α-Latrotoxin, Acting via Two Ca2+-dependent Pathways, Triggers Exocytosis of Two Pools of Synaptic Vesicles

Ashton, Anthony C.; Volynski, Kirill E.; Lelianova, Vera G.; Orlova, Elena V.; Renterghem, Catherine Van; Canepari, Marco; Seagar, Michael; Ushkaryov, Yuri A.

doi:10.1074/jbc.m108088200

Cited by 65 publications

(129 citation statements)

References 48 publications

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“…We previously showed that LTX N4C fails to form ionic pores in the membrane of synaptosomes and LPH-transfected BHK cells (8). Here, we confirmed and extended these observations by comparing the abilities of the recombinant toxins to induce 45 Ca 2ϩ influx into COS7 cells expressing exogenous LPH or NRX.…”

Section: Molecular Basis Of the Inability Of The Mutant Toxin To Insesupporting

confidence: 80%

“…LTX N4C -evoked Release of [ 14 C]Glu from Rat Synaptosomes-As mentioned above, LTX N4C stimulates receptor-mediated exocytosis of amino acid neurotransmitters from synaptosomes only in the presence of Ca 2ϩ (8). In the current work, we substituted Ca 2ϩ with Sr 2ϩ .…”

Section: Fig 2 Ltx N4c Is Unable To Form Ionic Pores a Influx Ofmentioning

confidence: 95%

“…Interestingly, the receptor-mediated effect of LTX N4C in central synapses is Ca 2ϩ -dependent (8), reasonably suggesting that the Ca 2ϩ -dependent receptors (i.e. NRXs) may be responsible for mediating this LTX action.…”

mentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

“…Lacking the major (ionophore) activity, LTX N4C was originally thought to be altogether inactive (12,17). However, we have discovered that, in fact, LTX N4C strongly stimulates receptor-mediated transmitter release (8), providing an ideal tool for investigating exocytotic signals transduced by LTX receptors.…”

mentioning

confidence: 99%

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Mutant α-Latrotoxin (LTXN4C) Does Not Form Pores and Causes Secretion by Receptor Stimulation

Volynski

Capogna

Ashton

et al. 2003

Journal of Biological Chemistry

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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...

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Section: Molecular Basis Of the Inability Of The Mutant Toxin To Insesupporting

confidence: 80%

Section: Fig 2 Ltx N4c Is Unable To Form Ionic Pores a Influx Ofmentioning

confidence: 95%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Mutant α-Latrotoxin (LTXN4C) Does Not Form Pores and Causes Secretion by Receptor Stimulation

Volynski

Capogna

Ashton

et al. 2003

Journal of Biological Chemistry

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Signal Transduction Mediated through Adhesion-GPCRs

Mizuno¹,

Itoh

2010

Advances in Experimental Medicine and Biology

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The Latrophilins, “Split-Personality” Receptors

Silva¹,

Ushkaryov

2010

Advances in Experimental Medicine and Biology

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Latrophilin, a neuronal "adhesion-G protein-coupled receptor", is the major brain receptor for alpha-latrotoxin, a black widow spidertoxin which stimulates strong neuronal exocytosis in vertebrates. Latrophilin has an unusual structure consisting of two fragments that are produced by the proteolytic cleavage of the parental molecule and that behave independently in the plasma membrane. On binding an agonist, the fragments reassociate and send an intracellular signal. This signal, transduced by a heterotrimeric G protein, causes release of calcium from intracellular stores and massive release of neurotransmitters. Latrophilin represents a phylogenetically conserved family of receptors, with orthologues found in all animals and up to three homologues present in most chordate species. From mammalian homologues, latrophilins 1 and 3 are expressed in neurons, while latrophilin 2 is ubiquitous. Latrophilin 1 may control synapse maturation and exocytosis, whereas latrophilin 2 may be involved in breast cancer. Latrophilins may play different roles during development and in adult animals: thus, LAT-1 determines cell fate in early embryogenesis in Caenorhabditis elegans and controls neurotransmitter release in adult nematodes. This diversity suggests that the functions of latrophilins may be determined by their interactions with respective ligands. The finding of the ligand of latrophilin 1, the large postsynaptic protein lasso, is the first step in the quest for the physiological functions of latrophilins.

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α-Latrotoxin, Acting via Two Ca2+-dependent Pathways, Triggers Exocytosis of Two Pools of Synaptic Vesicles

Cited by 65 publications

References 48 publications

Mutant α-Latrotoxin (LTXN4C) Does Not Form Pores and Causes Secretion by Receptor Stimulation

Mutant α-Latrotoxin (LTXN4C) Does Not Form Pores and Causes Secretion by Receptor Stimulation

Signal Transduction Mediated through Adhesion-GPCRs

The Latrophilins, “Split-Personality” Receptors

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