Marion L. Loughnan

Novel omega -Conotoxins from Conus catus Discriminate among Neuronal Calcium Channel Subtypes

ω-Conotoxins selective for N-type calcium channels are useful in the management of severe pain. In an attempt to expand the therapeutic potential of this class, four new ω-conotoxins (CVIA–D) have been discovered in the venom of the piscivorous cone snail, Conus catus, using assay-guided fractionation and gene cloning. Compared with other ω-conotoxins, CVID has a novel loop 4 sequence and the highest selectivity for N-type over P/Q-type calcium channels in radioligand binding assays. CVIA−D also inhibited contractions of electrically stimulated rat vas deferens. In electrophysiological studies, ω-conotoxins CVID and MVIIA had similar potencies to inhibit current through central (α1B-d) and peripheral (α1B-b) splice variants of the rat N-type calcium channels when coexpressed with rat β3 in Xenopus oocytes. However, the potency of CVID and MVIIA increased when α1B-d and α1B-b were expressed in the absence of rat β3, an effect most pronounced for CVID at α1B-d (up to 540-fold) and least pronounced for MVIIA at α1B-d (3-fold). The novel selectivity of CVID may have therapeutic implications. 1H NMR studies reveal that CVID possesses a combination of unique structural features, including two hydrogen bonds that stabilize loop 2 and place loop 2 proximal to loop 4, creating a globular surface that is rigid and well defined.

Isolation, Structure, and Activity of GID, a Novel alpha 4/7-Conotoxin with an Extended N-terminal Sequence

Using assay-directed fractionation ofConus geographus crude venom, we isolated α-conotoxin GID, which acts selectively at neuronal nicotinic acetylcholine receptors (nAChRs). Unlike other neuronally selective α-conotoxins, α-GID has a four amino acid N-terminal tail, γ-carboxyglutamate (Gla), and hydroxyproline (O) residues, and lacks an amidated C terminus. GID inhibits α7 and α3β2 nAChRs with IC50values of 5 and 3 nm, respectively and is at least 1000-fold less potent at the α1β1γδ, α3β4, and α4β4 combinations. GID also potently inhibits the α4β2 subtype (IC50 of 150 nm). Deletion of the N-terminal sequence (GIDΔ1–4) significantly decreased activity at the α4β2 nAChR but hardly affected potency at α3β2 and α7 nAChRs, despite enhancing the off-rates at these receptors. In contrast, Arg12 contributed to α4β2 and α7 activity but not to α3β2 activity. The three-dimensional structure of GID is well defined over residues 4–19 with a similar motif to other α-conotoxins. However, despite its influence on activity, the tail appears to be disordered in solution. Comparison of GID with other α4/7-conotoxins which possess an NN(P/O) motif in loop II, revealed a correlation between increasing length of the aliphatic side-chain in position 10 (equivalent to 13 in GID) and greater α7 versus α3β2 selectivity.

Identification of a novel class of nicotinic receptor antagonists : Dimeric conotoxins VxXIIA, VxXIIB, and VxXIIC from conus vexillum

The venoms of predatory marine snails (Conus spp.) contain diverse mixtures of peptide toxins with high potency and selectivity for a variety of voltage-gated and ligand-gated ion channels. Here we describe the chemical and functional characterization of three novel conotoxins, αD-VxXIIA, αD-VxXIIB, and αD-VxXIIC, purified from the venom of Conus vexillum. Each toxin was observed as an ∼11-kDa protein by LC/MS, size exclusion chromatography, and SDS-PAGE. After reduction, the peptide sequences were determined by Edman degradation chemistry and tandem MS. Combining the sequence data together with LC/MS and NMR data revealed that in solution these toxins are pseudo-homodimers of paired 47-50-residue peptides. The toxin subunits exhibited a novel arrangement of 10 conserved cystine residues, and additional post-translational modifications contributed heterogeneity to the proteins. Binding assays and two-electrode voltage clamp analyses showed that αD-VxXIIA, αD-VxXIIB, and αD-VxXIIC are potent inhibitors of nicotinic acetylcholine receptors (nAChRs) with selectivity for α7 and β2 containing neuronal nAChR subtypes. These dimeric conotoxins represent a fifth and highly divergent structural class of conotoxins targeting nAChRs.

