Conantokin Peptides Research Articles

Classifying neuronal subclasses of the cerebellum through constellation pharmacology.

A pressing need in neurobiology is the comprehensive identification and characterization of neuronal subclasses within the mammalian nervous system. To this end, we used constellation pharmacology as a method to interrogate the neuronal and glial subclasses of the mouse cerebellum individually and simultaneously. We then evaluated the data obtained from constellation-pharmacology experiments by cluster analysis to classify cells into neuronal and glial subclasses, based on their functional expression of glutamate, acetylcholine, and GABA receptors, among other ion channels. Conantokin peptides were used to identify N-methyl-d-aspartate (NMDA) receptor subtypes, which revealed that neurons of the young mouse cerebellum expressed NR2A and NR2B NMDA receptor subunits. Additional pharmacological tools disclosed differential expression of α-amino-3-hydroxy-5-methyl-4-isoxazloepropionic, nicotinic acetylcholine, and muscarinic acetylcholine receptors in different neuronal and glial subclasses. Certain cell subclasses correlated with known attributes of granule cells, and we combined constellation pharmacology with genetically labeled neurons to identify and characterize Purkinje cells. This study illustrates the utility of applying constellation pharmacology to classify neuronal and glial subclasses in specific anatomical regions of the brain.

Synthetic conantokin peptides potently inhibit N-methyl-D-aspartate receptor-mediated currents of retinal ganglion cells.

Retinal ganglion cells (RGCs), which are the sole output neurons of the retina, express N-methyl-D-aspartate receptors (NMDARs), rendering these cells susceptible to glutamate excitotoxicity, with implications for loss of normal RGC excitatory responses in disorders such as glaucoma and diabetic retinopathy. Therefore, antagonists that inhibit NMDAR-mediated currents specifically by targeting the GluN2B component of the ion channel have the potential to serve as a basis for developing potential therapeutics. The roles of peptidic conantokins, which are potent brain neuronal NMDAR inhibitors, were studied. By using patch-clamp whole-cell analyses in dissociated RGCs and retinal whole-mount RGCs, we evaluated the effects of synthetic conantokin-G (conG) and conantokin-T (conT), which are small γ-carboxyglutamate-containing peptides, on NMDA-mediated excitatory responses in mouse RGCs. Both conG and conT inhibited the NMDA-mediated currents of dark-adapted dissociated and whole-mount RGCs in a dose-dependent, reversible, noncompetitive manner. Inhibition of NMDA-mediated steady-state currents by NMDAR nonsubunit-selective conT was approximately threefold greater than GluN2B-selective conG or ifenprodil, demonstrating its potential ability to inhibit both GluN2A- and GluN2B-containing ion channels in RGCs. Because the extent of inhibition of NMDA-evoked currents by conG and the pharmacologic GluN2B-selective inhibitor ifenprodil were similar (40-45%) to that of the GluN2A-selective antagonist NVP-AAM0077, we conclude that the levels of GluN2A and GluN2B subunits are similar in RGCs. These results provide a novel basis for developing effective neuroprotective agents to aid in the prevention of undesired glutamatergic excitotoxicity in neurodegenerative diseases of the retina and demonstrate functional assembly of NMDARs in RGCs.

The crystal structure of the calcium-bound con-G[Q6A] peptide reveals a novel metal-dependent helical trimer.

The ability to form and control both secondary structure and oligomerization in short peptides has proven to be challenging owing to the structural instability of such peptides. The conantokin peptides are a family of γ-carboxyglutamic acid containing peptides produced in the venoms of predatory sea snails of the Conus family. They are examples of short peptides that form stable helical structures, especially in the presence of divalent cations. Both monomeric and dimeric conantokin peptides have been identified and represent a new mechanism of helix association, "the metallozipper motif" that is devoid of a hydrophobic interface between monomers. In the present study, a parallel/antiparallel three-helix bundle was identified and its crystal structure determined at high resolution. The three helices are almost perfectly parallel and represent a novel helix-helix association. The trimer interface is dominated by metal chelation between the three helices, and contains no interfacial hydrophobic interactions. It is now possible to produce stable monomeric, dimeric, or trimeric metallozippers depending on the peptide sequence and metal ion. Such structures have important applications in protein design.

