Molecular Mechanisms and Probe-Dependent Effects of Clinically Relevant GABA_A Receptor Modulators

Modulators of γ-aminobutyric acid type A (GABA_A) receptor, including benzodiazepines, anaesthetics, and Z-drugs, act through distinct allosteric mechanisms that remain incompletely understood at the molecular level. Here, we investigate probe-dependent pharmacology in GABA_A receptor modulation by integrating molecular dynamics simulations, deep learning-based enhanced sampling, dynamic pharmacophores, and our recently developed MDPath approach for mapping allosteric communication networks. We demonstrate that benzodiazepines and Z-drugs leverage physiological GABA for activity through long-range allosteric coupling. These modulators bind in the transmembrane domain (TMD), establishing allosteric commmunication to the extracellular domain (ECD) GABA binding sites, stabilizing GABA receptor interactions and explaining their probe-dependent positive allosteric modulation. In contrast, anaesthetics forms additional interactions that enable direct binding-driven channel activation independent of GABA, distinguishing ago-PAMs from PAMs despite binding at the same sites. Remarkably, the flumazenil antidote acts by globally disrupting interdomain allosteric communication, selectively preventing the action of ECD-dependent modulators (benzodiazepines and Z-drugs) while sparing anaesthetics' direct effects. Our integrated computational approach reveals the structural determinants of receptor state transitions, providing a framework for understanding probe-dependent pharmacology in ligand-gated ion channels with implications for rational drug design.