beta-Leptinotarsin-h, purified from the hemolymph of the beetle Leptinotarsa haldemani, is a potent ( approximately 1 nM) neuroactive protein that rapidly (few seconds) stimulates Ca(2+) influx and
neurotransmitter release. Our goals were to further characterize beta-leptinotarsin-h and to test the hypothesis that it stimulates Ca(2+) influx through
presynaptic Ca(2+) 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-leptinotarsin-h on Ca(2+) current, Ca(2+) uptake, Ca(2+) 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(2+) flux for their effects on beta-leptinotarsin-h-stimulated Ca(2+) uptake in rat
brain synaptosomes. The
non-selective Ca(2+)
channel blockers flunarizine, Ni(2+),
ruthenium red, high-concentration
thapsigargin, and SKF 96365 inhibited beta-leptinotarsin-h's activity, but none of the tested
selective blockers of voltage-operated Ca(2+) channels (omega-agatoxin IVA, omega-conotoxin GVIA, omega-conotoxin MVIIC,
nicardipine,
nifedipine, SNX-482) was
inhibitory.
Selective inhibitors of ligand-operated, store-operated, and transduction-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 Ca(2+) uptake through
presynaptic Ca(2+) 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.