Ca2+-induced self-assembly in designed peptides with optimally spaced gamma-carboxyglutamic acid residues

We have previously elucidated a new paradigm for the metal ion-induced helix–helix assembly in the natural γ-carboxyglutamic acid (Gla)-containing class of conantokin (con) peptides, typified by con-G and a variant of con-T, con-T[K7Gla], independent of the hydrophobic effect. In these “metallo-zipper” structures, Gla residues spaced at i, i+4, i+7, i+11 intervals, which is similar to the arrangement of a and d residues in typical heptads of coiled-coils, coordinate with Ca2+ and form specific antiparallel helical dimers. In order to evaluate the common role of Gla residues in peptide self-assembly, we extend herein the same Gla arrangement to designed peptides: NH2–(γLSγEAK)3–CONH2 (peptide 1) and NH2–γLSγEAKγLSγQANγLSγKAE–CONH2 (peptide 2). Peptide 1 and peptide 2 exhibit no helicity alone, but undergo structural transitions to helical conformations in the presence of a variety of divalent cations. Sedimentation equilibrium ultracentrifugation analyses showed that peptide 1 and peptide 2 form helical dimers in the presence of Ca2+, but not Mg2+. Folding and thiol-disulfide rearrangement assays with Cys-containing peptide variants indicated that the helical dimers are mixtures of antiparallel and parallel dimers, which is different from the strict antiparallel strand orientations of con-G and con-T[K7γGla] dimers. These findings suggest that the Gla arrangement, i, i+4, i+7, i+11, i+14, plays a key role in helix formation, without a strict adherence to strand orientation of the helical dimer.

Conantokin-Br from Conus brettinghami and Selectivity Determinants for the NR2D Subunit of the NMDA Receptor

Conantokins are venom peptides from marine cone snails that are NMDA receptor antagonists. Here, we report the characterization of a 24 AA conantokin from Conus brettinghami Coomans , H. E. , Moolenbeek , R. G. and Wils , E. ( 1982 ) Basteria 46 ( 1/4 ), 3 - 67 , conantokin-Br (con-Br), the first conantokin that does not have the conserved glutamate residue at position 2. Molecular modeling studies suggest that con-Br has a helical structure between residues 2-13. In contrast to other characterized conantokins, con-Br has a high potency for NMDA receptors with NR2D subunits. To identify determinants for NR2D potency, we synthesized chimeras of con-Br and conantokin-R (con-R); the latter has a approximately 30-fold lower potency for the NR2D subtype. The characterization of two reciprocal chimeras (con-Br/R and con-R/Br), comprising the first 9-10 N-terminal AAs of each conantokin followed by the corresponding C-terminal AAs of the other conantokin demonstrates that determinants for NR2D selectivity are at the N-terminal region. Additional analogues comprising 1-3 amino acid substitutions from each peptide into the homologous region of the other led to the identification of a key determinant; a Tyr residue in position 5 increases potency for NR2D, while Val at this locus causes a decrease. The systematic definition of key determinants in the conantokin peptides for NMDA receptor subtype selectivity is an essential component in the development of conantokin peptides that are highly selective for each specific NMDA receptor subtype.

Non-strict strand orientation of the Ca 2+-induced dimerization of a conantokin peptide variant with sequence-shifted γ-carboxyglutamate residues

We have previously found a new mode of metal ion-induced helix–helix assembly for the γ-carboxyglutamate (Gla)-containing, neuroactive conantokin (con) peptides that is independent of the hydrophobic effect. In these unique “metallo-zipper” assemblies of con-G and con-T[K7γ], interhelical Ca 2+ coordination induces dimer formation with strictly antiparallel chain orientation in conantokin peptides in which Gla residues are positioned at “i, i + 4, i + 7, i + 11” intervals. In order to probe the property of self-assembly in conantokin peptides with an extended Gla network, a con-T variant (con-T-tri) was synthesized that contains five Gla residues spaced at “i, i + 4, i + 7, i + 11, i + 14” intervals. Sedimentation equilibrium analyses showed that Ca 2+, but not Mg 2+, was capable of promoting con-T-tri self-assembly. Oxidation and rearrangement assays with Cys-containing con-T-tri variants revealed that the peptide strands in the complex can orient in both parallel and antiparallel forms. Stable parallel and antiparallel dimeric forms of con-T-tri were modeled using disulfide-linked peptides and the biological viability of these species was confirmed by electrophysiology. These findings suggest that small changes within the helix–helix interface of the conantokins can be exploited to achieve desired modes of strand alignment.

