Elsevier

Neuroscience

Volume 10, Issue 3, October 1983, Pages 997-1009
Neuroscience

The effect of α-latrotoxin on the neurosecretory PC12 cell line: Studies on toxin binding and stimulation of transmitter release

https://doi.org/10.1016/0306-4522(83)90238-5Get rights and content

Abstract

α-Latrotoxin of black widow spider venom was found to bind with high affinity(KA = 1.8·109M−1) to specific sites present in discrete number (∼6300/cell, ∼12/aμn2) at the surface membrane of PC12 cells. This binding correlated with (and therefore, probably caused) the secretory response produced by the toxin. Binding was enhanced (∼ 2-fold) in the presence of mM concentrations of various divalent cations (Ca2+, Mn2+ and Co2+) while Ba2+ and Sr2+ had a smaller effect and Mg2+ was inactive. Hypertonicity, concanavalin A and trypsin pretreatment of the cells blocked the binding interaction. The α-latrotoxin-induced stimulation of3H-dopamine release was massive and occurred very rapidly when cells were exposed to the toxin in a Ca2+-containing Krebs-Ringer medium, whereas it occurred at a much slower rate in a Ca2+-free, Mg2+-containing Ringer. Introduction of Ca2+ into the latter medium resulted in a shift of the release rate from slow to fast. In contrast, in divalent cation-free medium the response was abolished. The toxin-induced secretory response was unaffected by Na2+ and Ca2+ channel blockers (tetrodotoxin and D600) as well as by calmodulin inhibitors (calmidazolium and trifluoperazine). The effects of Ca2+ and Mg2+ were found to be concentration-dependent, with half maximal responses occurring at approximately 0.3 and l.5mM for the two divalent cations, respectively. Other divalent cations could substitute for Ca2+ and Mg2+, the relative efficacy being Sr2+ > Ca2+ ⩾> Ba2+ > Mn2+ > Mg2+ > Co2+. Moreover, the response occurring at suboptimal concentration of Ca2+ (0.4 mM) was potentiated by the concomitant addition of either Mg2+, Mn2+ or Co2+. The effect(s) of divalent cations in supporting the α-latrotoxin-induced release response seem(s) to occur primarily at step(s) beyond toxin binding because

  • (a)

    the stimulatory effects of the various cations on release were not matched by parallel effects on binding

  • (b)

    Ca2+ maintained its ability to stimulate fast release even when toxin binding had occurred in a Ca2+-free medium. Delays in the release responses were observed when cells were exposed to αLTx in Na2+-free, glucosamine or methylamine-based media, or depolarized with high K2+ (in the presence of D600) before toxin treatment. Moreover, in these two conditions the ability of Mg2+ to support the αLTx response was considerably decreased.

Taken together with previous data of the literature these results appear consistent with the hypothesis that αLTx acts by activating a ‘non-conventional’ cation channel of low ionic specificity in the plasma membrane. This would result in a stimulated influx of various divalent cations, not only Ca2+ but also Mg2+ and the others investigated, which would then be able to stimulate the release response, although with different efficacy.

Reference (39)

  • GreeneL.A. et al.

    Release, storage and uptake of catecholamines by a clonal cell line of nerve growth factor (NGF) responsive pheochromocytoma cells

    Brain Res.

    (1977)
  • WatanabeO. et al.

    The effect of α-latrotoxin on the neurosecretory PC12 cell line: electron microscopy and cytotoxicity studies

    Neuroscience

    (1983)
  • BabaA. et al.

    The action of black widow spider venom on cholinergic mechanisms in synaptosomes

    J. Neurochem.

    (1980)
  • BakerP.F. et al.

    Calcium dependence of catecholamine release from bovine adrenal medullary cells after exposure to intense electric field

    J. Memb. Biol.

    (1982)
  • BakerP.F. et al.

    Calcium Movement in Excitable Cells

    (1975)
  • CeccarelliB. et al.

    Freeze-fracture studies of frog neuromuscular junction during intense release of neurotransmitter

    J. Cell Biol.

    (1979)
  • CeccarelliB. et al.

    Ca2+-dependent recycling of synaptic vesicles of the frog neuromuscular junction

    J. Cell Biol.

    (1980)
  • ClarkA.W. et al.

    Changes in the fine structure of the neuromuscular junction of the frog caused by black widow spider venom

    J. Cell Biol.

    (1972)
  • Del CastilloJ. et al.

    Discrete and discontinuous action of brown widow spider venom on the presynaptic nerve terminals of frog muscle

    J. Physiol., Lond.

    (1975)
  • FinkelsteinA. et al.

    Black widow spider venom: effect of the purified toxin on lipid bilayer membranes

    Science, N.Y.

    (1976)
  • FrontaliN. et al.

    Purification from black widow spider venom of a protein factor causing the depletion of synaptic vesicles of neuromuscular junction

    J. Cell Biol.

    (1976)
  • GorioA. et al.

    Acetylcholine compartments in mouse diaphragm. Comparison of the effects of black widow spider venom, electrical stimulation and high concentration of potassium

    J. Cell Biol.

    (1978)
  • GrassoA. et al.

    Black widow spider toxin-induced calcium fluxes and transmitter release in a neurosecretory cell line

    Nature, Lond.

    (1980)
  • GrassoA. et al.

    A toxin purified from the venom of black widow spider affects uptake and release of radioactive γ aminobutyrate and N-epinephrine from rat brain synaptosomes

    Ear. J. Biochem.

    (1979)
  • GreeneL.A. et al.

    Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor

  • HerrupK. et al.

    Properties of the nerve growth factor receptor of a clonal line of rat pheochromocytoma (PC12) cell

    Expl. Cell Res.

    (1978)
  • HowardB.D. et al.

    Effects and mechanisms of polypeptide neurotoxins that act presynaptically

    A. Rev. Pharmac. Toxicol.

    (1980)
  • HurlbutW.P. et al.

    Use of black widow spider venom to study the release of neurotransmitters

  • HurlbutW.P. et al.

    Effects of calcium and magnesium on the frequency of miniature end plate potentials during prolonged tetanization

    J. Physiol., Lond.

    (1971)
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