ω-Conotoxin CVID Inhibits a Pharmacologically Distinct Voltage-sensitive Calcium Channel Associated with Transmitter Release from Preganglionic Nerve Terminals

Abstract

Neurotransmitter release from preganglionic parasympathetic neurons is resistant to inhibition by selective antagonists of L-, N-, P/Q-, R-, and T-type calcium channels. In this study, the effects of different -conotoxins from genus Conus were investigated on current flow-through cloned voltage-sensitive calcium channels expressed in Xenopus oocytes and nerve-evoked transmitter release from the intact preganglionic cholinergic nerves innervating the rat submandibular ganglia. Our results indicate that -conotoxin CVID from Conus catus inhibits a pharmacologically distinct voltage-sensitive calcium channel involved in neurotransmitter release, whereas -conotoxin MVIIA had no effect. Venom of the predatory marine gastropods of the genus Conus (cone snails) contain a unique array of peptides whose pharmaceutical potential remains largely unexploited (1). These peptides have been classified based on their pharmacological target and structure (2, 3). One important class, the -conotoxins, utilizes a four-loop framework to selectively inhibit "Ntype" voltage-sensitive calcium channels (VSCCs) 1 found in the central and peripheral nervous systems of vertebrates (4).Neurotransmitter release from preganglionic parasympathetic neurons has been found to be resistant to inhibition by a range of selective calcium channel antagonists of L-, N-, P/Q-, R-, and T-type calcium channels (5-8). A recently discovered -conotoxin from Conus catus (CVID) is highly selective for N-type over P/Q-type VSCCs (9) and shows potent analgesic activity in rats (10). In this study, we investigated the effects of CVID on autonomic neurotransmission using conventional intracellular microelectrode recording techniques. -Conotoxin CVID was found to be a potent inhibitor of neurally evoked transmitter release from the intact preganglionic cholinergic nerves innervating the rat submandibular ganglia, whereas -conotoxin MVIIA had no effect.The orientation and nature of the residues in loop 2 of -conotoxins have been shown to be crucial for selective binding to the N-type VSCC (11-15). Since the only sequence difference in loop 2 between CVID and MVIIA is at position 10, we investigated the importance of this position for -conotoxin structure and ability to block neurotransmitter release from preganglionic parasympathetic neurons. The inhibition of preganglionic transmitter release was favored in -conotoxins with a Lys at position 10.

Analgesic ω-Conotoxins CVIE and CVIF Selectively and Voltage-Dependently Block Recombinant and Native N-Type Calcium Channels

“…This is consistent with the previous structure-activity relationship studies, in which at least four residues in addition to Tyr13 (mostly positively charged residues) from loop 2 and loop 4 were found to contribute to N-type VGCC affinity (Nadasdi et al, 1995;Nielsen et al, 2000). Furthermore, the sequence analysis of -conotoxins reveals a highly conserved positively charged patch at position 10, a position previously shown to affect VGCC-selectivity and voltage-dependent recovery from block (Adams et al, 2003). It seems that subtle changes in side-chain length can influence how -conotoxins interact with N-type VGCCs, with C. catus evolving -conoAnalgesic CVIE and CVIF Block N-Type Calcium Channels toxins using both Arg10 (CVIA, CVIE, and CVIF) and Lys10 (CVIB and CVID).…”

“…The highly conserved Tyr13 on the neutral side of the -conotoxins is oriented in an almost identical manner, consistent with it playing a key role in binding to N-type VGCCs Adams et al, 2003), presumably through a hydrogen bond from the hydroxyl group on Tyr13 to the channel. Second, the positively charged face of -conotoxins is mostly distributed across loop 2 and loop 4 residues and is likely to contribute to VGCC binding via ionic and/or electrostatic interactions.…”

“…Molecular models of -conotoxins CVIE and CVIF were built in the program Modeler 9v2 using MVIIA (Protein Data Bank identification number 1ttk) (Adams et al, 2003) as the template because they share a high degree of protein sequence identity (ϳ80%). The sequence alignment was generated using ClustalW (Larkin et al, 2007).…”

The Physiology, Pathology, and Pharmacology of Voltage-Gated Calcium Channels and Their Future Therapeutic Potential

“…These peptides generally share a similar disulfide bridge arrangement and act as pore blockers. Although most of them act selectively on Ca V 2.2 channels (e.g., v-conotoxins SIA, FVIA, or CVID) (Smith et al, 2002;Adams et al, 2003;Lee et al, 2010), others also block Ca V 2.1 channels (e.g., v-conotoxins SIB and MVIIC) (Hillyard et al, 1992;Adams et al, 1993;Woppmann et al, 1994). Some of these v-conotoxins are effective systemically in a mouse model of inflammatory pain (Sadeghi et al, 2013).…”

Les canaux calciques dépendants du voltage au cœur de la douleur

“…Bien que difficile à gérer en termes de dosage, le ziconotide reste cependant une molécule intéressante. Actuellement, l'ω-conotoxine CVID (AM-336) [15], isolée à partir du venin Les sous-unités régulatrices des canaux HSA sont également représentées. Ces sous-unités auxiliaires modulent les propriétés biophysiques de la sous-unité α 1 .…”