NR2B-selective conantokin peptide inhibitors of the NMDA receptor display enhanced antinociceptive properties compared to non-selective conantokins

NR2B-selective inhibitors show lower side-effects in preclinical pain models than non-selective NMDA receptor (NMDAR) antagonists, but it is unclear whether the improved safety of NR2B-selective inhibitors is due to their subtype selectivity or to a unique mode of inhibition of the receptor. In this study, the analgesic effects of intracerebral bolus injections of conantokin peptides with different NMDAR subunit selectivity were determined in mice by the standard hot-plate test, and following stimuli with acetic acid, formalin and complete Freund's adjuvant (CFA). In the standard hot-plate model, con-G[S16Y], a NR2B-selective inhibitor, showed the highest analgesic activity among conantokin peptides tested. In the acetic acid- and CFA-induced pain models, con-G[S16Y] and, to a lesser extent, con-G exhibited higher analgesic activity compared to non-selective inhibitors, such as con-R[1-17]. In the formalin test, while all conantokin peptides could partially suppress the first phase response, only con-G[S16Y] and con-G significantly inhibited the second phase response and suppressed paw edema. Our results suggest that the antinociceptive action of the conantokins may be related to their NR2B-selectivity and that these peptides may be useful as both neurobiological tools for probing mechanisms of nociception and as therapeutic agents for pain relief.

Novel Conantokins from Conus parius Venom Are Specific Antagonists of N-Methyl-D-aspartate Receptors

We report the discovery and characterization of three conantokin peptides from the venom of Conus parius. Each peptide (conantokin-Pr1, -Pr2, and -Pr3) contains 19 amino acids with three gamma-carboxyglutamate (Gla) residues, a post-translationally modified amino acid characteristic of conantokins. The new peptides contain several amino acid residues that differ from previous conantokin consensus sequences. Notably, the new conantokins lack Gla at the 3rd position from the N terminus, where the Gla residue is replaced by either aspartate or by another post-translationally modified residue, 4-trans-hydroxyproline. Conantokin-Pr3 is the first conantokin peptide to have three different post-translational modifications. Conantokins-Pr1 and -Pr2 adopt alpha-helical conformations in the presence of divalent cations (Mg2+ and Ca2+) but are generally unstructured in the absence of divalent cations. Conantokin-Pr3 adopts an alpha-helical conformation even in the absence of divalent cations. Like other conantokins, the new peptides induced sleep in young mice and hyperactivity in older mice upon intracranial injection. Electrophysiological assays confirmed that conantokins-Pr1, -Pr2, and -Pr3 are N-methyl-d-aspartate (NMDA) receptor antagonists, with highest potency for NR2B-containing NMDA receptors. Conantokin-Pr3 demonstrated approximately 10-fold selectivity for NR2B-containing NMDA receptors. However, conantokin-Pr2 showed minimal differences in potency between NR2B and NR2D. Conantokins-Pr1, -Pr2, and -Pr3 all demonstrated high specificity of block for NMDA receptors, when tested against various ligand-gated ion channels. Conus parius conantokins allow for a better definition of structural and functional features of conantokins as ligands targeting NMDA receptors.

