Electrophysiological Characterization of a Novel Conotoxin That Blocks Molluscan Sodium Channels

A novel peptide toxin, PnIVB, isolated from the venom of Conus pennaceus blocks voltage-gated sodium current in Aplysia neurons. Complete blockade is obtained at a PnIVB concentration of 80±2.2 nM and 50% blockade at 16±0.86 nM. The potency of PnIVB in blocking Aplysia sodium current is four orders of magnitude larger than that of tetrodotoxin. The toxin has no paralytic activity when injected into fish. The rapid blockade of sodium current by PnIVB is not associated with a change in the activation or inactivation kinetics of the current, or with the reversal potential. Sodium current blockade is reversible after a 30 min wash with 50 times the bath volume. The novel conotoxin PnlVB can be used as a powerful tool for mollusc neurobiological research and as a molecular probe to explore the structure-function relations of voltage-gated sodium channel subtypes.     

A novel conotoxin inhibiting vertebrate voltage-sensitive potassium channels.

Toxins from cone snail (Conus species) venoms are multiple disulfide bonded peptides. Based on their pharmacological target (ion channels, receptors) and their disulfide pattern, they have been classified into several toxin families and superfamilies. Here, we report a new conotoxin, which is the first member of a structurally new superfamily of Conus peptides and the first conotoxin affecting vertebrate K+ channels. The new toxin, designated conotoxin ViTx, has been isolated from the venom of Conus virgo and comprises a single chain of 35 amino acids cross-linked by four disulfide bridges. Its amino acid sequence (SRCFPPGIYCTSYLPCCWGICCSTCRNVCHLRIGK) was partially determined by Edman degradation and deduced from the nucleotide sequence of the toxin cDNA. Nucleic acid sequencing also revealed a prepropeptide comprising 67 amino acid residues and demonstrated a posttranslational modification of the protein by releasing a six-residue peptide from the C-terminal. Voltage clamp studies on various ion channels indicated that the toxin inhibits the vertebrate K+ channels Kv1.1 and Kv1.3 but not Kv1.2. The chemically synthesized product exhibited the same physiological activity and identical molecular mass (3933.7 Da) as the native toxin.     

Functional profiling of neurons through cellular neuropharmacology

We describe a functional profiling strategy to identify and characterize subtypes of neurons present in a peripheral ganglion, which should be extendable to neurons in the CNS. In this study, dissociated dorsal-root ganglion neurons from mice were exposed to various pharmacological agents (challenge compounds), while at the same time the individual responses of >100 neurons were simultaneously monitored by calcium imaging. Each challenge compound elicited responses in only a subset of dorsal-root ganglion neurons. Two general types of challenge compounds were used: agonists of receptors (ionotropic and metabotropic) that alter cytoplasmic calcium concentration (receptor-agonist challenges) and compounds that affect voltage-gated ion channels (membrane-potential challenges). Notably, among the latter are K-channel antagonists, which elicited unexpectedly diverse types of calcium responses in different cells (i.e., phenotypes). We used various challenge compounds to identify several putative neuronal subtypes on the basis of their shared and/or divergent functional, phenotypic profiles. Our results indicate that multiple receptor-agonist and membrane-potential challenges may be applied to a neuronal population to identify, characterize, and discriminate among neuronal subtypes. This experimental approach can uncover constellations of plasma membrane macromolecules that are functionally coupled to confer a specific phenotypic profile on each neuronal subtype. This experimental platform has the potential to bridge a gap between systems and molecular neuroscience with a cellular-focused neuropharmacology, ultimately leading to the identification and functional characterization of all neuronal subtypes at a given locus in the nervous system.     

Identification of a novel S-superfamily conotoxin from vermivorous Conus caracteristicus

Conotoxins have been classified into several different superfamilies based on the highly conserved signal peptide sequences of their precursors. However, little is known about the five disulfide bonds containing S-superfamily conotoxins. Only two S-superfamily conotoxins have been identified but their cDNAs are not reported. In this work, we identified a novel S-superfamily conotoxin ca8a from vermivorous Conus caracteristicus. Its sequence shares no homology with those of two other previously reported toxins of the same superfamily, but they have the same cysteine framework, in particular the CX(3)CXC-CXC-CXCXC pattern at the C-terminal part. This implies that these toxins might have the same spatial scaffold, but different local conformation or residue side chains may be the cause of their different biological functions. Furthermore, the cDNA of ca8a was cloned with the RACE method. ca8a has a signal peptide sequence different from those of other conotoxins. This gives a defining feature of S-superfamily conotoxins and led to the cloning of more S-superfamily conotoxins from cone snails of different prey types, which indicates that S-superfamily conotoxins widely exist. These results will certainly enrich our understanding of the highly diversified S-superfamily conotoxins.     

