Calcium channel diversity and neurotransmitter release: the omega-conotoxins and omega-agatoxins.
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Amino Acid Sequence↗
Biomedical subjects
B M Olivera
Publications and source records attributed to B M Olivera.
Toxityping rat brain calcium channels with omega-toxins from spider and cone snail venoms.
Different types of voltage-sensitive Ca2+ channels in the brain can be defined by specific ligands: L-type Ca2+ channels are uniquely sensitive to dihydropyridines, and N-type Ca2+ channels are selectively blocked by the Conus peptide omega-CTX-GVIA. Cloning data have revealed additional calcium channel types in mammalian brain for which selective ligands would be desirable. We describe binding experiments involving three newer ligands that block dihydropyridine- and omega-CTX-GVIA-resistant Ca channels: omega-Aga-IIIA and omega-Aga-IVA from venom of the spider Agelenopsis aperta and omega-CTX-MVIIC from Conus magus. [125I]omega-Aga-IVA binds with high affinity (IC50 = approximately 50 nM) to receptors in rat brain which may correspond to P-like calcium channels. A second high-affinity site (IC50 = approximately 1 nM) is defined by [125I]omega-CTX-MVIIC, proposed here to be on an "O"-type calcium channel. [125I]omega-Aga-IIIA targets homologous binding sites present on multiple Ca channel types. The IIIA sites overlap with the binding sites for [125I]omega-CTX-GVIA and [125I]omega-CTX-MVIIC. The IIIA sites do not overlap with the site defined by omega-Aga-IVA. Thus toxin ligands may be highly specific for a particular Ca channel (i.e., GVIA for the N-type channel) or exhibit broader specificity (i.e., omega-Aga-IIIA, which appears to bind L-, N-, P-, and O-type Ca2+ channels).
Agatoxins↗
Conantokin-G antagonism of the NMDA receptor subtype expressed in cultured cerebellar granule cells.
Conantokins are peptide antagonists of the N-methyl-D-aspartate (NMDA) subclass of excitatory amino acid receptors. We compared conantokin-G and AP5 antagonism of NMDA receptor activity expressed in cultures of neonatal rat cerebellar granule cells, using the fluorescent calcium indicator dye fura-2. The results were consistent with the binding of two molecules of agonist (NMDA) for channel activation and one antagonist molecule (AP5) for inhibition. However, conantokin-G antagonism was more complex: the peptide inhibited only approximately 70% of the elevation of intracellular free calcium produced by NMDA. These results, when combined with previous ones [8], suggest that conantokin-G may have different affinities for, and functional effects on, different subtypes of NMDA receptor complexes expressed in the mammalian CNS.
2-Amino-5-phosphonovalerate↗
Localization of [125I]omega-conotoxin GVIA binding in human hippocampus and cerebellum.
The peptide toxin omega-conotoxin GVIA (omega-CgTx) has been shown to be a high affinity ligand for N-type calcium channels in the brain. We have employed [125I]omega-CgTx to localize N-type channels in human hippocampus and cerebellum using autoradiography. Ten micron thick slide-mounted tissue sections of human cerebellum and hippocampus were labeled with [125I]omega-CgTx under various conditions. Specific binding to human cerebellum was virtually irreversible and saturable. It was displaceable by the N-channel antagonist, omega-conotoxin MVIIA, but not by L- or P-channel ligands. Binding sites were heterogeneously distributed with denser binding in the molecular layer than the granule cell layer of cerebellum and with specific laminar patterns evident in the hippocampus. [125I]omega-CgTx should be a useful tool for the study of N-type calcium channels in human brain tissue.
Autoradiography↗
A new Conus peptide ligand for Ca channel subtypes.
