title: Inhibition of neuronal high voltage-activated calcium channels by insect peptides: a comparison with the actions of omega-conotoxin GVIA.
abstract: The whole cell variant of the patch clamp technique was used to investigate the actions of two novel insect peptides on high voltage-activated Ca2+ currents in cultured dorsal root ganglion (DRG) neurones. The insect peptides (PMP-D2 and PMP-C) were isolated originally from insect brains and fat bodies, and have been found to have similar three-dimensional structures to the N-type Ca2+ channel inhibitor omega-conotoxin GVIA (omega-CgTx GVIA). High voltage-activated Ca2+ currents were activated from a holding potential of -90 mV by depolarizing step commands to 0 mV. Extracellular application of synthetic PMP-D2 or PMP-C (1 microM) attenuated high voltage-activated Ca2+ currents. The effects of PMP-C were strongly dependent on the frequency of current activation, but inhibition was apparent and reached a steady state after 20 steps when currents were evoked for 30 msec at 0.1 Hz. The actions of the two insect peptides overlapped both with each other and with omega-CgTx GVIA, suggesting that N-type Ca2+ current was predominantly sensitive to these peptides. Low voltage-activated T-type current and 1,4-dihydropyridine sensitive L-type Ca2+ currents were insensitive to 1 microM PMP-D2 and PMP-C, which indicates a degree of selectivity. The presence of a fucose group on PMP-C abolished the ability of this peptide to attenuate high voltage-activated Ca2+ currents, which may reflect a mechanism by which peptide function could be regulated in insects. The electrophysiological data are supported by studies on 45Ca2+ influx into rat cerebrocortical synaptosomes. Both PMP-D2 (10 microM), PMP-C (10 microM) and omega-CgTx GVIA (1 microM) attenuated a proportion of 45Ca2+ influx into the synaptosomes, but additive effects of these peptides were not observed. We conclude that these naturally occurring peptides obtained from invertebrate preparations have inhibitory effects on N-type Ca2+ channels. Although the peptides have related three-dimensional structures, they have distinct amino acid sequences and appear to have different mechanisms of action to produce inhibition of mammalian neuronal high voltage-activated Ca2+ channels.
authors.affiliation: Department of Biomedical Sciences, Aberdeen University, Foresterhill, UK. rod.scott@abdn.ac.uk
authors.forename: R H
authors.initials: RH
authors.lastname: Scott
authors.forename: V J
authors.initials: VJ
authors.lastname: Gorton
authors.forename: L
authors.initials: L
authors.lastname: Harding
authors.forename: D
authors.initials: D
authors.lastname: Patel
authors.forename: S
authors.initials: S
authors.lastname: Pacey
authors.forename: C
authors.initials: C
authors.lastname: Kellenberger
authors.forename: H
authors.initials: H
authors.lastname: Hietter
authors.forename: I
authors.initials: I
authors.lastname: Bermudez
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D002132:Calcium Radioisotopes; D002478:Cells, Cultured; D047169:Cyclotides; D004594:Electrophysiology; D005727:Ganglia, Spinal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006110:Grasshoppers / Q000378:metabolism*; D066298:In Vitro Techniques; D019476:Insect Proteins / Q000494:pharmacology; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D008969:Molecular Sequence Data; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism; D020866:omega-Conotoxin GVIA
chemical_list: D002121:Calcium Channel Blockers; D002132:Calcium Radioisotopes; D047169:Cyclotides; D019476:Insect Proteins; D010455:Peptides; D015842:Serine Proteinase Inhibitors; C072986:PMP-D2 peptide, insect; C072987:PMP-C peptide, insect; D020866:omega-Conotoxin GVIA