Open-State Dynamics and Allosteric Modulation of the α1β3γ2 GABA_A Receptor Stabilized by L9'T/S Substitutions

Pentameric GABA type A receptors (GABA_ARs) mediate fast inhibitory neurotransmission and are major targets of clinically important neuroactive drugs, yet the open state of heteropentameric receptors has remained difficult to define structurally and mechanistically. Here, we establish an experimentally supported, atomistic characterization that links pore hydration, ion-permeation energetics, conductance, and global conformational rearrangements in open-like α1β3γ2 GABA_AR ensembles and delineate how positive and negative allosteric modulators bias the gating landscape. We use in-silico mutagenesis and Gaussian-accelerated molecular dynamics to stabilize open-like ensembles of the α1β3γ2 receptor via hydrophilic substitutions at the conserved 9' hydrophobic gate (L9'T/L9'S). The mutants expand the pore at 9' and 20' (pore entry), increase hydration and water flux, and lower Cl^- permeation barriers at 9' and -2' (desensitization gate) from ∼ 19/∼9 kcal/mol in wild-type receptors to ∼ 1.3-1.6/∼3.0-3.4 kcal/mol, yielding ohmic conductance of ∼10-30 pS in computational electrophysiology. Conformationally, the mutants exhibit reduced global twist, outward M2 helix tilts, and orthosteric C-loop closure, consistent with activation-like signatures. On this open-like background, positive allosteric modulators (diazepam, ganaxolone) primarily tune the residual -2' constriction, whereas the orthosteric antagonist bicuculline drives a time-ordered, bottom-to-top closing sequence (-2' first, followed by 9'/20' and global twist) via an asymmetric collapse of the M2 bundle that approaches closed-state landmarks while sampling desensitized-like coupling of extracellular and transmembrane domains. Two-electrode voltage-clamp recordings confirm spontaneous activity in L9'T-containing receptors, supporting the mutant-stabilized open-like ensembles. Together, these results provide a coherent atomistic framework for gating and allosteric modulation in α1β3γ2 GABA_ARs and establish a tractable platform for state-selective structure-based design.