Isolation and Structure-Activity of μ-Conotoxin TIIIA, A Potent Inhibitor of Tetrodotoxin-Sensitive Voltage-Gated Sodium Channels

μ-Conotoxins are three-loop peptides produced by cone snails to inhibit voltage-gated sodium channels during prey capture. Using polymerase chain reaction techniques, we identified a gene sequence from the venom duct of Conus tulipa encoding a new μ-conotoxin-TIIIA (TIIIA). A 125I-TIIIA binding assay was established to isolate native TIIIA from the crude venom of Conus striatus. The isolated peptide had three post-translational modifications, including two hydroxyproline residues and C-terminal amidation, and <35% homology to other μ-conotoxins. TIIIA potently displaced [3H]saxitoxin and 125I-TIIIA from rat brain (Nav1.2) and skeletal muscle (Nav1.4) membranes. Alanine and glutamine scans of TIIIA revealed several residues, including Arg14, that were critical for high-affinity binding to tetrodotoxin (TTX)-sensitive Na+ channels. We were surprised to find that [E15A]TIIIA had a 10-fold higher affinity than TIIIA for TTX-sensitive sodium channels (IC50, 15 vs. 148 pM at rat brain membrane). TIIIA was selective for Nav1.2 and -1.4 over Nav1.3, -1.5, -1.7, and -1.8 expressed in Xenopus laevis oocytes and had no effect on rat dorsal root ganglion neuron Na+ current. 1H NMR studies revealed that TIIIA adopted a single conformation in solution that was similar to the major conformation described previously for μ-conotoxin PIIIA. TIIIA and analogs provide new biochemical probes as well as insights into the structure-activity of μ-conotoxins.

α-Conotoxins EpI and AuIB switch subtype selectivity and activity in native versus recombinant nicotinic acetylcholine receptors

The Xenopus laevis oocyte expression system was used to determine the activities of α‐conotoxins EpI and the ribbon isomer of AuIB, on defined nicotinic acetylcholine receptors (nAChRs). In contrast to previous findings on intracardiac ganglion neurones, α‐EpI showed no significant activity on oocyte‐expressed α3β4 and α3β2 nAChRs but blocked the α7 nAChR with an IC50 value of 30 nM. A similar IC50 value (103 nM) was obtained on the α7/5HT3 chimeric receptor stably expressed in mammalian cells. Ribbon AuIB maintained its selectivity on oocyte‐expressed α3β4 receptors but unlike in native cells, where it was 10‐fold more potent than native α‐AuIB, had 25‐fold lower activity. These results indicate that as yet unidentified factors influence α‐conotoxin pharmacology at native versus oocyte‐expressed nAChRs.

Allosteric α1-Adrenoreceptor antagonism by the conopeptide ρ-TIA

A peptide contained in the venom of the predatory marine snail Conus tulipa, ρ-TIA, has previously been shown to possess α1-adrenoreceptor antagonist activity. Here, we further characterize its pharmacological activity as well as its structure-activity relationships. In the isolated rat vas deferens, ρ-TIA inhibited α1-adrenoreceptor-mediated increases in cytosolic Ca2+ concentration that were triggered by norepinephrine, but did not affect presynaptic α2-adrenoreceptor-mediated responses. In radioligand binding assays using [125I]HEAT, ρ-TIA displayed slightly greater potency at the α1B than at the α1A or α1D subtypes. Moreover, although it did not affect the rate of association for [3H]prazosin binding to the α1B-adrenoreceptor, the dissociation rate was increased, indicating non-competitive antagonism by ρ-TIA. N-terminally truncated analogs of ρ-TIA were less active than the full-length peptide, with a large decline in activity observed upon removal of the fourth residue of ρ-TIA (Arg4). An alanine walk of ρ-TIA confirmed the importance of Arg4 for activity and revealed a number of other residues clustered around Arg4 that contribute to the potency of ρ-TIA. The unique allosteric antagonism of ρ-TIA resulting from its interaction with receptor residues that constitute a binding site that is distinct from that of the classical competitive α1-adrenoreceptor antagonists may allow the development of inhibitors that are highly subtype selective.

