INTRODUCTION: GABAA receptors (GABAARs) are pentameric ligand-gated ion channels that are crucial in fast inhibitory transmission in adult CNS. The activation process of GABAAR couples agonist binding to the binding site in the extracellular domain, with an opening of the channel gate in the far-distant transmembrane domain. GABAAR gating is a complex process that includes preactivation (flipping), which is a step of bound receptor that remains closed and precedes opening. Our recent study shows that flipping is modulated by benzodiazepines. A key element in GABAARs activation is loop C capping, an inward movement of the β2 subunit loop C upon ligand binding; however, the exact role of loop C in relation to GABAAR binding and gating remains elusive. AIM(S): This study aims to explain how a mutation of the β2F200 residue in loop C affects functioning of the GABAAR in reference to receptor binding and gating, including modulation of preactivation by flurazepam (benzodiazepine). METHOD(S): β2F200 mutated receptors (Tyr, Ile, Cys) were expressed in HEK293 cells. Patch clamp was used to record macroscopic currents elicited by saturating [GABA] (combined with ultrafast perfusion system) and single channel currents. Kinetic analysis was preformed and followed by kinetic modeling. RESULTS: β2F200 mutants exhibited a shift in dose‑response relationship. The mutation significantly slowed down current onset and desensitization, but deactivation was accelerated. Flurazepam potentiated currents evoked by saturating [GABA] in contrast to WT receptors. Single‑channel analysis showed a significant change in all shut time distributions components and shortening of open time distributions. CONCLUSIONS: Kinetic modeling of macroscopic and single channel currents confirmed alteration in all considered gating properties. In silico ligand docking indicated a drop in the binding affinity for each mutant. GABAAR loop C plays a critical role in receptor binding and gating. FINANCIAL SUPPORT: NCN grant 2015/18/A/NZ1/ 00395.