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

Abstract

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-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 (␣ 1 Arg67, ␣ 1 Arg132, and ␤ 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 ␣ 1 R67A or ␤ 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, ␣ 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 antagonistbound complex. In contrast, ␤ 2 R207A and ␣ 1 R132A sped antagonist unbinding, indicating that these arginines stabilize the antagonist-bound state. ␣ 1 R132A also conferred a new longlived 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.

Epileptic encephalopathy de novo GABRB mutations impair γ‐aminobutyric acid type A receptor function

“…The LGS-associated GABRB3 mutations are at the b1/a2 interface, which is directly coupled with the ligand binding channel gating pathway of the receptor. 30,31 These mutations could be more disruptive to channel function than the IS-associated mutations at the a1/b2 and c1/b2 interfaces (which are indirectly coupled by rearrangements throughout the b sheets/a helices of the receptor 26 ). For the mutations located in the signal peptide, GABRB3(P11S, S15F), 6 and at the c1/b2 interface, GABRB3(G32R) 7 and GABRG2(R82Q, P83S), 32 the reductions in GABA A receptor currents were smaller (reduced to $42, $48, $50-62, $34, and $12% of the wt currents, respectively) than those caused by the LGS-associated GABRB3(D120N, E180G, Y302C) mutations (reduced to $24, $1, and $5% of the wt currents, respectively) located at the b1/a2 interface.…”

Pentameric ligand-gated ion channel ELIC is activated by GABA and modulated by benzodiazepines

“…We find that the α-amino nitrogen of glutamate occupies a position that is almost identical (<1.0 Å) to the γ-amino nitrogen of GABA and is involved in similar interactions, including cation-π interactions with Y151 and Y200 (homologous to F133 and F188 in ELIC). Arginine residues of loop D, which may stabilize the carboxyl tail of GABA in vertebrate receptors (9,39), are not conserved in ELIC (R67 in α1, R104 in ρ1, R56 in GluCl, and V40 in ELIC). This difference likely explains the different binding pose of the γ-carboxylate group of glutamate and carboxyl tail of GABA ( Fig.…”

GABA A Receptor Family: Overview on Structural Characterization

“…[ 93,290] • Estabiliza la unión de antagonistas. [290] • No participa de la apertura del canal. [93] β 2 S209…”

“…[ 94,290] • Se ha propuesto que obstruye la unión de antagonistas. [290] • No está involucrado en la activación del canal. [290] • Este residuo se encuentra conservado en todas las subunidades α, π y ρ de receptores GABA A .…”