A novel conotoxin inhibitor of Kv1.6 channel and nAChR subtypes defines a new superfamily of conotoxins

Using assay-directed fractionation of the venom from the vermivorous cone snail Conus planorbis, we isolated a new conotoxin, designated p114a, with potent activity at both nicotinic acetylcholine receptors and a voltage-gated potassium channel subtype. p114a 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 α-helix and two 3₁₀-helices present. Analysis of a cDNA clone encoding the prepropeptide precursor of p114a revealed a novel signal sequence, indicating that p114a belongs to a new gene superfamily, the J-conotoxin superfamily. Five additional peptides in the J-superfamily were identified. Intracranial injection of p114a in mice elicited excitatory symptoms that included shaking, rapid circling, barrel rolling, and seizures. Using the oocyte heterologous expression system, p114a was shown to inhibit both a K⁺ channel subtype (Kv1.6, IC₅₀ = 1.59 μM) and neuronal (IC₅₀ = 8.7 μM for α3β4) and neuromuscular (IC₅₀ = 0.54 μM for α1β1εα) subtypes of the nicotinic acetylcholine receptor (nAChR). Similarities in sequence and structure are apparent between the middle loop of p114a and the second loop of a number of α-conotoxins. This is the first conotoxin shown to affect the activity of both voltage-gated and ligand-gated ion channels.