endogenous ligands have now become centerpieces of their own areas of research, with a potential of uncovering new mechanisms of synapse formation and regulation that may have medical implications. However, any future success of α-Latrotoxin research will require a better understanding of this unusual natural tool and a more precise dissection of its multiple mechanisms.
-
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.
-
α Latrotoxin forms calcium permeable membrane pores via interactions with latrophilin or neurexin
European Journal of Neuroscience, 2000Co-Authors: Catherine Van Renterghem, Yuri A. Ushkaryov, Vera G. Lelianova, Cécile Iborra, Nicole Martinmoutot, Michael SeagarAbstract:In order to explore the mechanisms by which alpha-Latrotoxin activates neurotransmitter release, we have characterized its effects by patch-clamp methods on cells heterologously expressing its receptors, latrophilin-1 or neurexin-Ialpha. Application of alpha-Latrotoxin (1 nM) to cells expressing rat latrophilin or neurexin, but not mock-transfected cells, induced a cationic conductance. In cells expressing latrophilin, current development was slow in the absence of divalent cations, but was accelerated by Ca2+ or Mg2+. In cells expressing neurexin, alpha-Latrotoxin did not elicit currents in the absence of Ca2+. The toxin-induced conductance was rectifying, persistent, permeable to monovalent and divalent cations, but blocked by La3+. Single-channel recording revealed a permanently open state, with the same unitary conductance irrespective of whether cells expressed latrophilin or neurexin. Therefore, while pore formation displayed differences consistent with the reported properties of alpha-Latrotoxin binding to latrophilin and neurexin, the pores induced by alpha-Latrotoxin had identical properties. These results suggest that after anchoring to either of its nerve terminal receptors, alpha-Latrotoxin inserts into the membrane and constitutes a single type of transmembrane ion pore.
N.h. Himmelreich - One of the best experts on this subject based on the ideXlab platform.
-
Involvement of membrane GABA transporter in α-Latrotoxin-stimulated [3H]GABA release
Neurochemistry international, 2004Co-Authors: M.v Linetska, L.g. Storchak, A.s Tarasenko, N.h. HimmelreichAbstract:Abstract α-Latrotoxin evokes massive [ 3 H]GABA release from rat brain synaptosomes by stimulating exocytosis and outflow from non-vesicular pool. In the present study, GABA transporter-mediated [ 3 H]GABA release was shown to be involved in α-Latrotoxin-triggered release of [ 3 H]GABA from non-vesicular pool. The following agents have been exploited as tools: (1) a protonophore carbonyl cyanide- p -trifluoromethoxyphenyl-hydrazon (FCCP) and bafilomycin A 1 for evoking depletion of synaptic vesicle [ 3 H]GABA and enlargement of non-vesicular pool; (2) a non-substrate high-affinity GABA transport blocker NO-711 for determining participation of GABA carrier in the toxin-stimulated GABA release; (3) a competitive inhibitor of GABA reuptake nipecotic acid for heteroexchange [ 3 H]GABA release. As shown by the experiments with nipecotic acid, FCCP and bafilomycin A 1 considerably increase the content of non-vesicular [ 3 H]GABA. The treatment of the synaptosomes with these agents modified the response to α-Latrotoxin, particularly to its subnanomolar concentrations: the lack or substantial lowering of the toxin-evoked release during the first 2 min after the toxin addition and substantial enhancement of release up to the 5th minute were observed. Only the step of enhanced release was sensitive to GABA transporter blocker NO-711. Distinct sensitivity to NO-711 was shown to be characteristic for different steps of α-Latrotoxin-stimulated [ 3 H]GABA release from the control, untreated synaptosomes: lack of any effect of NO-711 during the first 2 min and powerful inhibition in 10 min after the toxin application. Taken together these data appear to indicate that the toxin non-simultaneously from vesicular and non-vesicular origins releases the neurotransmitter, the first rapid step reflects exocytosis stimulation, and the second tardy step is at least in part due to the release mediated by GABA transporters. The incomplete inhibition with NO-711 of the tardy step of the release evoked by nanomolar toxin concentrations suggests the participation not only of the GABA transporters.