Inhibition of Neuronal Nicotinic Acetylcholine Receptor Subtypes by α-Conotoxin GID and Analogues

α-Conotoxins are small disulfide-rich peptides from the venom of the Conus species that target the nicotinic acetylcholine receptor (nAChR). They are valuable pharmacological tools and also have potential therapeutic applications particularly for the treatment of chronic pain. α-Conotoxin GID is isolated from the venom of Conus geographus and has an unusual N-terminal tail sequence that has been shown to be important for binding to the α4β2 subtype of the nAChR. To date, only four conotoxins that inhibit the α4β2 subtype have been characterized, but they are of considerable interest as it is the most abundant nAChR subtype in the mammalian brain and has been implicated in a range of diseases. In this study, analysis of alanine-scan and truncation mutants of GID reveals that a conserved proline in α-conotoxins is important for activity at the α7, α3β2, and α4β2 subtypes. Although the proline residue was the most critical residue for activity at the α3β2 subtype, Asp3, Arg12, and Asn14 are also critical at the α7 subtype. Interestingly, very few of the mutations tested retained activity at the α4β2 subtype indicating a tightly defined binding site. This lack of tolerance to sequence variation may explain the lack of selective ligands discovered for the α4β2 subtype to date. Overall, our findings contribute to the understanding of the structure-activity relationships of α-conotoxins and may be beneficial for the ongoing attempts to exploit modulators of the neuronal nAChRs as therapeutic agents.

Isolation and Characterization of Conopeptides by High‐performance Liquid Chromatography Combined with Mass Spectrometry and Tandem Mass Spectrometry

The sensitivity of mass spectrometry combined with the separatory power of high-performance liquid chromatography was used to investigate the venom of individual cone shells, This analysis has revealed that cone venoms contain a complex mixture of peptides which vary quantitatively and qualitatively both between and within species, A differential alkylation procedure followed by tandem mass spectrometric analysis can be used to determine the disulfide bond connectivity in these small, cysteine-rich peptides.

Novel alpha D-conopeptides and their precursors identified by cDNA cloning define the D-conotoxin superfamily.

AlphaD-conotoxins are peptide inhibitors of nicotinic acetylcholine receptors (nAChRs) first described from Conus vexillum (alphaD-VxXIIA-C and renamed here to alphaD-VxXXA, alphaD-VxXXB, and alphaD-VxXXC). In this study, we report cDNA sequences encoding D-superfamily conopeptides identified in the Clade XII Conidae Conus vexillum, Conus capitaneus, Conus mustelinus, and Conus miles, together with partial sequences of corresponding peptides from this family. The D-superfamily signal peptide sequences display greater heterogeneity than reported for other conotoxin superfamilies. Phylogenetic analysis of the relationships among alphaD-conotoxin precursors reveals two distinct groups containing either an EMM or AVV signal peptide sequence motif. Homodimer and heterodimer combinations of predicted mature toxin sequences likely account for the partial amino acid sequences and mass values observed for several of the native dimeric peptide components identified in C. capitaneus, C. miles, and C. mustelinus venom. The discovery of the precursors and several novel conotoxins from different species defines this large conotoxin family and expands our understanding of sequence diversification mechanisms in Conus species.

Neuronally Selective μ-Conotoxins from Conus striatus Utilize an α-Helical Motif to Target Mammalian Sodium Channels

μ-Conotoxins are small peptide inhibitors of muscle and neuronal tetrodotoxin (TTX)-sensitive voltage-gated sodium channels (VGSCs). Here we report the isolation of μ-conotoxins SIIIA and SIIIB by 125I-TIIIA-guided fractionation of milked Conus striatus venom. SIIIA and SIIIB potently displaced 125I-TIIIA from native rat brain Nav1.2 (IC50 values 10 and 5 nm, respectively) and muscle Nav1.4 (IC50 values 60 and 3 nm, respectively) VGSCs, and both inhibited current through Xenopus oocyte-expressed Nav1.2 and Nav1.4. An alanine scan of SIIIA-(2–20), a pyroglutamate-truncated analogue with enhanced neuronal activity, revealed residues important for affinity and selectivity. Alanine replacement of the solvent-exposed Trp-12, Arg-14, His-16, Arg-18 resulted in large reductions in SIIIA-(2–20) affinity, with His-16 replacement affecting structure. In contrast, [D15A]SIIIA-(2–20) had significantly enhanced neuronal affinity (IC50 0.65 nm), while the double mutant [D15A/H16R]SIIIA-(2–20) showed greatest Nav1.2 versus 1.4 selectivity (136-fold). 1H NMR studies revealed that SIIIA adopted a single conformation in solution comprising a series of turns and anα-helical motif across residues 11–16 that is not found in larger μ-conotoxins. The structure of SIIIA provides a new structural template for the development of neuronally selective inhibitors of TTX-sensitive VGSCs based on the smaller μ-conotoxin pharmacophore.