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

Abstract

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.

Conus Venom Peptide Pharmacology

“…The high-affinity block of Ca v 2.2 by CVID was found to be reversible in heterologous systems expressing only the pore-forming ␣-subunit, but irreversible in DRG neurons, an effect that is mimicked in expression systems where the ␣-subunit is coexpressed with auxiliary ␤3 and ␣2␦1 subunits (Lewis et al, 2000;Mould et al, 2004;Motin et al, 2007). Likewise, inhibition of Ca v 2.2 by CVIE and CVIF was found to be virtually irreversible in the presence of ␤3 subunits but reversible in systems coexpressing the ␤2 subunit (Berecki et al, 2010). Intriguingly, these effects parallel modification of channel characteristics by auxiliary subunits, suggesting that altered block characteristics of -conotoxins in the presence of auxiliary subunits reflect altered channel kinetics.…”

“…However, reversibility can be induced by hyperpolarizations to potentials more negative than the minimum of the steady-state inactivation curve; i.e., -conotoxins bind with higher affinity to the inactivated state of the N-channel than the resting state (Stocker et al, 1997). Such voltage-dependent recovery from block varies for different -conotoxins and is strongly influenced for some by the ␣2␦-and ␤-subunits, in particular ␣2␦1, ␤2a, and ␤3, coexpressed with the ␣-subunit (Mould et al, 2004;Berecki et al, 2010). Reversibility of GVIA and MVIIA is only weakly influenced by ␣2␦1-and ␤2a-or ␤3-subunit coexpression, and both appear to have a narrower therapeutic index than for CVID, which is strongly influenced by the presence of these subunits (Mould et al, 2004;Berecki et al, 2010).…”

“…Such voltage-dependent recovery from block varies for different -conotoxins and is strongly influenced for some by the ␣2␦-and ␤-subunits, in particular ␣2␦1, ␤2a, and ␤3, coexpressed with the ␣-subunit (Mould et al, 2004;Berecki et al, 2010). Reversibility of GVIA and MVIIA is only weakly influenced by ␣2␦1-and ␤2a-or ␤3-subunit coexpression, and both appear to have a narrower therapeutic index than for CVID, which is strongly influenced by the presence of these subunits (Mould et al, 2004;Berecki et al, 2010). Two more recently identified -conotoxins from Conus catus, CVIE and CVIF, show greater voltage-dependent reversal in the presence of ␣2␦1-and ␤-subunits than CVID (Berecki et al, 2010).…”

“…Intriguingly, these effects parallel modification of channel characteristics by auxiliary subunits, suggesting that altered block characteristics of -conotoxins in the presence of auxiliary subunits reflect altered channel kinetics. In addition to altered binding kinetics, many -conotoxins also have lower affinity for Ca v 2.2 in the presence of auxiliary subunits, especially Ca v ␣2␦1 (Lewis et al, 2000;Berecki et al, 2010). However, the precise mechanisms contributing to these intriguing characteristics of -conotoxins, as well as the effect of these pharmacological differences on the therapeutic potential of these peptides, remain to be elucidated.…”

“…It is now appreciated that -conotoxins represent some of the most selective known inhibitors for the neuronal Ca v isoforms 2.2 and 2.1. Specifically, -conotoxins CVID, CVIE, CVIF, GVIA, and MVIIA are particularly selective for Ca v 2.2, whereas MVIIC and MVIID show a preference for Ca v 2.1 underlying P/Q-type calcium currents (Monje et al, 1993;Woppmann et al, 1994;Nadasdi et al, 1995;Nielsen et al, 1996;Flinn et al, 1999;Lewis et al, 2000;Berecki et al, 2010). The binding site of -conotoxins to the Ca v subtypes has been mapped primarily to the external vestibule of the channel in the domain III S5-S6 region (Ellinor et al, 1994), consistent with preliminary results obtained from docking of -MVIIA to a homology model of Ca v 2.2 ( Fig.…”