Pharmacological dissection of calcium channel subtype-related components of strontium inflow in large mossy fiber boutons of mouse hippocampus

Several subtypes of voltage-dependent calcium channels (VDCCs) are present in the presynaptic terminals. In the mammalian hippocampus, P/Q-, N-, and R- but not L-type VDCCs are involved in the fast transmitter release from large mossy fiber (MF) boutons, which are associated with CA3 pyramidal cell dendrites. We investigated whether L-type VDCCs are indeed absent in these large MF boutons. With the use of Sr2+ as the Ca2+ substitute, the stimulus-evoked Sr2+ increment (delta[Sr2+]pre) was evaluated fluorometrically. Delta[Sr2+]pre appeared to be proportional to Sr2+ inflow through VDCCs and was specifically attenuated by conventional VDCC subtype-selective antagonists. The P/Q-type selective omega-agatoxin IVA (AgTx(IVA)) blocked delta[Sr2+]pre with an IC50 of 28 nM and by 30-35% at its maximum effective concentration of 0.5 microM. The N-type selective omega-conotoxin GVIA (CgTx(GVIA)) blocked delta[Sr2+]pre with an IC50 of 15 nM and by 20-25% at its maximum effective concentration of 1 microM. The R-type selective SNX-482 blocked delta[Sr2+]pre with an IC50 of 79 nM and by 20-25% at its maximum effective concentration of 1 microM. The effects of these toxins did not overlap at their maximum effective concentrations and about 70-80% of delta[Sr +]pre was blocked by the simultaneous exposure to these toxins. delta[Sr2+]pre component that is resistant to AgTx(IVA), CgTx(IVA), and SNX-482 was significantly potentiated by an L-type agonist, (S)-(-)-Bay K8644, and attenuated by an L-type antagonist, nimodipine, suggesting that L-type VDCCs are present in large MF terminals. The L-type agonist, (+/-)-Bay K8644, also potentiated Sr2+ inflow into individual boutons identified as large MF boutons under confocal microscopy. Almost similar results were observed for Ca2+ inflow-dependent fluorescence increments. L-type VDCCs appear to be present in large MF boutons and mediate a substantial Ca2+ inflow into presynaptic terminals during action potentials.     

Refined solution structure of ω-conotoxin GVIA: implications for calcium channel binding

The polypeptide ω-conotoxin GVIA (GVIA) is an N-type calcium channel blocker from the venom of Conus geographus, a fish-hunting cone shell. Here we describe a high-resolution solution structure of this member of the ‘inhibitor cystine knot’ protein family. The structure, based on NMR data acquired at 600 MHz, has mean pairwise RMS differences of 0.25 ± 0.06 and 1.07 ± 0.14 Å over the backbone heavy atoms and all heavy atoms, respectively. The solvent-accessible side chains are better defined than in previously published structures and provide an improved basis for docking GVIA with models of the calcium channel. Moreover, some side chain interactions important in GVIA folding in vitro and in stabilizing the native structure are defined clearly in the refined structure. Two qualitatively different backbone conformations in the segment from Thr11 to Asn14 persisted in the restrained simulated annealing calculations until a small number of lower bound constraints was included to prevent close contacts from occurring that did not correspond with peaks in the NOESY spectrum. It is possible that GVIA is genuinely flexible at this segment, spending a finite time in the alternative conformation, and this may influence its interaction with the calcium channel.     

From the identification of gene organization of α conotoxins to the cloning of novel toxins

In the venoms of cone snails, alpha conotoxins are competitive antagonists of nicotinic acetylcholine receptors. Eleven novel cDNA and eight partial gene sequences (including two pseudogenes) of alpha conotoxins were identified from five species of cone snail. As expected, every cDNA encodes a precursor of prepropeptide. In all the partial genes of alpha conotoxins identified, there is a long intron inserted at a fixed position in the pro-region, dividing the encoding region into two exons. The mutation rate in exon I (encoding the signal peptide and a part of pro-region) is much lower than that in exon II (encoding the other part of pro-region, the mature peptide and 3' untranslational region). Interestingly, the sequences at the 5' and 3' end of introns are highly conserved. In addition, in the identified introns exist long dinucleotide (e.g. "GT", "CA") or trinucleotide ("CAT") repeats. In the special case of Pu 1.1, there are five almost identical repeats of a 150 bp sequence in the long intron. Taking advantage of the conserved 3' end sequence of intron, 16 alpha conotoxins, as well as a pseudogene and three kappa A conotoxins, were identified from their genomic DNAs. Based on the comparison of these cDNA and gene sequences, a hypothesis of the alpha conotoxin evolution was proposed.     

