Programme Contents

Differential action of ω-conotoxins CVID and CVIB on voltage-gated calcium channels in rat sensory neurons

Selective antagonists of voltage-gated calcium channels (VGCCs) are of considerable interest both as research tools and potential therapeutic agents. The selectivity of VGCC antagonists is essential for dissecting the various Ca²⁺ channel types underlying the whole-cell Ca²⁺ current whereas potency and reversibility play an important role in the use of a VGCC antagonist as a pharmaceutical agent. ω-Conotoxins GVIA, MVIIA and MVIIC have been used routinely as selective blockers of N- and P/Q-types of VGCCs in excitable cells. However, the newly discovered ω-conotoxins from Conus catus, CVID has been shown to have the highest selectivity for N-type over P/Q-type VGCCs among the other N-type selective VGCC antagonists (Lewis et al., 2000). The present study investigated the selectivity, potency and reversibility of action of ω-conotoxins CVID and CVIB in isolated sensory neurons dissociated from rat dorsal root ganglia (DRG) and on recombinant VGCCs expressed in Xenopus oocytes. Bath application of either CVID or CVIB inhibited depolarization-activated whole cell Ba²⁺ currents in DRG neurons with pIC₅₀ values of -8.12 ± 0.05 and -7.64 ± 0.08, respectively. The block of Ba²⁺ currents in DRG neurons by CVID appeared to be irreversible after >30 min washout whereas Ba²⁺ currents exhibited rapid recovery from block by CVIB (>80% within 3 min). ω-Conotoxin CVIB inhibited more of the whole-cell Ba²⁺ current in DRG neurons than CVID and the recoverable component of the Ba²⁺ current inhibited by CVIB was mediated by the N-type VGCC. The potency of CVID and CVIB block of N- and P/Q-type VGCCs was compared with the ω-conotoxins, GVIA, MVIIA and MVIIC. ω-Conotoxins GVIA and MVIIA inhibited Ba²⁺ currents in DRG neurons to a similar degree as CVID. The residual current amplitude obtained in the presence of maximally effective concentrations of the ω-conotoxins was: GVIA, 54 ± 0.2%; MVIIA, 41 ± 0.04% and CVID, 34 ± 1%. The residual current after block by CVIB was 3 ± 5% of control level reflecting non-selective N- and P/Q- action of the toxin. ω-Conotoxin CVIB reversibly inhibited Ba²⁺ currents mediated by N- (Ca_v2.2) and P/Q- (Ca_v2.1) type VGCCs expressed in Xenopus oocytes. The α₂δ₁ auxiliary subunit coexpressed with Ca_ν2.2 and Ca_ν2.1 reduced the potency of CVIB as reported previously for CVID at recombinant N-type VGCCs (Mould et al., 2004). The present study demonstrates that ω-conotoxins CVID and CVIB can be successfully used for pharmacological isolation of N- and P/Q- components of the Ca²⁺ conductance in rat DRG neurons. CVID selectively and irreversibly blocked the N-type component of the whole cell Ba²⁺ current in DRG neurons but blocked reversibly the recombinant N-type (Ca_v2.2) VGCC. In contrast, CVIB blocked reversibly the N-type component and blocked irreversibly P/Q- component of the whole cell Ba²⁺ current in DRG neurons. ω-Conotoxins CVID and CVIB may be useful as antagonists of N- and P/Q-type VGCCs in sensory neurons involved in processing primary nociceptive information.

Lewis, R.J., Nielsen, K.J., Craik, D.J., Loughnan, M.L., Adams, D.A., Sharpe, I.A., Luchian T., Adams, D.J., Bond, T., Thomas, L., Jones, A., Matheson, J-L., Drinkwater, R., Andrews, P.R. & Alewood, P.F. (2000) Journal of Biological Chemistry 275, 35335–35344.

Mould, J., Yasuda, T., Schroeder, C.I., Beedle, A.M., Doering, C.J., Zamponi, G.W., Adams, D.J. and Lewis, R.J. (2004) Journal of Biological Chemistry 279, 34705-34714.