Characterization of β-leptinotarsin-h and the effects of calcium flux antagonists on its activity

beta-Leptinotarsin-h, purified from the hemolymph of the beetle Leptinotarsa haldemani, is a potent (similar to 1 nM) neuroactive protein that rapidly (few seconds) stimulates Ca2+ influx and neurotransmitter release. Our goals were to further characterize beta-leptinotarsin-h and to test the hypothesis that it stimulates Ca2+ influx through presynaptic Ca2+ channels. Analysis of partial amino acid sequences revealed that beta-leptinotarsin-h is a unique protein with significant similarity to only one other protein, the juvenile hormone esterase of Leptinotarsa decemlineata, commonly known as the Colorado potato beetle. We have examined the effect of beta-Ieptinotarsin-h on Ca2+ current, Ca2+ uptake, Ca2+ levels, and neurotransmitter release in synaptosomes, cell lines, and neuronal systems. We found that its preferred site of action appears to be mammalian presynaptic nerve terminals. We tested antagonists of Ca2+ flux for their effects on beta-leptinotarsin-h-stimulated Ca2+ uptake in rat brain synaptosomes. The non-selective Ca2+ channel blockers flunarizine, Ni2+, ruthenium red, high-concentration thapsigargin, and SKF 96365 inhibited beta-leptinotarsin-h's activity, but none of the tested selective blockers of voltage-operated Ca2+ channels (omega-agatoxin IVA, omega-conotoxin GVIA, omega-conotoxin MVIIC, nicardipine, nifedipine, SNX-482) was inhibitory. Selective inhibitors of ligand-operated, store-operated, and transduct ion-operated channels were also not inhibitory. beta-Leptinotarsin-h did not stimulate Na+ uptake, ruling out Na+ channels and many non-selective cation channels as targets. We conclude that beta-leptinotarsin-h stimulated Ca2+ uptake through presynaptic Ca2+ channels; which channel is yet to be determined. beta-Leptinotarsin-h may prove to be a useful tool with which to investigate calcium channels and calcium flux. Published by Elsevier Ltd.