The analgesic α-conotoxins Vc1.1, RgIA, and PeIA attenuate nociceptive transmission via activation of G protein-coupled GABA_B receptors (GABA_BRs) to modulate N-type calcium channels in primary afferent neurons and recombinantly coexpressed human GABA_BR and Cav2.2 channels in human embryonic kidney 293T cells. Here, we investigate the effects of analgesic α-conotoxins following the mutation of amino acid residues in the Venus flytrap (VFT) domains of the GABA_BR subunits predicted through computational peptide docking and molecular dynamics simulations. Our docking calculations predicted that all three of the α-conotoxins form close contacts with VFT residues in both B1 and B2 subunits, comprising a novel GABA_BR ligand-binding site. The effects of baclofen and α-conotoxins on the peak Ba²⁺ current (I_Ba) amplitude were investigated on wild-type and 15 GABA_BR mutants individually coexpressed with human Cav2.2 channels. Mutations at the interface of the VFT domains of both GABA_BR subunits attenuated baclofen-sensitive I_Ba inhibition by the analgesic α-conotoxins. In contrast, mutations located outside the putative peptide-binding site (D380A and R98A) did not. The key GABA_BR residues involved in interactions with the α-conotoxins are K168 and R207 on the B2 subunit and S130, S153, R162, E200, F227, and E253 on the B1 subunit. The double mutant, S130A + S153A, abolished inhibition by both baclofen and the α-conotoxins. Depolarization-activated I_Ba mediated by both wild-type and all GABA_BR mutants were inhibited by the selective GABA_BR antagonist CGP 55845. This study identifies specific residues of GABA_BR involved in the binding of the analgesic α-conotoxins to the VFT domains of the GABA_BR.

This study defines the binding site of the analgesic α-conotoxins Vc1.1, RgIA, and PeIA on the human GABA_B receptor to activate Gi/o proteins and inhibit Cav2.2 channels. Computational docking and molecular dynamics simulations of GABA_BR identified amino acids of the Venus flytrap (VFT) domains with which the α-conotoxins interact. GABA_BR alanine mutants attenuated baclofen-sensitive Cav2.2 inhibition by the α-conotoxins. We identify an allosteric binding site at the interface of the VFT domains of the GABA_BR subunits for the analgesic α-conotoxins.