Subtype-selective antagonism of N-methyl- d-aspartate receptor ion channels by synthetic conantokin peptides

Conantokin-G (con-G), conantokin-T (con-T), a truncated conantokin-R (con-R[1–17]), that functions the same as wild-type con-R, and variant sequences of con-T, were chemically synthesized and employed to investigate their selectivities as antagonists of glutamate/glycine-evoked ion currents in human embryonic kidney-293 cells expressing various combinations of NMDA receptor (NMDAR) subunits (NR), viz., NR1a/2A, NR1a/2B, NR1b/2A and NR1b/2B. Con-G did not substantially affect ion flow into NR1a,b/NR2A-transfected cells, but potently inhibited cells expressing NR1a,b/NR2B, showing high NR2B selectivity. Con-T and con-R served as non-selective antagonists of all of four NMDAR subunit combinations. C-terminal truncation variants of the 21-residue con-T were synthesized and examined in this regard. While NMDAR ion channel antagonist activity, and the ability to adopt the Ca 2+-induced α-helical conformation, diminished as a function of shortening the COOH-terminus of con-T, NMDAR subtype selectivity was enhanced in the con-T[1–11], con-T[1–9], and con-T[1–8] variants toward NR2A, NR1b, and NR1b/2A, respectively. Receptor subtype selectivity was also obtained with Met-8 sequence variants of con-T. Con-T[M8A] and con-T[M8Q] displayed selectivity with NR2B-containing subunits, while con-T[M8E] showed enhanced activity toward NR1b-containing NMDAR subtypes. Of those studied, the most highly selective variant was con-T[M8I], which showed maximal NMDAR ion channel antagonism activity toward the NR1a/2A subtype. These studies demonstrate that it is possible to engineer NMDAR subtype antagonist specificity into con-T. Since the subunit composition of the NMDAR varies temporally and spatially in developing brain and in various disease states, conantokins with high subtype selectivities are potentially valuable drugs that may be used at specific stages of disease and in selected regions of the brain.

Helix-Helix Interactions between Homo- and Heterodimeric γ-Carboxyglutamate-containing Conantokin Peptides and Their Derivatives

The conantokins are a family of small, naturally occurring gamma-carboxyglutamate (Gla)-rich peptides that specifically antagonize the N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptor. One member of this family, conantokin-G (con-G), undergoes Ca(2+)-mediated self-assembly to form an antiparallel helical dimer. Subunit interactions in this complex are incumbent upon intermolecular Ca(2+) bridging of Gla residues spaced at i, i + 4, i + 7, i + 11 intervals within the monomer. Herein, we further probe the molecular determinants governing such helix-helix interactions. Select variants were synthesized to evaluate the contributions of non-Gla residues to conantokin self-association. Con-G dimerization was shown to be exothermic and accompanied by positive heat capacity changes. Using positional Gla variants of conantokin-R (con-R), a non-dimerizing conantokin, i, i + 4, i + 7, i + 11 Gla spacing alone was shown to be insufficient for self-assembly. The Ca(2+)-dependent antiparallel heterodimerization of con-G and con-T(K7 gamma), two peptides that harbor optimal Gla spacing, was established. Last, the effects of covalently constrained con-G dipeptides on NMDA-evoked current in HEK293 cells expressing combinations of NR1a, NR1b, NR2A, and NR2B subunits of the NMDA receptor were investigated. The antiparallel dipeptide was unique in its ability to potentiate current at NR1a/2A receptors and, like monomeric con-G, was inhibitory at NR1a/2B and NR1b/2B combinations. In contrast, the parallel species was completely inactive at all subunit combinations tested. These results suggest that, under physiological Ca(2+) concentrations, equilibrium levels of con-G dimer most likely exist in an antiparallel orientation and exert effects on NMDA receptor activity that differ from the monomer.

