Analgesic a-Conotoxin Binding Site on the Human GABAB ReceptorS

The analgesic a-conotoxins Vc1.1, RgIA, and PeIA attenuate noci-ceptive transmission via activation of G protein-coupled GABAB receptors (GABABRs) to modulate N-type calcium channels in primary afferent neurons and recombinantly coexpressed human GABABR and Cav2.2 channels in human embryonic kidney 293T cells. Here, we investigate the effects of analgesic a-conotoxins following the mutation of amino acid residues in the Venus flytrap (VFT) domains of the GABABR subunits predicted through compu-tational peptide docking and molecular dynamics simulations. Our docking calculations predicted that all three of the a-conotoxins form close contacts with VFT residues in both B1 and B2 subunits, comprising a novel GABABR ligand-binding site. The effects of bac-lofen and a-conotoxins on the peak Ba2+ current (IBa) amplitude were investigated on wild-type and 15 GABABR mutants individu-ally coexpressed with human Cav2.2 channels. Mutations at the interface of the VFT domains of both GABABR subunits attenuated baclofen-sensitive IBa inhibition by the analgesic a-conotoxins. In contrast, mutations located outside the putative peptide-binding site (D380A and R98A) did not. The key GABABR residues involved in interactions with the a-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 a-conotoxins. Depolarization-activated IBa mediated by both wild-type and all GABABR mutants were inhibited by the selective GABABR antagonist CGP 55845. This study identifies specific residues of GABABR involved in the binding of the analgesic a-conotoxins to the VFT domains of the GABABR.SIGNIFICANCE STATEMENT This study defines the binding site of the analgesic a-conotoxins Vc1.1, RgIA, and PeIA on the human GABAB receptor to activate Gi/o proteins and inhibit Cav2.2 channels. Computational dock-ing and molecular dynamics simulations of GABABR identified amino acids of the Venus flytrap (VFT) domains with which the a-conotoxins interact. GABABR alanine mutants attenuated bac-lofen-sensitive Cav2.2 inhibition by the a-conotoxins. We identify an allosteric binding site at the interface of the VFT domains of the GABABR subunits for the analgesic a-conotoxins.