Abstract
γ-Aminobutyric acid type A receptors (GABA(A)Rs) are pentameric ligand-gated ion channels mediating fast inhibitory neurotransmission in the mammalian brain. Although recent structural and kinetic studies have advanced understandings regarding their activation mechanisms, the molecular determinants coupling agonist binding to channel gating remain unclear. We investigated the contribution of the β(2)E153 residue, located on loop B of the extracellular domain, to the activation of α(1)β(2)γ(2) GABA(A)Rs. Macroscopic and single-channel patch clamp recordings were used to characterize two β(2)E153-mutants: charge reversal (β(2)E153K) and hydrophobic substitution (β(2)E153A). Both substitutions disrupted normal receptor kinetics, with β(2)E153K selectively accelerating deactivation and β(2)E153A affecting both deactivation and desensitization. Single-channel analysis showed that β(2)E153A reduced open probability and mean open times, consistent with altered gating transitions inferred from kinetic modeling. Structural inspection suggested that β(2)E153 forms electrostatic interactions with β(2)K196 and β(2)R207 to stabilize loop C and maintain the agonist-bound conformation. The disruption of this interaction likely destabilizes loop C, leading to weakened agonist binding and modified gating. Overall, our results identify β(2)E153 as a key element in the long-range allosteric network linking the binding site to the channel gate in GABA(A)Rs.