Kinetic and mechanistic characterization of NMDA receptor antagonism by replacement and truncation variants of the conantokin peptides

The characterization of conantokin-T (con-T), conantokin-R (con-R), and variants thereof, using the whole-cell patch clamp technique, was undertaken to evaluate the contribution of various residues towards the onset and recovery of N-methyl- d-aspartate (NMDA) receptor inhibition in cultured embryonic murine hippocampal neurons. The results obtained indicate that the two most C-terminal γ-carboxyglutamic acid (Gla) residues of the conantokins, while not essential for activity, provided for more tenacious binding to the receptor. Specifically, con-T[γ10K/γ14K] and con-R[γ11A/γ15A] displayed 5.6- and 8.4-fold decreases in τ off, respectively, compared to the parent peptides. For the truncated con-T variants, con-T[1-9/Q6G], and a sarcosine (Src)-containing species, con-T[1-9/G1Src/Q6G], the τ off was over 80- and 40-fold faster, respectively, compared to con-T. For the latter peptide, the coapplication of 300 μM spermine enhanced the onset rate constant from 3.1×10 3 M −1 s −1 to 12.6×10 3 M −1 s −1. From analysis of equilibrium dose–inhibition curves using the Cheng–Prusoff equation, a K i value of 1.1 μM for the peptide was obtained. Con-T[1-9/G1Src/Q6G] demonstrated an apparent competitive mode of inhibition relative to NMDA. Schild analysis of the data yielded an equilibrium dissociation constant of 2.4 μM for the interaction of con-T[1-9/G1Src/Q6G] with the receptor.

Structure-function relationships of the NMDA receptor antagonist conantokin peptides.

The three members of the conantokin peptide family identified to date are conantokin(con)-G, -T and -R. Their defining attributes include a high relative content of gamma-carboxyglutamic acid (Gla), N-terminal sequence identity, as well as considerable overall sequence homology, and antagonism of the N-methyl-D-aspartate receptor (NMDAR). As promising templates for the design of neuroprotective agents, a thorough evaluation of structure-function relationships in these peptides will be invaluable in aiding rational drug modeling. To this end, a comprehensive assessment of the contributions of individual residues to conantokin structure and function is required. The current review summarizes recent efforts in this area, and also includes the effects of peptide length, as well as structural-stabilization and -destabilization on the structural and inhibitory profiles of an extensive panel ofconantokin derivatives.

The Amino Acid Residue at Sequence Position 5 in the Conantokin Peptides Partially Governs Subunit-selective Antagonism of RecombinantN-Methyl-d-aspartate Receptors

Whole cell voltage clamp recordings were performed to assess the ability of conantokin-G (con-G), conantokin-T (con-T), and a 17-residue truncated form of conantokin-R (con-R[1-17]) to inhibit N-methyl-d-aspartate (NMDA)-evoked currents in human embryonic kidney 293 cells transiently expressing various combinations of NR1a, NR1b, NR2A, and NR2B receptor subunits. Con-T and con-R[1-17] attenuated ion currents in cells expressing NR1a/NR2A or NR1a/NR2B. Con-G did not affect NMDA-evoked ionic currents in cells expressing NR1a/NR2A, but it showed inhibitory activity in cells expressing NR1a/NR2B receptors and the triheteromeric combination of NR1a/NR2A/NR2B. An Ala-rich con-G analog, con-G[Q6G/gamma7K/N8A/gamma10A/gamma14A/K15A/S16A/N17A] (Ala/con-G, where gamma is Gla), in which all nonessential amino acids were altered to Ala residues, manifested subunit specificity similar to that of con-G, suggesting that the replaced residues are not responsible for selectivity in the con-G framework. A sarcosine-containing con-T truncation analog, con-T[1-9/G1Src/Q6G], inhibited currents in NR1a/NR2A and NR1a/NR2B receptors, eliminating residues 10-21 as mediators of the broad subunit selectivity of con-T. In contrast to the null effects of con-G and Ala/con-G at a NR1a/NR2A-containing receptor, some inhibition ( approximately 40%) of NMDA-evoked currents was effected by these peptides in cells expressing NR1b/NR2A. This finding suggests that the presence of exon 5 in NR1b plays a role in the activity of the conantokins. Analysis of various conantokin analogs demonstrated that Leu(5) of con-G is an important determinant of conantokin selectivity. Taken as a whole, these results suggest that the important molecular determinants on conantokins responsible for NMDA receptor activity and specificity are discretely housed in specific residues of these peptides, thus allowing molecular manipulation of the NMDA receptor inhibitory properties of the conantokins.

Binding of cations to individual gamma-carboxyglutamate residues of conantokin-G and conantokin-T.

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.