Molecular Mechanisms and Probe‐Dependent Effects of Clinically Relevant GABAA Receptor Modulators

ABSTRACT Modulators of γ‐aminobutyric acid type A (GABAA) 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 GABAA 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.