PT  - JOURNAL ARTICLE
AU  - Anuja R. Bony
AU  - Jeffrey R. McArthur
AU  - Akari Komori
AU  - Ann R. Wong
AU  - Andrew Hung
AU  - David J. Adams
TI  - <strong>Analgesic α</strong><strong>-Conotoxin Binding Site on the Human GABA<sub>B</sub> Receptor</strong>
AID  - 10.1124/molpharm.122.000543
DP  - 2022 Jan 01
TA  - Molecular Pharmacology
PG  - MOLPHARM-AR-2022-000543
4099  - http://molpharm.aspetjournals.org/content/early/2022/08/08/molpharm.122.000543.short
4100  - http://molpharm.aspetjournals.org/content/early/2022/08/08/molpharm.122.000543.full
AB  - The analgesic α-conotoxins Vc1.1, RgIA, and PeIA attenuate nociceptive transmission via activation of G protein-coupled GABAB receptors (GABABR) to modulate N-type calcium channels in primary afferent neurons and recombinantly co-expressed human GABABR and Cav2.2 channels in HEK293T cells. Here, we investigated the effects of analgesic α-conotoxins following the mutation of amino acid residues in the Venus Flytrap (VFT) domains of the GABABR 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 GABABR ligand-binding site. The effects of baclofen and α-conotoxins on the peak Ba2+ current (IBa) amplitude were investigated on wild-type and 15 GABABR mutants individually co-expressed 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 α-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 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 IBa mediated by both wild-type and all GABAαBR mutants were inhibited by the selective GABABR antagonist CGP 55845. This study identifies specific residues of GABABR involved in the binding of the analgesic α-conotoxins to the VFT domains of the GABABR. Significance Statement This study defines the binding site of analgesic α-conotoxins Vc1.1, RgIA, and PeIA on the human GABAB receptor to activate Gi/o proteins and inhibit Cav2.2 channels. Computational docking and MD simulations of GABABR identified amino acids of the Venus flytrap (VFT) domains with which the α-conotoxins interact. GABABR 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 GABABR subunits for the analgesic α-conotoxins.