Three Arginines in the GABAA Receptor Binding Pocket Have Distinct Roles in the Formation and Stability of Agonist- versus Antagonist-Bound Complexes

Binding of the agonist GABA to the GABAA receptor causes channel gating, whereas competitive antagonists that bind at the same site do not. The details of ligand binding are not well understood, including which residues interact directly with ligands, maintain the structure of the binding pocket, or transduce the action of binding into opening of the ion channel gate. Recent work suggests that the amine group of the GABA molecule may form a cation-π bond with residues in a highly conserved “aromatic box” within the binding pocket. Although interactions with the carboxyl group of GABA remain unknown, three positively charged arginines (α1Arg67, α1Arg132, and β2Arg207) just outside of the aromatic box are likely candidates. To explore their roles in ligand binding, we individually mutated these arginines to alanine and measured the effects on microscopic ligand binding/unbinding rates and channel gating. The mutations α1R67A or β2R207A slowed agonist binding and sped unbinding with little effect on gating, demonstrating that these arginines are critical for both formation and stability of the agonist-bound complex. In addition, α1R67A sped binding of the antagonist 2-(3-carboxypropyl)-3-amino-6-(4 methoxyphenyl)pyridazinium bromide (SR-95531), indicating that this arginine poses a barrier to formation of the antagonist-bound complex. In contrast, β2R207A and α1R132A sped antagonist unbinding, indicating that these arginines stabilize the antagonist-bound state. α1R132A also conferred a new long-lived open state, indicating that this arginine influences the channel gate. Thus, each of these arginines plays a unique role in determining interactions with agonists versus antagonists and with the channel gate