A cDNA clone encoding a new omega-conotoxin was identified from Conus magus. The predicted peptide was chemically synthesized using a novel strategy that efficiently yielded the biologically active disulfide-bonded isomer. This peptide, omega-conotoxin MVIID, targets other voltage-gated calcium channels besides the N-subtype and exhibits greater discrimination against the N-channel subtype than any other omega-conotoxin variant to date. Consequently, omega-conotoxin MVIID may be a particularly useful ligand for calcium channel subtypes that are not of the L- or N-subclasses. Of the eight major sequence variants of omega-conotoxins that have been elucidated, four come from Conus magus venom. We suggest that sequence variants from the same venom may be designed to optimally interact with different molecular variants of calcium channels; such omega-conotoxin sets from a single venom may therefore be useful for helping to identify novel calcium channel subtypes.
Amino Acid Sequence↗
Biotinylated derivatives of omega-conotoxins GVIA and MVIID: probes for neuronal calcium channels.
The omega-conotoxins are small, disulfide-rich peptides which inhibit voltage-sensitive calcium channels. Biotinylated omega-conotoxins are potentially useful reagents for characterizing distinct subsets of calcium channels. We describe the preparation and characterization of biotinylated derivatives of two specific omega-conotoxins, GVIA and MVIID, which bind different calcium channel subtypes. Eight biotinylated derivatives were tested; all specifically displaced binding of the radiolabeled unbiotinylated omega-conotoxin. In general, the addition of one biotin moiety decreased the apparent affinity for the receptor target site by only approximately 10-fold. However, derivatization of omega-conotoxin MVIID at the Lys10 residue caused a much more marked effect, a ca 500-fold decrease in affinity. These results indicate that the vicinity of the Lys10 residue of omega-conotoxin MVIID may be more critical for binding to the receptor target site than regions around other amino groups in omega-conotoxins GVIA and MVIID. Thus, high affinity biotinylated omega-conotoxin GVIA and MVIID derivatives have been chemically defined; the biotin groups have been shown to be accessible to streptavidin. Given the commercial availability of streptavidin coupled to various reporter groups, the biotinylated omega-conotoxin derivatives described here should be widely useful for fluorescence, electron microscopic or immunological applications.
Amino Acid Sequence↗
Presence of serotonin in the venom of Conus imperialis.
The bioactive components in the venoms of cone snails are largely small peptides which target to a diverse set of ion channels and neuronal receptors. We report here the presence of substantial levels of serotonin in the venom of the imperial cone snail, Conus imperialis. This venom also contains many small bioactive peptides. The identification of serotonin has been confirmed by mass spectroscopy, NMR, HPLC mobility and UV spectroscopy. Although serotonin has been reported in arthropod venoms, it was previously reported to be absent in Conus venoms. We examined a total of fourteen different Conus species, and found detectable serotonin only in the venom of C. imperialis. Conus imperialis specializes in feeding upon the amphinomid polychaete Eurythoe complanata. We speculate that serotonin may play a role in the capture of this prey.
Chromatography, High Pressure Liquid↗
Inhibition of Escherichia coli chemotaxis by omega-conotoxin, a calcium ion channel blocker.
Escherichia coli chemotaxis was inhibited by omega-conotoxin, a calcium ion channel blocker. With Tris-EDTA-permeabilized cells, nanomolar levels of omega-conotoxin inhibited chemotaxis without loss of motility. Cells treated with omega-conotoxin swam with a smooth bias, i.e., tumbling was inhibited.
Calcium Channel Blockers↗
Autoradiographic localization of the binding of calcium channel antagonist, [125I]omega-agatoxin IIIA, in rat brain.
The calcium channel antagonists omega-agatoxin IIIA (omega-Aga-IIIA) and omega-conotoxin GVIA (omega-CgTx) were radioiodinated and used to locate binding sites in the rat brain by receptor autoradiography. While patterns of regional binding to sagittal sections of rat brain were generally similar for the 2 toxins, notable differences in the cerebellum and hippocampus were observed. Specific [125I]omega-Aga-IIIA binding was greatest in the granule cell layers of the cerebellum and of the dentate gyrus. In contrast, binding of [125I]omega-CgTx was most intense in the molecular layers of these structures. Less than one-third of [125I]omega-Aga-IIIA binding in rat brain slices was inhibited by pre-exposure to 250 nM omega-CgTx, while 40 nM omega-Aga-IIIA virtually eliminated the binding of [125I]omega-CgTx under the same conditions. The P-type calcium channel antagonist omega-Aga-IVA blocked only a small fraction of [125I]omega-Aga-IIIA and [125I]omega-CgTx binding. These autoradiographic data are consistent with membrane binding experiments and indicate that the combined use of agatoxins and conotoxins may be useful in the characterization of separate types of neuronal calcium channels.