-
Phenylarsine oxide inhibits α-Latrotoxin-stimulated [3H]GABA release from rat brain synaptosomes
Neurochemistry international, 2003Co-Authors: M.v Linetska, L.g. Storchak, N.h. HimmelreichAbstract:Abstract Phosphatidylinositol 4,5-biphosphate has been implicated in a variety of membrane-trafficking processes, including exocytosis of neurotransmitters. However, there are contradictory findings concerned ability of phenylarsine oxide (PAO), an inhibitor of phosphatidylinositol 4-kinase, to affect exocytotic release of different types of neurotransmitters. We bent our efforts to a detailed analysis of action of PAO on Ca 2+ -dependent and Ca 2+ -independent [ 3 H ]GABA release produced by exposure of rat brain synaptosomes to different concentrations of α-Latrotoxin. We also compared PAO action on α-Latrotoxin- and 4-aminopyridine (4-AP)-evoked [ 3 H ]GABA release. The experiments have shown that release of [ 3 H ]GABA evoked by the depolarization with 4-AP was decreased by 80% as a result of action of 3 μM PAO and the complete inhibition of release was observed with 10 μM PAO. When α-Latrotoxin as a stimulant was applied, release of [ 3 H ]GABA was increased as toxin concentration used was elevated from 0.5 to 3.0 nM, however, concomitantly, the response of the toxin-induced [ 3 H ]GABA release to PAO became attenuated: 10 μM PAO led to almost complete inhibition of the effect of 0.5 nM α-Latrotoxin and only partly decreased (by 40%) the response to 3.0 nM α-Latrotoxin. To test whether the efficacy of PAO depended on the toxin-induced outflow of cytosolic [ 3 H ]GABA, synaptosomes with depleted cytosolic [ 3 H ]GABA pool were also exploited. Depletion was performed by means of heteroexchange of cytosolic [ 3 H ]GABA with nipecotic acid. The experiments have shown that treatment of loaded synaptosomes with nipecotic acid resulted in some increase of [ 3 H ]GABA release evoked by 0.5 nM α-Latrotoxin, but in the two-fold decrease of the response to 3.0 nM α-Latrotoxin. PAO essentially inhibited [ 3 H ]GABA release from depleted synaptosomes irrespective of α-Latrotoxin concentration used. Therefore, the amount of [ 3 H ]GABA released from cytosolic pool determined, in considerable degree, the insensitivity of α-Latrotoxin action to PAO. Thus, our data show that subnanomolar concentrations of α-Latrotoxin may be used for stimulation of exocytotic release of [ 3 H ]GABA. Exposure of synaptosomes with nanomolar toxin concentrations leads not only to stimulation of exocytosis, but also to leakage of [ 3 H ]GABA from cytosolic pool. PAO potently inhibits exocytotic release of [ 3 H ]GABA and its inhibitory effectiveness is diminished as far as the outflow of [ 3 H ]GABA is elevated.
-
Does extracellular calcium determine what pool of GABA is the target for α-Latrotoxin?
