Characterization of Beta-leptinotarsin-h and the Effects of Calcium Flux Antagonists on its Activity
ARMY MEDICAL RESEARCH INST OF INFECTIOUS DISEASES FORT DETRICK MD
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Beta-Leptinotarsin-h, purifed from the hemolymph of the beetle Leptinotarsa haldemani, is a potent neuroactive protein that stimulates Ca2 influx and neurotransmitter release. Our goals were to further characterize beta-leptinotarsin-h and to test the hypthesis 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 examined the effect of beta-leptinotarsin-h on neurotransmitter release, Ca2 current, Ca2 uptake, and Ca2 levels in several 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. We found that the nonselective Ca2 channel blockers flunarizine, Ni2, ruthenium red, high-concentration thapsigargin, and SKF 96365 inhibited beta-leptinotarsin-hs activity, but that non of the selective blockers of voltage-operated Ca2 channels w-agatoxin, IVA, w-conotoxin GVIA, w-conotoxin MVIIC, nicardipine, SNX 482 that we tested 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 nonselective cation channels as targets. We conclude that beta-leptinotarsin-h stimulated Ca2 channels, which channel is yet to be determined.
- Inorganic Chemistry