Agatoxins↗
Novel alpha- and omega-conotoxins from Conus striatus venom.
Three neurotoxic peptides from the venom of Conus striatus have been purified, biochemically characterized, and chemically synthesized. One of these, an acetylcholine receptor blocker designated alpha-conotoxin SII, has the sequence GCCCNPACGPNYGCGTSCS. In contrast to all other alpha-conotoxins, SII has three disulfide bonds (instead of two), has no net positive charge, and has a free C-terminus. The other two paralytic peptides are Ca channel-targeted omega-conotoxins, SVIA and SVIB. omega-SVIA is the smallest natural omega-conotoxin so far characterized and has the sequence CRSSGSPCGVTSICCGRCYRGKCT-NH2. Although omega-conotoxin SVIA is a potent paralytic toxic in lower vertebrate species, it was much less effective in mammals. The third toxin, omega-conotoxin SVIB, has the sequence CKLKGQSCRKTSYDCCSGSCGRSGKC-NH2. This peptide has a different pharmacological specificity from other omega-conotoxins previously purified from Conus venoms; only omega-conotoxin SVIB has proven to be lethal to mice upon ic injection. Binding competition experiments with rat brain synaptosomal membranes indicate that the high-affinity binding site for omega-conotoxin SVIB is distinct from the high-affinity omega-conotoxin GVIA or MVIIA site.
Amino Acid Sequence↗
Heterodimeric structure of the spider toxin omega-agatoxin IA revealed by precursor analysis and mass spectrometry.
We report the first molecular characterization of a precursor sequence for a small, Ca2+ channel blocking, peptide spider toxin, omega-agatoxin IA. By integrating information generated from a molecular genetic approach using agatoxin cDNAs with data provided from mass spectrometry of the mature toxin, we were able to deduce the likely mechanisms by which the toxin precursor peptide is processed to its mature heterodimeric form. A particularly interesting feature of the prepropeptide is the occurrence of two glutamate-rich sequences interposed between the signal sequences, the major peptide toxin, and the minor toxin peptide. Excision of the more distal glutamate-rich region appears to be signaled by flanking arginine residues but likely occurs only after a disulfide linkage has formed between the major and minor chains of the mature toxin. Our molecular genetic approach toward characterizing this toxin will allow us to quickly generate a series of spider sequences from which mature toxin structures can be deduced and eventually expressed. Additionally, this approach will provide insights into the evolutionary divergence observed among spider peptide toxins.
Agatoxins↗
Conantokin-G selectively inhibits N-methyl-D-aspartate-induced currents in Xenopus oocytes injected with mouse brain mRNA.
The conantokins are a family of peptides isolated from the venom of predatory marine snails of the genus Conus. Here we demonstrate that one of these peptides, conantokin-G, specifically inhibits the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors that are expressed in mouse brain mRNA-injected Xenopus oocytes. Increasing the concentration of conantokin-G causes the NMDA dose-response curve to shift to progressively higher concentrations. We therefore conclude that conantokin-G interacts with the glutamate binding site of the receptor. In contrast, the peptide does not compete with glycine, and this indicates that conantokin-G does not act at the binding site of this co-agonist of the NMDA receptor. Furthermore, the inhibitory effects of conantokin-G appear to be insensitive to membrane potential.
