A New ω-Conotoxin That Targets N-Type Voltage-Sensitive Calcium Channels with Unusual Specificity

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Abstract
A new specific voltage-sensitive calcium channel (VSCC) blocker has been isolated from the venom of the fish-hunting cone snail Conus consors. This peptide, named ω-Ctx CNVIIA, consists of 27 amino acid residues folded by 3 disulfide bridges. Interestingly, loop 4, which is supposed to be crucial for selectivity, shows an unusual sequence (SSSKGR). The synthesis of the linear peptide was performed using the Fmoc strategy, and the correct folding was achieved in the presence of guanidinium chloride, potassium buffer, and reduced/oxidized glutathione at 4 °C for 3 days. Both synthetic and native toxin caused an intense shaking activity, characteristic of ω-conotoxins targeting N-type VSCC when injected intracerebroventricularly to mice. Binding studies on rat brain synaptosomes revealed that the radioiodinated ω-Ctx CNVIIA specifically and reversibly binds to high-affinity sites with a Kd of 36.3 pM. Its binding is competitive with ω-Ctx MVIIA at low concentration (Ki = 2 pM). Moreover, ω-Ctx CNVIIA exhibits a clear selectivity for N-type VSCCs versus P/Q-type VSCCs targeted respectively by radioiodinated ω-Ctx GVIA and ω-Ctx MVIIC. Although ω-Ctx CNVIIA clearly blocked N-type Ca2+ current in chromaffin cells, this toxin did not inhibit acetylcholine release evoked by nerve stimuli at the frog neuromuscular junction, in marked contrast to ω-Ctx GVIA. ω-Ctx CNVIIA thus represents a new selective tool for blocking N-type VSCC that displays a unique pharmacological profile and highlights the diversity of voltage-sensitive Ca2+ channels in the animal kingdom.