GABA receptors (GABARs) play a crucial role in mammalian adult brain inhibition. The dysfunction of GABAergic drive is related to such disorders as epilepsy, schizophrenia, and depression. Substantial progress has recently been made in describing the static structure of GABARs, but the molecular mechanisms that underlie the activation process remain elusive. The C loop of the GABAR structure shows the largest movement upon ligand binding to the orthosteric binding site, a phenomenon that is referred to as “capping.” The C loop is known to be involved in agonist binding, but its role in the gating of Cys-loop receptors is still debated. Herein, we investigated this issue by analyzing the impact of a βF200 residue mutation of the C loop on gating properties of αβγ GABARs. Extensive analyses and the modeling of current responses to saturating agonist application demonstrated that this mutation strongly affected preactivation, opening, closing and desensitization, i.e. all considered gating steps. Single-channel analysis revealed that the βF200 mutation slowed all shut time components, and open times were shortened. Model fitting of these single-channel data further confirmed that the βF200 mutation strongly affected all of the gating characteristics. We also found that this mutation altered receptor sensitivity to the benzodiazepine flurazepam, which was attributable to a change in preactivation kinetics. In silico analysis indicated that the βF200 mutation resulted in distortion of the C loop structure, causing the movement of its tip from the binding site. Altogether, we provide the first evidence that C loop critically controls GABAR gating.Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.