Analgesic α-Conotoxin Binding Site on the Human GABA B Receptor.

The analgesic α -conotoxins Vc1.1, RgIA, and PeIA attenuate nociceptive transmission via activation of G protein-coupled GABA _B receptors (GABA _B Rs) to modulate N-type calcium channels in primary afferent neurons and recombinantly coexpressed human GABA _B R 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 _B R 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 _B R ligand-binding site. The effects of baclofen and α -conotoxins on the peak Ba ²⁺ current (I _Ba ) amplitude were investigated on wild-type and 15 GABA _B R mutants individually coexpressed with human Cav2.2 channels. Mutations at the interface of the VFT domains of both GABA _B R 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 _B R 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 _B R mutants were inhibited by the selective GABA _B R antagonist CGP 55845. This study identifies specific residues of GABA _B R involved in the binding of the analgesic α -conotoxins to the VFT domains of the GABA _B R. SIGNIFICANCE STATEMENT: 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 _B R identified amino acids of the Venus flytrap (VFT) domains with which the α -conotoxins interact. GABA _B R 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 _B R subunits for the analgesic α -conotoxins.
(Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)