A Novel Conotoxin Inhibitor of Kv1.6 Channel and nAChR Subtypes Defines a New Superfamily of Conotoxins^,

Abstract

Using assay-directed fractionation of the venom from the vermivorous cone snail Conus planorbis, we isolated a new conotoxin, designated pl14a, with potent activity at both nicotinic acetylcholine receptors and a voltage-gated potassium channel subtype. pl14a contains 25 amino acid residues with an amidated C-terminus, an elongated N-terminal tail (six residues), and two disulfide bonds (1-3, 2-4 connectivity) in a novel framework distinct from other conotoxins. The peptide was chemically synthesized, and its three-dimensional structure was demonstrated to be well-defined, with an alpha-helix and two 3(10)-helices present. Analysis of a cDNA clone encoding the prepropeptide precursor of pl14a revealed a novel signal sequence, indicating that pl14a belongs to a new gene superfamily, the J-conotoxin superfamily. Five additional peptides in the J-superfamily were identified. Intracranial injection of pl14a in mice elicited excitatory symptoms that included shaking, rapid circling, barrel rolling, and seizures. Using the oocyte heterologous expression system, pl14a was shown to inhibit both a K+ channel subtype (Kv1.6, IC50 = 1.59 microM) and neuronal (IC50 = 8.7 microM for alpha3beta4) and neuromuscular (IC50 = 0.54 microM for alpha1beta1 epsilondelta) subtypes of the nicotinic acetylcholine receptor (nAChR). Similarities in sequence and structure are apparent between the middle loop of pl14a and the second loop of a number of alpha-conotoxins. This is the first conotoxin shown to affect the activity of both voltage-gated and ligand-gated ion channels.

Tyrosine-rich Conopeptides Affect Voltage-gated K+ Channels

“…zin-S1 (24, 25) is a Kunitz domain K ϩ channel inhibitor from Conus striatus. The investigation of C. planorbis venom revealed not only K ϩ channel-targeted peptides unrelated to the three classes above but also two different toxins unrelated to each other that both preferentially target Kv1.6: J-conotoxin PlXIVA, a peptide with two disulfide bonds and previously called pl14a (9,21), and CPY-Pl1, the peptide characterized in this study. It is not unprecedented for venom from one species to contain unrelated peptides that antagonize the same ion channel complex.…”

“…The CPY peptide that has been most well characterized, CPY-Pl1, is highly selective for the Kv1.6 subtype. It is notable that CPY-Pl1 is ϳ18-fold more potent on Kv1.6 than pl14a, a disulfide-rich peptide that was isolated previously from the same venom (9,21).…”

Conus Venom Peptide Pharmacology

“…pl14a, a member of the J superfamily, is an unusual conotoxin in that it inhibits both K v 1.6 and several isoforms of the nicotinic acetylcholine receptor (nAChR) (Imperial et al, 2006). Homology modeling suggests that this peptide possesses both the putative functional dyad, formed by residues Lys18 and Tyr19, and a ring of basic residues comprising Arg3, Arg5, Arg12, and Arg25, reminiscent of the pharmacophore found in the M-conotoxin RIIIK (Mondal et al, 2007).…”

“…It is noteworthy that although this peptide has not been tested across the various subtypes of nAChRs, lt14a produced dose-dependent analgesia in a hot-plate assay in mice, suggesting that it may target therapeutically relevant nAChRs. Further atypical ␣-conotoxins include the ␣J-pl14a, which was recently identified with an unusual dual activity at two different classes of ion channels (Imperial et al, 2006). Indeed, this 25-amino acid peptide inhibits both K v 1.6 and muscle and ␣3␤2 nAChRs (see section III.D).…”

Functional profiling of neurons through cellular neuropharmacology

“…One of these antagonists, a conopeptide from Conus planorbis (pl14a), is selective for K V 1.6 (27), whereas a kunitz domain small protein from mamba venom, Dendrotoxin-K (Dtx-K), is reported to be highly selective for K V 1.1 (28). In previous reports, the targeting selectivities of κM-RIIIJ, pl14a, and Dtx-K were assessed by tests on heterologously expressed homomeric channels (26)(27)(28).…”