The C loop at the orthosteric binding site is critically involved in GABAA receptor gating

Mutations at the M2 and M3 Transmembrane Helices of the GABAARs α1 and β2 Subunits Affect Primarily Late Gating Transitions Including Opening/Closing and Desensitization

“…As already mentioned in the Introduction , mutation of the α 1 F45 residue (loop G) 22 affected practically all gating features of the receptor including preactivation, opening/closing, and desensitization providing another example that structural determinants for these gating transitions are shared. Similar conclusions were drawn in our recent study on the role of the loop C (β 2 F200) 24 in which the impact on all gating characteristics, including desensitization, was reported. Perhaps the most surprising was our recent observation that the mutation of α 1 F14 and β 2 F31 residues, located nearly at the “top” of the ECD, had again a clear impact on all gating transitions including desensitization.…”

“…In the case of the α 1 G258V and β 2 L296V mutations, the major manifestations of the altered kinetic phenotype with respect to the WT receptors (slower onset and reduced rate and extent of rapid desensitization) could be observed within the time window of approximately 30 ms. We thus restricted our analysis for this time window and fitted the respective traces (for WT and the mutants) with a simplified model with one open and one (rapid) desensitized state ( Figure 4 A, exemplary fits in part Ba–c, simulated traces in part Ca). Using this approach, the values of the rate constants for the WT receptors ( Figure 4 Da) were comparable to those obtained in our previous studies 24 , 26 and the kinetic features of current responses mediated by these receptors were well-reproduced by curves obtained from modeling within the considered time range (data not shown). Also, in the case of the considered mutants, fitting with the simplified model to approximately 30 ms intervals of current responses well-reproduced the time course of recorded currents.…”

“…The major point that, in our opinion, deserves a particular emphasis, is that the region of M2 and M3 segments is not unique for determining either opening/closing (“efficacy”) or desensitization. Several studies by our group and by others clearly indicated that the mutations located close to the agonist binding site may have a strong impact on receptor gating, including the specific effect on efficacy: β 2 F200 (loop C), 24 α 1 F45 (loop G), 22 α 1 F64A (loop D), 38 β 2 E155 (loop B). 25 , 41 Another intriguing consequence of the mutations at the proximity of the GABA A R agonist binding sites is increased spontaneous activity, further underscoring long-range structural interactions underlying receptor openings (e.g., β 2 E155).…”

Shisa7-dependent regulation of GABAA receptor single-channel gating kinetics

“…We find that compared to control, Shisa7 reduces E1 and E3, which represent transitions from the final close state directly proceeding channel opening states (Figure 6B, Table 2). We do not find significant differences between control and Shisa7 in the pre-activation step, ϕ, a short-lived, close-close state transition that ultimately progresses towards channel activation (Gielen et al, 2012, Kaczor et., 2021, Terejko et al, 2019, Dixon et al, 2014, Gielen et al, 2018, Lape et al, 2008) nor in Σ, a close-close transition that leads the channel further away from opening (Lema and Auerbach, 2008). Furthermore, there are no significant differences in E2 (Figure 6B), representing the occupancy of the shortest open state and is consistent with no changes in time nor proportion spent in the presence of Shisa7 for this state (Figure 5E, 5F).…”

8-Substituted Triazolobenzodiazepines: In Vitro and In Vivo Pharmacology in Relation to Structural Docking at the α1 Subunit-Containing GABAA Receptor

“…The Figures show the α+γ-binding interface of the α1GABA A receptor, with the α1 interface in aquamarine and the γ2 interface in orchid, with the ligands posed to the C-loop of the α1 subunit, a component of the receptor well-established as critical to ligand-mediated binding and gating (cf. Terejko et al, 2020). The triazolam structure was docked in a similar conformation as bound alprazolam in the CryoER structure (6HUO).…”