Neurochemistry international, 2002Co-Authors: L.g. Storchak, M.v Linetska, N.h. HimmelreichAbstract:Abstract Presynaptic neurotoxin α-Latrotoxin, from the venom of Latrodectus mactans tredecimguttatus , causes massive [ 3 H ]GABA release from rat brain synaptosomes, irrespective of calcium presence in the extracellular medium. Whether the binding of α-Latrotoxin to Ca 2+ -dependent (neurexin 1α) or to Ca 2+ -independent (latrophilin) receptor triggers [ 3 H ]GABA release by the same mechanisms or different ones, inducing either exocytotic process or outflow by mobile membrane GABA transporter, is unknown. We examined α-Latrotoxin-evoked [ 3 H ]GABA release from synaptosomes which cytosolic [ 3 H ]GABA pool was depleted either by applying competitive inhibitors of the GABA transporter, nipecotic acid and 2,4-diaminobutyric acid, or by permeation with digitonin. We also compared the effect of the GABA transporter inhibitors on depolarisation-evoked and α-Latrotoxin-evoked [ 3 H ]GABA release using as depolarising agents 4-aminopyridine and high KCl in the Ca 2+ -containing and in Ca 2+ -free medium, respectively. Incubation of synaptosomes with nipecotic acid induced the essential acceleration of unstimulated [ 3 H ]GABA release and deep inhibition of high KCl-evoked Ca 2+ -independent [ 3 H ]GABA release. In contrast, at the similar conditions the effect of α-Latrotoxin was greatly augmented with respect to the control response. Another way to assay what GABA pool was involved in α-Latrotoxin-induced release lays in an analysis of the effects of depolarisation and α-Latrotoxin in consecutive order. The preliminary 4-aminopyridine-stimulated [ 3 H ]GABA release attenuated the toxin effect. But when depolarisation occurred in Ca 2+ -free medium, no influence on α-Latrotoxin effect was revealed. Employing digitonin-permeated synaptosomes, we have shown that α-Latrotoxin could stimulate [ 3 H ]GABA release in the medium with 1 mM EGTA, this effect of the toxin was blocked by concanavalin A and was ATP-dependent. The latter suggests that α-Latrotoxin-released neurotransmitter has the vesicular nature. We assume that the type of the toxin membrane receptor does not determine the mechanisms of [ 3 H ]GABA release evoked by α-Latrotoxin.
-
[20] - Presynaptic Activity of α-Latrotoxin: Purification and Properties
Methods in Neurosciences, 1992Co-Authors: Ya. T. Terletskaya, N.h. Himmelreich, Yu.v. SokolovAbstract:Publisher Summary This chapter presents the presynaptic activity of α-Latrotoxin (LTX), its purification procedures, and its properties. The venom of black widow spider Latrodectus mactans tredecimguttatus contains, as its major protein component, the toxin called α-Latrotoxin. The main effect of LTX consists of a massive stimulation of transmitter release from the neuromuscular junctions of vertebrates that eventually leads to blocking of the synaptic transmission. α -Latrotoxin is an acidic protein, with a molecular weight of about 130,000. It consists of only a single polypeptide chain, and is devoid of any proteolytic and lipolytic activity. Numerous studies have been undertaken to determine the nature of the neurotoxic activity of LTX. The chapter summarizes the characteristics of the neurotoxin—(1) α-Latrotoxin binds to the high-affinity receptor on presynaptic plasma membranes and pheochromocytoma cells PC-12, (2) it has the ability to promote an influx of divalent cations through selective channels in presynaptic membranes, (3) it can penetrate, in a specific way, into liquid bilayers, and (4) it has the ability to induce the fusion of artificial phospholipid structures.
Jessica E. Garb - One of the best experts on this subject based on the ideXlab platform.
-
Additional file 1: of House spider genome uncovers evolutionary shifts in the diversity and expression of black widow venom proteins associated with extreme toxicity
2017Co-Authors: Kerry Gendreau, Robert Haney, Evelyn Schwager, Torsten Wierschin, Mario Stanke, Stephen Richards, Jessica E. GarbAbstract:Table describing house spider Latrotoxin sequence features. (XLSX 34Â kb
-
Additional file 8: of House spider genome uncovers evolutionary shifts in the diversity and expression of black widow venom proteins associated with extreme toxicity
2017Co-Authors: Kerry Gendreau, Robert Haney, Evelyn Schwager, Torsten Wierschin, Mario Stanke, Stephen Richards, Jessica E. GarbAbstract:Manual adjustments to Augustus predicted house spider Latrotoxin sequences. (XLSX 47Â kb
-
Gene structure, regulatory control, and evolution of black widow venom Latrotoxins
FEBS Letters, 2014Co-Authors: Kanaka Varun Bhere, Nadia A Ayoub, Robert A. Haney, Jessica E. GarbAbstract:Black widow venom contains α-Latrotoxin, infamous for causing intense pain. Combining 33 kb of Latrodectus hesperus genomic DNA with RNA-Seq, we characterized the α-Latrotoxin gene and discovered a paralog, 4.5 kb downstream. Both paralogs exhibit venom gland specific transcription, and may be regulated post-transcriptionally via musashi-like proteins. A 4 kb intron interrupts the α-Latrotoxin coding sequence, while a 10 kb intron in the 3′ UTR of the paralog may cause non-sense-mediated decay. Phylogenetic analysis confirms these divergent Latrotoxins diversified through recent tandem gene duplications. Thus, Latrotoxin genes have more complex structures, regulatory controls, and sequence diversity than previously proposed.
