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 GABA(A) 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 (α(1)Arg67, α(1)Arg132, and β(2)Arg207) 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 α(1)R67A or β(2)R207A 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, α(1)R67A 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, β(2)R207A and α(1)R132A sped antagonist unbinding, indicating that these arginines stabilize the antagonist-bound state. α(1)R132A 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.