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Leptinotoxin‐h Action in Synaptosomes, Neurosecretory Cells, and Artificial Membranes: Stimulation of Ion Fluxes
Author(s) -
Madeddu Luisa,
Pozzan Tullio,
Robello Mauro,
Rolandi Ranieri,
Hsiao Ting H.,
Meldolesi Jacopo
Publication year - 1985
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1985.tb10526.x
Subject(s) - depolarization , tetrodotoxin , neurotoxin , biophysics , toxin , verapamil , membrane , synaptosome , chemistry , pertussis toxin , phospholipid , biochemistry , membrane potential , calcium , biology , g protein , receptor , organic chemistry
Leptinotoxin‐h (LPTx), a neurotoxin (otherwise designated β‐leptinotarsin‐h) known to stimulate the release of neurotransmitters from synapses, was purified from the hemolymph of the potato beetle, Leptinotarsa haldemani , by a simplification of the procedure originally developed by Crosland et al. [ Biochemistry 23 , 734–741, (1984)]. Highly and partially purified preparations of the toxin were applied to guinea pig synaptosomes and neurosecretory (PC12) cells. When applied in a Ca 2+ ‐containing Ringer medium, at concentrations in the 10 −11 ‐10 −10 M range, the toxin induced: (a) rapid depolarization of the plasma membrane, which was not inhibited by organic blockers of voltage‐dependent Na + and Ca 2+ channels (tetrodotoxin or verapamil); (b) large 45 Ca influx: and (c) increased free cytosolic Ca 2+ concentration. These latter two effects were unaffected by verapamil. In Ca 2+ ‐free media the effects of the toxin were different in the two systems investigated. In synaptosomes, depolarization was still observed, even if the toxin concentrations needed were higher (10X) than those effective in the complete medium. In contrast, in PC12 cells no effect of the toxin on membrane potential was observed. Binding of LPTx to its cellular targets could not be investigated directly because the toxin was inactivated by the procedures used for its labeling. Indirect evidence suggested however that Ca 2+ is necessary for toxin binding to PC12 cells. Interaction of LPTx with air/water interfaces, as well as with cholesterol/phospholipid mono‐ and bilayer membranes was investigated. The results indicate that the toxin has affinity for hydrophobic surfaces, but lacks the capacity to insert across membranes unless transpositive voltage is applied. Our results are inconsistent with the previous conclusion of Crosland et al. (1984), who suggested opening of the Ca 2+ channel as the mechanism of action of LPTx. The effects of the toxin resemble those of α‐latrotoxin (α‐LTx) of the black widow spider venom, and therefore the two toxins might act by similar mechanisms. However, the sites recognized by the two toxins might be different, because LPTx does not inhibit α‐LTx binding.