Neuronal (N)-type Ca2ϩ channel-selective -conotoxins have emerged as potential new drugs for the treatment of chronic pain. In this study, two new -conotoxins, CVIE and CVIF, were discovered from a Conus catus cDNA library. Both conopeptides potently displaced 125 I-GVIA binding to rat brain membranes. In Xenopus laevis oocytes, CVIE and CVIF potently and selectively inhibited depolarizationactivated Ba 2ϩ currents through recombinant N-type (␣1 B-b / ␣ 2 ␦1/␤ 3 ) Ca 2ϩ channels. Recovery from block increased with membrane hyperpolarization, indicating that CVIE and CVIF have a higher affinity for channels in the inactivated state. The link between inactivation and the reversibility of -conotoxin action was investigated by creating molecular diversity in ␤ subunits: N-type channels with ␤ 2a subunits almost completely recovered from CVIE or CVIF block, whereas those with ␤ 3 subunits exhibited weak recovery, suggesting that reversibility of the -conotoxin block may depend on the type of ␤-subunit isoform. In rat dorsal root ganglion sensory neurons, neither peptide had an effect on low-voltageactivated T-type channels but potently and selectively inhibited high voltage-activated N-type Ca 2ϩ channels in a voltage-dependent manner. In rat spinal cord slices, both peptides reversibly inhibited excitatory monosynaptic transmission between primary afferents and dorsal horn superficial lamina neurons. Homology models of CVIE and CVIF suggest that -conotoxin/voltage-gated Ca 2ϩ channel interaction is dominated by ionic/electrostatic interactions. In the rat partial sciatic nerve ligation model of neuropathic pain, CVIE and CVIF (1 nM) significantly reduced allodynic behavior. These N-type Ca 2ϩ channel-selective -conotoxins are therefore useful as neurophysiological tools and as potential therapeutic agents to inhibit nociceptive pain pathways.

Scorpion α-toxins are invaluable pharmacological tools for studying voltage-gated sodium channels, but few structure-function studies have been undertaken due to their challenging synthesis. To address this deficiency, we report a chemical engineering strategy based upon native chemical ligation. The chemical synthesis of α-toxin OD1 was achieved by chemical ligation of three unprotected peptide segments. A high resolution X-ray structure (1.8 Å) of synthetic OD1 showed the typical βαββ α-toxin fold and revealed important conformational differences in the pharmacophore region when compared with other α-toxin structures. Pharmacological analysis of synthetic OD1 revealed potent α-toxin activity (inhibition of fast inactivation) at Nav1.7, as well as Nav1.4 and Nav1.6. In addition, OD1 also produced potent β-toxin activity at Nav1.4 and Nav1.6 (shift of channel activation in the hyperpolarizing direction), indicating that OD1 might interact at more than one site with Nav1.4 and Nav1.6. Investigation of nine OD1 mutants revealed that three residues in the reverse turn contributed significantly to selectivity, with the triple OD1 mutant (D9K, D10P, K11H) being 40-fold more selective for Nav1.7 over Nav1.6, while OD1 K11V was 5-fold more selective for Nav1.6 than Nav1.7. This switch in selectivity highlights the importance of the reverse turn for engineering α-toxins with altered selectivity at Nav subtypes.

