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

GABA type A receptors (GABAARs) mediate inhibitory neurotransmission, and their dysfunction contributes to epilepsy, anxiety, and depression. Although closed and desensitized structures of heteropentameric GABAARs are known, an open-state conformation has been difficult to capture. Here we use in-silico mutagenesis and Gaussian-accelerated MD to stabilize open-like ensembles of the α1β3γ2 receptor via hydrophilic substitutions at the hydrophobic 9' gate (L9'T/L9'S). The mutants expand the pore at 9' and 20' (extracellular pore entry), increase hydration and water flux, and lower Cl⁻ permeation barriers at 9' and -2' (desensitization gate) from ~19/~9 kcal/mol (WT) to ~1.3-1.6/~3.0-3.4 kcal/mol, yielding ohmic conductance ~10-30 pS in computational electrophysiology. Conformationally, the mutants show reduced twist, outward M2 tilts, and C-loop closure, consistent with activation-like signatures. On this open-like background, PAMs (diazepam, ganaxolone) primarily tune the residual -2' constriction, whereas bicuculline (orthosteric antagonist) drives a time-ordered, bottom-to-top closing sequence (-2' first, then 9'/20', then twist) via an asymmetric collapse of the M2 bundle that approaches closed-state landmarks while sampling desensitized-like ECD-TMD coupling. Two-electrode voltage clamp confirms spontaneous activity in L9'T-containing receptors, supporting the mutant-stabilized open-like ensembles. These results provide a coherent atomistic framework for gating and allosteric modulation in α1β3γ2 GABAARs and establish a tractable, ligand-responsive platform for state-selective structure-based design.