Fast synaptic inhibition in the mammalian central nervous system is mediated primarily via activation of the ␥-aminobutyric acid type A receptor (GABA A -R). Upon agonist binding, the receptor undergoes a structural transition from the closed to the open state. This transition, known as gating, is thought to be associated with a sequence of conformational changes originating at the agonist-binding site, ultimately resulting in opening of the channel. Using site-directed mutagenesis and several different GABA A -R agonists, we identified a number of highly conserved charged residues in the GABA A -R ␤ 2 subunit that appear to be involved in receptor activation. We then used charge reversal double mutants and disulfide trapping to investigate the interactions between these flexible loops within the ␤ 2 subunit. The results suggest that interactions between an acidic residue in loop 7 (Asp 146 ) and a basic residue in pretransmembrane domain-1 (Lys 215 ) are involved in coupling agonist binding to channel gating.Fast synaptic inhibition is a major determinant of network dynamics in the central nervous system (1). In the mammalian brain, this is mediated primarily via activation of the ␥-aminobutyric acid type A receptor (GABA A -R) 1 (2, 3). Each GABA A -R is composed of five subunits arranged around a central ion-conducting pore (4), with each subunit consisting of a large intracellular domain, four transmembrane domains (TM1-TM4), and a larger N-terminal extracellular domain (2). Experimental evidence suggests that the GABA-binding site lies within an asymmetric pocket formed at the interface between the ␣ and ␤ receptor subunits (5-8). The channel "gate" in the GABA A -R is believed to be formed by charged residues in the TM1-TM2 loop (9 -11). The binding of agonist triggers a complex structural transition that results in the opening of the gate, allowing ions to flow through the channel. The mechanisms by which this occurs remain poorly defined.The nature of the coupling between binding and channel opening in this receptor family has been recently investigated in several laboratories. Interactions between charged residues in the flexible loops 2 and 7 in the extracellular domain and those in the short linker between the second and third transmembrane domains (TM2-3L) of the GABA A -R ␣ 1 subunit have been implicated in the process of receptor activation (12). In addition, a recent report suggests that the pre-TM1 region is critical for receptor activation in the closely related serotonin (5-HT 3 ) receptor (13). In this study, we used site-directed mutagenesis and a number of GABA A -R agonists of varying efficacies to examine the contribution of the corresponding flexible loops in the GABA A -R ␤ 2 subunit to receptor activation. Based on previous studies (12-14) and the presence of highly conserved charged residues (see Fig. 1), we hypothesized that interactions between charged residues in these domains are crucial for coupling agonist binding to channel gating. We tested this hypothesis using a charge revers...