Animals↗
alpha-Conotoxins, small peptide probes of nicotinic acetylcholine receptors.
alpha-Conotoxins, a family of small peptides from the venoms of the Conus marine moluscs, are selective, snake alpha-neurotoxin-competitive antagonists of the nicotinic acetylcholine receptor. A new alpha-conotoxin, SIA, has been purified, sequenced, and synthesized. Cross-linking with bivalent reagents and photoaffinity labeling of the acetylcholine receptor with alpha-conotoxin yield covalent adducts. Surprisingly, cross-linking to other subunits is considerably more efficient than to the alpha subunit. The relative efficiency of photoactivatable cross-linking to different subunits of the receptor is a function of placement of the photoactivatable group on the toxin. Since the structures of alpha-conotoxins can be solved by 2D NMR [see Pardi et al. (1989) Biochemistry 28, 5494-5508; Kobayashi et al. (1989) Biochemistry 28, 4853-4860], this family of toxins should provide a set of new ligands for probing the acetylcholine receptor with considerable precision.
Affinity Labels↗
A genetic characterization of the nadC gene of Salmonella typhimurium.
The nadC gene of Salmonella encodes the pyridine biosynthetic enzyme PRPP-quinolinate phosphoribosyltransferase. Using a combination of genetic techniques, a deletion map for the Salmonella nadC gene has been generated which includes over 100 point mutants and 18 deletion intervals. The nadC alleles obtained by hydroxylamine mutagenesis include those suppressed by either amber, ochre, or UGA nonsense suppressors as well as alleles suppressed by the missense suppressor, sumA. Deletions were obtained by three separate protocols including spontaneous selection for loss of the nearby aroP gene, recombination between aroP::MudA and nadC::MudA insertion alleles, and selection for spontaneous loss of tetracycline resistance in a nearby guaC::Tn10dTc insertion mutant allele. The nadC mutants comprise one complementation group and the nadC+ allele is dominant to simple, nadC auxotrophic mutant alleles. Intragenic complementation of two nadC alleles, nadC493 and nadC494, mapping to deletion intervals 17 and 18, respectively, suggests that nadC encodes a multimeric enzyme. Both nadC and the nearby aroP locus are transcribed counterclockwise on the standard genetic map of Salmonella, in opposite orientation to the direction of chromosome replication.
Chromosome Mapping↗
Calcium channel-targeted polypeptide toxins.
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Amino Acid Sequence↗
Activity of the nicotinamide mononucleotide transport system is regulated in Salmonella typhimurium.
Transport of nicotinamide mononucleotide (NMN) requires two functions, NadI(T) and PnuC. The PnuC protein is membrane associated, as judged by isolation of active TnphoA gene fusions and demonstration that the fusion protein is membrane associated. The PnuC function appears to be the major component of the transport system, since mutant alleles of the pnuC gene permit NMN transport in the absence of NadI(T) function. We present evidence that the activity of the NMN transport system varies in response to internal pyridine levels (presumably NAD). This control mechanism requires NadI(T) function, which is provided by a bifunctional protein encoded by the nadI gene (called nadR by Foster and co-workers [J. W. Foster, Y. K. Park, T. Fenger, and M. P. Spector, J. Bacteriol. 172:4187-4196]). The nadI protein regulates transcription of the nadA and nadB biosynthetic genes and modulates activity of the NMN permease; both regulatory activities respond to the internal pyridine nucleotide level.
Bacterial Proteins↗
Conantokin-G: a novel peptide antagonist to the N-methyl-D-aspartic acid (NMDA) receptor.
Conantokin-G is a 17 amino acid peptide isolated from the venom of the fish-eating snail Conus geographus which produces hyperactivity when injected into the brains of adult mice. We show that this peptide is a selective N-methyl-D-aspartate (NMDA) antagonist based on its ability to block NMDA-induced elevation of cGMP in rat cerebellar slices in vitro (IC50 = 171 nM), but not kainic acid-induced elevations. This inhibition could not be overcome by increasing the NMDA concentration, indicating non-competitive inhibition. Conantokin-G displayed no affinity for binding sites for thienylcyclohexylpiperidine, various glutamate subclasses or those for several other neurotransmitters/neuromodulators. This peptide, however, enhanced [3H]glycine binding to rat forebrain membranes but not to spinal cord membranes. The activity profile of the peptide in various assays indicates that it is a novel type of non-competitive NMDA antagonist.
Amino Acid Sequence↗