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

b-Leptinotarsin-h, purified from the hemolymph of the beetle Leptinotarsa haldemani, is a potent (w1 nM) neuroactive protein that rapidly (few seconds) stimulates Ca influx and neurotransmitter release. Our goals were to further characterize bleptinotarsin-h and to test the hypothesis that it stimulates Ca influx through presynaptic Ca channels. Analysis of partial amino acid sequences revealed that b-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 b-leptinotarsin-h on Ca current, Ca uptake, Ca 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 Ca flux for their effects on b-leptinotarsin-h-stimulated Ca uptake in rat brain synaptosomes. The non-selective Ca channel blockers flunarizine, Ni, ruthenium red, high-concentration thapsigargin, and SKF 96365 inhibited b-leptinotarsin-h’s activity, but none of the tested selective blockers of voltage-operated Ca channels (u-agatoxin IVA, u-conotoxin GVIA, u-conotoxin MVIIC, nicardipine, nifedipine, SNX-482) was inhibitory. Selective inhibitors of ligand-operated, store-operated, and transduction-operated channels were also not inhibitory. b-Leptinotarsin-h did not stimulate Na uptake, ruling out Na channels and many non-selective cation channels as targets. We conclude that bleptinotarsin-h stimulated Ca uptake through presynaptic Ca channels; which channel is yet to be determined. bLeptinotarsin-h may prove to be a useful tool with which to investigate calcium channels and calcium flux. Published by Elsevier Ltd.