Analgesic α-Conotoxin Binding Site on the Human GABA_B Receptor

Abstract

The analgesic α-conotoxins Vc1.1, RgIA, and PeIA attenuate nociceptive transmission via activation of G protein-coupled GABA B receptors (GABA B R) to modulate N-type calcium channels in primary afferent neurons and recombinantly co-expressed human GABA B R and Cav2.2 channels in HEK293T cells. Here, we investigated the effects of analgesic αconotoxins 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 αconotoxins 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 co-expressed 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 αconotoxins to the VFT domains of the GABA B R.

Dual Antagonism of α9α10 nAChR and GABAB Receptor-Coupled CaV2.2 Channels by an Analgesic αO-Conotoxin Analogue

“…Previous studies demonstrated that the VFT region of the GABA B R1 subunit is typically where agonists and antagonists interact with the GABA B R, whereas some positive allosteric modulators are reported to interact at the pore region of the transmembrane domain . However, the binding site of conotoxins on the GABA B R has not been precisely determined. , Until recently, the binding site for several α-conotoxins was determined based on docking and mutagenesis studies . Docking calculations predicted that α-conotoxins form close contacts with VFT residues in both B1 and B2 subunits, which comprise a novel GABA B R ligand-binding site .…”

“…15,21 Until recently, the binding site for several α-conotoxins was determined based on docking and mutagenesis studies. 41 Docking calculations predicted that α-conotoxins form close contacts with VFT residues in both B1 and B2 subunits, which comprise a novel GABA B R ligand-binding site. 40 GeX-2 could bind to a variety of positions at GABA B R according to peptide docking and results from clustering of the docked conformations suggested preferential interactions with the Nterminal or the VTF region of the R2 subunit.…”

Discovery and optimisation of conotoxin Vc1.1 and analogues with analgesic properties

“…43 More recently, the binding of Vc1.1 to the GABA B R was investigated by a combination of molecular docking and molecular dynamics (MD) simulations, alongside confirmatory mutagenesis, electrophysiology and immunocytochemistry. 60 That study reported that Vc1.1 interacts directly with both B1 and B2 subunits of the GABA B R , in particular residues S130 and S131 in B1, and K168 in B2. The study also demonstrated 60 The development of Vc1.1 and its analogues originally primarily focused on systemic delivery through intramuscular administration for neuropathic pain.…”

“…60 That study reported that Vc1.1 interacts directly with both B1 and B2 subunits of the GABA B R , in particular residues S130 and S131 in B1, and K168 in B2. The study also demonstrated 60 The development of Vc1.1 and its analogues originally primarily focused on systemic delivery through intramuscular administration for neuropathic pain. In 2017, a potentially new application for chronic visceral pain was proposed following the response observed for Vc1.1 in an ex vivo assay for chronic visceral hypersensitivity (CVH) when compared with established GABA B agonists.…”

Conotoxins Targeting Voltage-Gated Sodium Ion Channels

“…Cone snail venoms are a particularly rich source of peptides that target ion channels as well as other receptors (Bony et al, 2022;Liang et al, 2022;Ruelas-Callejas et al, 2022).…”