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

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