Conantokin-T

Binding of cations to individual gamma-carboxyglutamate residues of conantokin-G and conantokin-T.[Pubmed:10406223]

J Pept Res. 1999 Apr;53(4):453-64.

Conantokin-G (con-G) and Conantokin-T (con-T) are naturally occurring gamma-carboxyglutamate (Gla)-containing peptides that interact with multivalent cations in functionally relevant manners. Selective 13C-enrichment of Cgamma and Cdelta in each of the Gla residues has allowed metal binding affinities to be measured at individual side chains. Con-T possesses two metal binding sites, one with high affinity at Gla10/Gla14 and another with weak binding at Gla3/Gla4. Con-G contains two sites of comparable low affinity for Ca2+. Analysis of the 13C line-widths of con-G in the presence of Mg2+ allowed the order of metal binding to be determined, with Gla10/Gla14 loading before the Gla3/Gla4/Gla7 cluster. While the variant peptide, apo-con-T[Lys7Gla], was shown to have a very low alpha-helical content, this peptide binds a second metal with much greater affinity than wild-type con-T. This provides additional evidence that Gla7 in con-G is primarily responsible for destabilizing the apo-form, but is an important ligand for metal chelation. The residue-specific alpha-helical stabilities of con-G and con-T in their metal-free and metal-loaded states were estimated by determining rates of proton exchange from backbone peptide bond amides with deuterium atoms from 2H20-containing solvents. For both peptides, the lifetimes of protons on several peptide bond amides increased as metals of higher affinity were bound to the peptides, with the longest half-lives found in the region of the alpha-helical turn stabilized by the Gla10/Gla14 metal coordination site. We propose that Gla10 and Gla14 constitute the primary tight metal ion binding site in both peptides. This detailed analysis with physiologically relevant metal cations is crucial for deciphering the roles of critical amino acids in the bioactivity of the conantokin peptides.

Amino acid determinants for NMDA receptor inhibition by conantokin-T.[Pubmed:11331410]

J Neurochem. 2001 May;77(3):812-22.

Several derivatives of Conantokin-T (con-T), a naturally occurring, gamma-carboxyglutamate (Gla)-containing peptide with NMDA receptor (NMDAR) antagonist properties, were synthesized and evaluated for their ability to displace [(3)H]MK-801 from adult rat forebrain membranes. Analyses of progressive C-terminal truncation analogs of the parent 21-mer revealed gradual losses in activity with decreased chain length. In this series, con-T[1-8] was identified as the shortest variant capable of manifesting inhibitory activity (< 1% of the parent peptide). Ala substitution studies of individual residues identified Gly1, Gla3, Met8 and Leu12 as important for activity, while Glu2, Gla4 and Tyr5 were shown to be essential in this regard. The effect of side-chain length and charge in the N-terminal region was probed by single amino acid replacements. No correlation was observed between potencies and circular dichroism-derived helical contents of the con-T derivatives. Further elaboration of structure-function relationships in con-T was effected through the design and synthesis of helically constrained and destabilized analogs. The results of the current study were compared with those of a previous investigation on con-G, a related conantokin. Substantial differences in activity requirements were noted between the peptides, particularly in the C-terminal regions. Chimeras of con-T and con-G were generated and revealed virtually no interchangeability of residues between these two peptides. Finally, single amino acid substitutions that resulted in analogs with enhanced inhibitory properties were combined to yield superior conantokin-based NMDAR inhibitors.

Inhibition of NMDA-induced currents by conantokin-G and conantokin-T in cultured embryonic murine hippocampal neurons.[Pubmed:10608277]

Neuropharmacology. 1999 Dec;38(12):1819-29.

