UNRAVELING THE GATING MECHANISM AND ALLOSTERIC MODULATION OF GABAA RECEPTORS

Ayobami H Diyaolu

GABAA receptors are the principal mediators of inhibitory neurotransmission in the mammalian central nervous system. Hence, they are implicated in the pathophysiology of epilepsy, depression, anxiety, autism spectrum disorder, and schizophrenia. Despite decades of investigation, critical gaps remain in the mechanistic understanding of how GABA activates these receptors, how allosteric modulators influence gating transitions, and the principles governing efficacy at transmembrane sites. The background, significance, knowledge gaps, hypothesis, and specific aims of this dissertation are outlined in Chapter 1. Chapter 2 provides a comprehensive review of neurosteroid modulation of GABAA receptors, covering mechanisms, pharmacology, and structure-activity relationships, thereby establishing the mechanistic and therapeutic context for the experimental work that follows. Chapter 3 elucidates the structural and energetic basis of GABA binding at the two orthosteric sites of the α1β3γ2 GABAA receptor. Using well-tempered metadynamics, steered molecular dynamics, and bias-exchange umbrella sampling, we resolved GABA's entry and exit pathways, identified the R85-D163 salt bridge underlying the approximately three-fold higher affinity of site 2 relative to site 1. Chapter 4 generates and characterizes the first in silico model of a functionally open GABAA receptor using hydrophilic L9′T and L9′S gain-of-function substitutions at the 9′ hydrophobic gate, combined with Gaussian-accelerated molecular dynamics and computational electrophysiology. The mutant-stabilized open-like ensemble exhibited expanded pore geometry, sustained hydration, chloride conductance of 10-32 pS, and activation-like global untwisting and ECD compaction. Both ganaxolone and diazepam further stabilized this open ensemble through convergent effects at their respective binding sites. Bicuculline, an orthosteric antagonist induces closure of the functionally open L9′T receptor in a progressive manner with concurrent conformational changes.Chapter 5 employs microsecond-scale molecular dynamics and funnel metadynamics to compare, for the first time, the binding dynamics, absolute affinities, membrane lipid contacts, and bilayer partitioning profiles of steroidal and non-steroid modulators at the neurosteroid binding site (NBS). A central finding is that near-equivalent binding free energies between ligand classes do not predict equivalent pharmacological potency. Instead, neurosteroid-like membrane partitioning, in particular, asymmetric inner leaflet localization emerges as a co-determinant of functional efficacy. Building on this foundation, Chapter 6 deploys a two-stage virtual screening campaign combining pharmacophore screening of 25 million compounds with active learning deep docking across 1.3 billion ZINC20 molecules. This identified over 22 viable non-steroidal NBS modulators with neurosteroid-comparable interaction profiles and membrane permeability characteristics. This dissertation provides integrated mechanistic insights into GABAA receptor gating and transmembrane allosteric modulation, and establishes a physics-based, membrane-aware framework for the rational discovery of next-generation GABAergic therapeutics.

UNRAVELING THE GATING MECHANISM AND ALLOSTERIC MODULATION OF GABAA RECEPTORS

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