Characterization of beta-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 ( 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.
Crosland
Richard D
RD
Scientific Review Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892-9529, USA. crosland@nih.gov
Fitch
Richard W
RW
Hines
Harry B
HB
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002118:Calcium / Q000378:metabolism*; D002120:Calcium Channel Agonists / Q000494:pharmacology*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000378:metabolism*; D019476:Insect Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009498:Neurotoxins / Q000494:pharmacology*; D018377:Neurotransmitter Agents / Q000378:metabolism; D017729:Presynaptic Terminals / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012964:Sodium / Q000378:metabolism; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism
D000911:Antibodies, Monoclonal; D002120:Calcium Channel Agonists; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D019476:Insect Proteins; D009498:Neurotoxins; D018377:Neurotransmitter Agents; C023401:leptinotarsin protein, Leptinotarsa haldemani; D012964:Sodium; D002118:Calcium