Structure-Activity Studies Reveal the Molecular Basis for GABA(B)-Receptor Mediated Inhibition of High Voltage-Activated Calcium Channels by alpha-Conotoxin Vc1.1

alpha-Conotoxins are disulfide-bonded peptides from cone snail venoms and are characterized by their affinity for nicotinic acetylcholine receptors (nAChR). Several alpha-conotoxins with distinct selectivity for nAChR subtypes have been identified as potent analgesics in animal models of chronic pain. However, a number of alpha-conotoxins have been shown to inhibit N-type calcium channel currents in rodent dissociated dorsal root ganglion (DRG) neurons via activation of G protein-coupled GABA(B) receptors (GABA(B)R). Therefore, it is unclear whether activation of GABA(B)R or inhibition of alpha 9 alpha 10 nAChRs is the analgesic mechanism. To investigate the mechanisms by which alpha-conotoxins provide analgesia, we synthesized a suite of Vc1.1 analogues where all residues, except the conserved cysteines, in Vc1.1 were individually replaced by alanine (A), lysine (K), and aspartic acid (D). Our results show that the amino acids in the first loop play an important role in binding of the peptide to the receptor, whereas those in the second loop play an important role for the selectivity of the peptide for the GABA(B)R over alpha 9 alpha 10 nAChRs. We designed a cVc1.1 analogue that is >8000-fold selective for GABA(B)R-mediated inhibition of high voltage-activated (HVA) calcium channels over alpha 9 alpha 10 nAChRs and show that it is analgesic in a mouse model of chronic visceral hypersensitivity (CVH). cVc1.1[D11A,E14A] caused dose-dependent inhibition of colonic nociceptors with greater efficacy in ex vivo CVH colonic nociceptors relative to healthy colonic nociceptors. These findings suggest that selectively targeting GABA(B)R-mediated HVA calcium channel inhibition by alpha-conotoxins could be effective for the treatment of chronic visceral pain.