-
Dramatic expansion of the black widow toxin arsenal uncovered by multi-tissue transcriptomics and venom proteomics
BMC Genomics, 2014Co-Authors: Robert A. Haney, Nadia A Ayoub, Thomas H. Clarke, Cheryl Y. Hayashi, Jessica E. GarbAbstract:Background Animal venoms attract enormous interest given their potential for pharmacological discovery and understanding the evolution of natural chemistries. Next-generation transcriptomics and proteomics provide unparalleled, but underexploited, capabilities for venom characterization. We combined multi-tissue RNA-Seq with mass spectrometry and bioinformatic analyses to determine venom gland specific transcripts and venom proteins from the Western black widow spider ( Latrodectus hesperus) and investigated their evolution. Results We estimated expression of 97,217 L. hesperus transcripts in venom glands relative to silk and cephalothorax tissues. We identified 695 venom gland specific transcripts (VSTs), many of which BLAST and GO term analyses indicate may function as toxins or their delivery agents. ~38% of VSTs had BLAST hits, including Latrotoxins, inhibitor cystine knot toxins, CRISPs, hyaluronidases, chitinase, and proteases, and 59% of VSTs had predicted protein domains. Latrotoxins are venom toxins that cause massive neurotransmitter release from vertebrate or invertebrate neurons. We discovered ≥ 20 divergent Latrotoxin paralogs expressed in L. hesperus venom glands, significantly increasing this biomedically important family. Mass spectrometry of L. hesperus venom identified 49 proteins from VSTs, 24 of which BLAST to toxins. Phylogenetic analyses showed venom gland specific gene family expansions and shifts in tissue expression. Conclusions Quantitative expression analyses comparing multiple tissues are necessary to identify venom gland specific transcripts. We present a black widow venom specific exome that uncovers a trove of diverse toxins and associated proteins, suggesting a dynamic evolutionary history. This justifies a reevaluation of the functional activities of black widow venom in light of its emerging complexity.
-
Molecular evolution of α-Latrotoxin, the exceptionally potent vertebrate neurotoxin in black widow spider venom
Molecular biology and evolution, 2013Co-Authors: Jessica E. Garb, Cheryl Y. HayashiAbstract:Black widow spiders (members of the genus Latrodectus) are widely feared because of their potent neurotoxic venom. α-Latrotoxin is the vertebrate-specific toxin responsible for the dramatic effects of black widow envenomation. The evolution of this toxin is enigmatic because only two α-Latrotoxin sequences are known. In this study, ∼4 kb α-Latrotoxin sequences and their homologs were characterized from a diversity of Latrodectus species, and representatives of Steatoda and Parasteatoda, establishing the wide distribution of Latrotoxins across the mega-diverse spider family Theridiidae. Across black widow species, α-Latrotoxin shows ≥94% nucleotide identity and variability consistent with purifying selection. Multiple codon and branch-specific estimates of the nonsynonymous/synonymous substitution rate ratio also suggest a long history of purifying selection has acted on α-Latrotoxin across Latrodectus and Steatoda. However, α-Latrotoxin is highly divergent in amino acid sequence between these genera, with 68.7% of protein differences involving non-conservative substitutions, evidence for positive selection on its physiochemical properties and particular codons, and an elevated rate of nonsynonymous substitutions along α-Latrotoxin’s Latrodectus branch. Such variation likely explains the efficacy of red-back spider, L. hasselti, antivenom in treating bites from other Latrodectus species, and the weaker neurotoxic symptoms associated with Steatoda and Parasteatoda bites. Long-term purifying selection on α-Latrotoxin indicates its functional importance in black widow venom, even though vertebrates are a small fraction of their diet. The greater differences between Latrodectus and Steatoda α-Latrotoxin, and their relationships to invertebrate-specific Latrotoxins, suggest a shift in α-Latrotoxin toward increased vertebrate toxicity coincident with the evolution of widow spiders.