Use-dependent blockade of Cav2.2 voltage-gated calcium channels for neuropathic pain

The translocation of extracellular calcium (Ca(2+)) via voltage-gated Ca(2+) channels (VGCCs) in neurons is involved in triggering multiple physiological cell functions but also the abnormal, pathophysiological responses that develop as a consequence of injury. In conditions of neuropathic pain, VGCCs are involved in supplying the signal Ca(2+) important for the sustained neuronal firing and neurotransmitter release characteristic of these syndromes. Preclinical data have identified N-type VGCCs (Ca(v)2.2) as key participants in contributing to these Ca(2+) signaling events and clinical data with the peptide blocker Prialt have now validated Ca(v)2.2 as a bona fide target for future drug discovery efforts to identify new and novel therapeutics for neuropathic pain. Imperative for the success of such an endeavor will be the ability to identify compounds selective for Ca(v)2.2, versus other VGCCs, but also compounds which demonstrate effective blockade during the pathophysiological states of neuropathic pain without compromising channel activity associated with sustaining normal housekeeping cellular functions. An approach to obtain this research target profile is to identify compounds, which are more potent in blocking Ca(v)2.2 during higher frequencies of firing as compared to the slower more physiologically-relevant frequencies. This may be achieved by identifying compounds with enhanced potency for the inactivated state of Ca(v)2.2. This commentary explores the rationale and options for engineering a use-dependent blocker of Ca(v)2.2. It is anticipated that this use-dependent profile of channel blockade will result in new chemical entities with an improved therapeutic ratio for neuropathic pain.     

Synthesis and immunological studies of α‐conotoxin chimera containing an immunodominant epitope from the 268–284 region of HSV gD protein

Abstract: We have synthesized and characterized new chimeric peptides by inserting an epitope of the glycoprotein D (gD) of herpes simplex virus (HSV) serotype 1 as ‘guest’ sequence in the ‘host’ structure of α‐conotoxin GI, a 13‐residue peptide (ECCNPACGRHYSC) isolated from the venom of Conus geographus. The 276–284 region of HSV gD‐1 selected for these studies is highly hydrophilic and adopts a β‐turn. The α‐conotoxin GI also contains a β‐turn in the 8–12 region, stabilized by two disulfide bridges at positions 2–7 and 3–13. Thus, the tetramer sequence of α‐conotoxin, ⁸Arg‐His‐Tyr‐Ser¹² has been replaced by Asp‐Pro‐Val‐Gly (DPVG), identified previously as the epitope core. The syntheses were performed by Fmoc strategy on Rink resin and DTNB or air oxidation were applied for the formation of the first 3–13 disulfide bond in the presence of guanidinium hydrochloride. For the formation of the second disulfide Cys²‐Cys⁷ three different oxidation procedures [iodine in 95% acetic acid, air oxidation in dimethyl sulfoxide/1 m HCl or Tl(tfa)₃ in trifluoroacetic acid (TFE)] were compared. The high‐performance liquid chromatography purified peptides were characterized by electrospray mass spectrometry and amino acid analysis. The bicyclic HSV‐α‐[Tyr¹]‐conotoxin chimeric peptide and native α‐conotoxin GI showed similar circular dichroism spectra in phosphate‐buffered saline (PBS) and in a PBS‐TFE 1 : 1 (v/v) mixture, which might suggest that these compounds also share similar secondary structures. In immunologic studies the characteristics of the primary and of the memory immunoglobulin (Ig) M‐ and IgG‐type antibody responses showed that the bicyclic HSV‐α‐[Tyr¹]‐conotoxin chimera is capable to induce strong antibody responses in C57/Bl/6 mice but was poorly immunogenic in CBA and BALB/c mice. Data obtained with the C57/Bl/6 serum indicate that the polyclonal antibodies recognize the DPVG motif presented in the bicyclic HSV‐α‐[Tyr¹]‐conotoxin and some reactivity was also found with the monocyclic but not with the linear form of the chimera. Results with two IgM type monoclonal antibodies from a bicyclic HSV‐α‐[Tyr¹]‐conotoxin immunized C57/Bl/6 mouse also point to the specific interaction with the DPVG sequence. Taken together these studies suggest, that the relative intensity of DPVG‐specific responses was found to be dependent on the mouse strain and on the conformation of the chimeric molecules. We found that the IgM monoclonal antibodies are able to recognize the linear DPVG sequence, while the majority of IgG antibodies is directed to the same motif in a conformation stabilized by double cyclization.