A peptide contained in the venom of the predatory marine snail Conus tulipa, -TIA, has previously been shown to possess ␣ 1 -adrenoreceptor antagonist activity. Here, we further characterize its pharmacological activity as well as its structure-activity relationships. In the isolated rat vas deferens, -TIA inhibited ␣ 1 -adrenoreceptor-mediated increases in cytosolic Ca 2؉ concentration that were triggered by norepinephrine, but did not affect presynaptic ␣ 2 -adrenoreceptor-mediated responses. In radioligand binding assays using [125 I]HEAT, -TIA displayed slightly greater potency at the ␣ 1B than at the ␣ 1A or ␣ 1D subtypes. Moreover, although it did not affect the rate of association for [ 3 H]prazosin binding to the ␣ 1B -adrenoreceptor, the dissociation rate was increased, indicating non-competitive antagonism by -TIA. N-terminally truncated analogs of -TIA were less active than the full-length peptide, with a large decline in activity observed upon removal of the fourth residue of -TIA (Arg 4 ). An alanine walk of -TIA confirmed the importance of Arg 4 for activity and revealed a number of other residues clustered around Arg 4 that contribute to the potency of -TIA. The unique allosteric antagonism of -TIA resulting from its interaction with receptor residues that constitute a binding site that is distinct from that of the classical competitive ␣ 1 -adrenoreceptor antagonists may allow the development of inhibitors that are highly subtype selective.␣ 1 -Adrenoceptors, members of the G protein-coupled receptor superfamily, are the predominant mediators of the response to norepinephrine released from the sympathetic nerves that innervate resistance vessels (1). Norepinephrine release modulates vascular tone and, as such, ␣ 1 -adrenoreceptors are critically involved in circulatory homeostasis. Several ␣ 1 -adrenoreceptor antagonists, such as the quinazoline derivative, prazosin, are widely used for the treatment of hypertension. ␣ 1 -Adrenoreceptor antagonists are also used to treat bladder outlet obstruction in benign prostatic hyperplasia (for review, see Ref.2) because of their ability to relax smooth muscle.Nevertheless, the ␣ 1 -adrenoreceptor ligands developed to date interact largely with residues of the transmembrane segments that are homologous between the various receptor subtypes, rather than with residues forming the framework regions (the intra-and extracellular loops). It is not surprising, therefore, that available agonists, and also antagonists, show limited subtype selectivity (affinities differing by 50-fold or less between the various subtypes). For this reason, we sought to identify novel ligands that are likely to interact allosterically and, thus, more likely with the framework residues that are distinct between the three ␣ 1 -adrenoreceptor subtypes (␣ 1A , ␣ 1B , and ␣ 1D ).The venoms of cone snails (marine gastropods of the genus Conus) contain bioactive peptides that disrupt neurotransmission. These compounds are referred to generically as "conopeptides" or "conotoxins." Individual ...

The large diversity of peptides from venomous creatures with high affinity for molecules involved in the development and maintenance of neuropathic pain has led to a surge in venom-derived analgesic research. Some members of the α-conotoxin family from Conus snails which specifically target subtypes of nicotinic acetylcholine receptors (nAChR) have been shown to be effective at reducing mechanical allodynia in neuropathic pain models. We sought to determine if three such peptides, Vc1.1, AuIB and MII were effective following intrathecal administration in a rat neuropathic pain model because they exhibit different affinities for the major putative pain relieving targets of α-conotoxins. Intrathecal administration of α-conotoxins, Vc1.1, AuIB and MII into neuropathic rats reduced mechanical allodynia for up to 6 hours without significant side effects. In vitro patch-clamp electrophysiology of primary afferent synaptic transmission revealed the mode of action of these toxins was not via a GABA B -dependant mechanism, and is more likely related to their action at nAChRs containing combinations of α3, α7 or other subunits. Intrathecal nAChR subunitselective conotoxins are therefore promising tools for the effective treatment of neuropathic pain. AbstractThe large diversity of peptides from venomous creatures with high affinity for molecules involved in the development and maintenance of neuropathic pain has led to a surge in venom-derived analgesic research. Some members of the α-conotoxin family from Conus snails which specifically target subtypes of nicotinic acetylcholine receptors (nAChR) have been shown to be effective at reducing mechanical allodynia in neuropathic pain models. We sought to determine if three such peptides, Vc1.1, AuIB and MII were effective following intrathecal administration in a rat neuropathic pain model because they exhibit different affinities for the major putative pain relieving targets of α-conotoxins. Intrathecal administration of α-conotoxins, Vc1.1, AuIB and MII into neuropathic rats reduced mechanical allodynia for up to 6 hours without significant side effects. In vitro patch-clamp electrophysiology of primary afferent synaptic transmission revealed the mode of action of these toxins was not via a GABA B -dependant mechanism, and is more likely related to their action at nAChRs containing combinations of α3, α7 or other subunits. Intrathecal nAChR subunitselective conotoxins are therefore promising tools for the effective treatment of neuropathic pain.3

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