Biological activity of alanine-substituted analogues of α-conotoxin Vc1.1 on N-type calcium channels in rat sensory neurons

B.P. Callaghan,¹ J. Jensen,² R.J. Clark,² D.J. Craik² and D.J. Adams,¹ ¹Health Innovations Research Institute, RMIT University, Bundoora, VIC 3083, Australia and ²Institute for Molecular Biosciences, University of Queensland, St Lucia, QLD 4072, Australia.

α-Conotoxin Vc1.1 is a 16 amino acid disulfide peptide that is a selective antagonist of the α9α10 nicotinic acetylcholine receptor (nAChR) subtype but has recently been shown to be a more potent inhibitor of N-type Ca²⁺ channel currents in dissociated neurons from rat dorsal root ganglia (DRG). The inhibition of N-type Ca²⁺ channel currents was blocked by inhibitors of G_i/o and selective GABA_B receptor antagonists suggesting that Vc1.1 acted via GABA_B receptors (Callaghan et al., 2008). To further explore the structure-activity relationship for Vc1.1 inhibition of N-type Ca²⁺ channels in DRG neurons, the amino acids except the conserved cysteines contained in the sequence of Vc1.1 (Gly(1)-Ser(4)-Asp(5)-Pro(6)-Arg(7)-Asn(9)-Tyr(10)- Asp(11)-His(12)-Pro(13)-Glu(14)-Ile(15)), were sequentially replaced by Ala. These analogues have been characterised by NMR spectroscopy demonstrating that the structure of the peptide is not significantly changed (Halai et al., 2009). The present study examined the activity of the Vc1.1 analogues on high voltage-activated Ca²⁺ channel currents in rat DRG neurons using the whole-cell patch clamp technique. Analogues that resulted in significant shifts to the right of the concentration-response relationship for inhibition of Ca²⁺ channel currents included S4A (n=4), N9A (n=16) and P13A (n=2). In contrast, analogues with the least effect or unchanged compared to Vc1.1 were D11A (n=4), E14A (n=6) and I15A (n=3). Interestingly [N9A]Vc1.1 has been reported to be more potent than Vc1.1 at the α9α10 nAChR whereas it is inactive at inhibiting N-type Ca²⁺ channel currents. These findings contribute to an improved understanding of the molecular basis for the GABA_B receptor-mediated inhibition of the N-type calcium channel current by Vc1.1.

Callaghan B, Haythornthwaite A, Berecki G, Clark RJ, Craik DJ, Adams DJ. (2008) J. Neurosci. 28: 10943-51.

Halai R, Clark RJ, Nevin ST, Jensen JE, Adams DJ, Craik DJ. (2009) J. Biol. Chem. 284: 20275-84.