Conantokin-G (con-G) and Conantokin-T (con-T) are small (17 and 21 amino acids, respectively) gamma-carboxyglutamate (Gla) containing peptides derived from the venoms of marine cone snails that are potent and selective inhibitors of N-methyl-D-aspartate (NMDA) receptors. In this study, the effects of con-G and con-T on NMDA-evoked responses were evaluated in mouse primary hippocampal neuronal cultures using the whole-cell patch-clamp technique. Under equilibrium conditions, NMDA-induced currents were inhibited by con-G and con-T (10 nM-100 microM) in a dose-dependent manner while maintaining a holding potential of -70 mV. In the presence of saturating amounts of NMDA (100 microM) and glycine (1 microM), the IC50 values obtained were 487 +/- 85 nM for con-G and 1030 +/- 130 nM for con-T. NMDA (10 microM-1 mM) dose-response curves produced in the presence of con-G or con-T (1 or 3 microM) resulted in a downward shift of the current response at saturation with NMDA, without affecting the EC50. The maximum response obtainable in the absence of peptide could not be achieved by increasing concentrations of NMDA. The same effect was also observed for conantokin inhibition of spermine-potentiated responses. Association rate constants (k(on)) for the peptides were determined in the presence of NMDA and glycine, with and without the addition of spermine. Using a single binding site bimolecular model, k(on) values were 3.1 +/- 0.2 x 10(3) M(-1) s(-1) for con-G and 3.2 +/- 0.1 x 10(3) M(-1) s(-1) for con-T in the absence of spermine. The added presence of a saturating amount of spermine (300 microM) resulted in an approximate 60% increase in the k(on) values for both con-G and con-T. These results demonstrate that con-T and con-G inhibit NMDA-evoked currents, as well as the potentiation by spermine, in what appears to be a noncompetitive manner, and that spermine increases the rate of conantokin inhibition.

Inhibition of MK801 binding in adult rat brain sections by conantokin-G and conantokin-T.[Pubmed:10515186]

Neurosci Lett. 1999 Oct 8;273(3):171-4.

The functional interactions of conantokins with anatomical sites in rat brain have been assessed through displacement of the non-competitive N-methyl-D-aspartate receptor (NMDAR) antagonist, dizocilpine (MK801). The binding of (+)-3-[125I]-iodo-MK801 (1 nM) to coronal sections from adult rat brain was inhibited in a dose-dependent manner by Conantokin-T (con-T) and conantokin-G (con-G). Quantitative densitometry was used to determine IC50 values for conantokin inhibition of [125I]-MK801 binding in the cortex, thalamus and hippocampus. Con-T completely inhibited [125I]-MK801 specific binding in all brain regions at a saturating concentration of 100 microM. Con-G was able to completely displace [125I]-MK801 in the cortex and thalamus, but only inhibited this same binding up to approximately 90% in the hippocampus. Both peptides maintained their inhibitory properties in the presence of 1 mM EDTA, suggesting that divalent cations are not required for their action in this regard. The added presence of spermine (150 microM) resulted in a two-fold increase in [125I]-MK801 binding and a two-fold decrease in the IC50 values for both peptides. The data obtained in this investigation further demonstrate that [125I]-MK801 is a useful probe for the indirect determination of functional NMDAR ligand binding sites in rat brain sections.

Conantokin-T. A gamma-carboxyglutamate containing peptide with N-methyl-d-aspartate antagonist activity.[Pubmed:2180939]

J Biol Chem. 1990 Apr 15;265(11):6025-9.

Conantokin-T, a 21-amino acid peptide which induces sleep-like symptoms in young mice was purified from the venom of the fish-hunting cone snail, Conus tulipa. The amino acid sequence of the peptide was determined and verified by chemical synthesis. The peptide has 4 residues of the modified amino acid, gamma-carboxyglutamate (Gla). The sequence of the peptide is: Gly-Glu-Gla-Gla-Tyr-Gln-Lys-Met-Leu-Gla-Asn-Leu-Arg-Gla-Ala-Glu-Val-Lys- Lys-Asn-Ala-NH2. Conantokin-T inhibits N-methyl-D-aspartate (NMDA) receptor-mediated calcium influx in central nervous system neurons. This observation suggests that like conantokin-G (a homologous Conus peptide with recently identified NMDA antagonist activity) Conantokin-T has NMDA antagonist activity. A sequence comparison of conantokins-T and -G identifies the 4 Gla residues and the N-terminal dipeptide sequence as potential key elements for the biological activity of this peptide.