Programme Contents

Biological activity of α-conotoxins on N-type calcium channels in rat sensory neurons

Conotoxins are selective antagonists of a range of membrane receptors, ion channels and transporters associated with pain pathways. Previous studies have demonstrated the analgesic potential of several different α-conotoxins that competitively inhibit nAChRs with varying degrees of subtype selectivity. We have previously reported that the α9α10 nAChR-selective α-conotoxins Vc1.1 and RgIA inhibit N-type calcium channels in rat sensory neurons via a GABA_B receptor-dependent signalling mechanism, which may contribute to their pain relieving actions (Callaghan et al., 2008). We have also recently shown that the synthetic cyclisation of Vc1.1 produced an orally active peptide by improving in vivo stability (Clark et al., 2010). Here we report that α-conotoxin AuIB, a weak but selective α3β4 nAChR antagonist, also inhibits high voltage-activated (HVA) Ca²⁺ channels in DRG neurons via a GABA_B receptor dependent pathway whereas MII, a potent α3β2 nAChR antagonist does not. Native AuIB peptide found in the venom, has C1-C3, C2-C4 cystine globular connectivity whereas when AuIB is synthesized chemically, both the the globular and ribbon (C1-C4, C2-C3 connectivity) isomers are formed. The ribbon isomer of AuIB was also examined on GABA_B receptor /N-type Ca²⁺ channel. α-Conotoxins were assessed on Ca²⁺ channel currents in rat DRG neurons using the whole-cell patch clamp recording technique. AuIB (100 nM) reduced peak Ca²⁺ channel current amplitude to 64.6 ± 6.2% of control with an IC50 of 1.5 ± 0.3 nM (n = 17). Application of the ribbon isomer of AuIB (100 nM) did not affect Ca²⁺ channel current amplitude (92.9 ± 3% of control, n = 5), whereas this isomer inhibits α3β4 nAChRs (Grishan et al., 2010). Application of the selective N-type Ca²⁺ channel inhibitor, ω-conotoxin CVID, confirmed that AuIB targets the N-type component of the HVA Ca²⁺ channel currents. Preincubation with the receptor antagonist CGP 55845, blocked the effect of AuIB on HVA Ca²⁺ channel currents. MII at concentrations up to 1 μM did not inhibit depolarization-activated Ca²⁺ channel currents. The linker length of cyclised AuIB on Ca²⁺ channel current inhibition was also examined. Cyclised AuIB (100 nM) with 4 linking residues (GGAA) reduced Ca²⁺ channel current amplitude to 49.3± 8.3% of control with an IC50 of 5.9 ± 0.5 nM (n = 4) and cyclised AuIB (100 nM) with 5 linking residues (AGAGA) reduced Ca²⁺ channel current amplitude to 69.1± 8.8% of control with an IC50 of 21.3 ± 0.8 nM (n = 4). These findings demonstrate that α-conotoxins other than α9α10 nAChR-selective conotoxins inhibit N-type calcium channel currents via the GABA_B-mediated pathway and cyclisation of the peptide retains this inhibitory activity.

Callaghan B, Haythornthwaite A, Berecki G, Clark RJ, Craik DJ, Adams DJ. (2008) Journal of Neuroscience 28(43): 10943-51.

Clark RJ, Jensen J, Nevin S, Callaghan B, Adams D. Craik, D. (2010) Angewandte Chemie (International ed. in English) 49(37): 6545-8.

Grishin AA, Wang CI, Muttenthaler M, Alewood PF, Lewis RJ, Adams DJ. (2010) Journal of Biological Chemistry 285(29): 22254-63.