Interaction between GABAA receptor α1 and β2 subunits at the N-terminal peripheral regions is crucial for receptor binding and 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 extensively discussed in our previous reports, the desensitization onset can be clearly observed upon rapid agonist applications, offering a unique opportunity to characterize respective rate constants, whereas in the stationary recordings, this dynamic phase of the desensitization process is not visible; therefore, the reliability of estimation of the desensitization rates is limited. 21 , 38 Nevertheless, in the single-channel modeling we reproduced well, at the qualitative level, changes in desensitization observed in the macroscopic recordings (for α 1 G258V, α 1 L300V, β 2 L296V =reduction of desensitization and for β 2 G254V= enhancement). For the α 1 G258V and α 1 L300V mutants, the lack of effect on the flipping rate δ 2 was consistent in the macroscopic and single-channel modeling, but there were differences for the unflipping rate γ 2 ( Figures 4 , 5 , and 8 ).…”

“…Moreover, in our recent study, we provided evidence that mutation of the F45 residue, located close to the agonist binding site at the loop G of the α 1 subunit, profoundly altered desensitization kinetics. Unexpectedly, we also found that mutation of the α 1 F14 and β 2 F31 residues, located “above” the orthosteric binding site in ECD, also had a strong impact on the desensitization transitions . Thus, our studies suggest that desensitization might structurally depend on vast fragments of the GABA A R macromolecule, raising a concept of a “diffuse” desensitization gating mechanism rather than its well defined localization.…”

“…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. 21 Moreover, it was shown that the involvement of these residues in the receptor gating depends on intersubunit interactions mediated by these phenylalanine residues, further underscoring long-range interactions in the structural mechanisms underlying conformational transitions. All these observations point to the concept of “diffuse gating mechanism” for all conformational transitions, including desensitization.…”

“…Unexpectedly, we also found that mutation of the α 1 F14 and β 2 F31 residues, located “above” the orthosteric binding site in ECD, also had a strong impact on the desensitization transitions. 21 Thus, our studies suggest that desensitization might structurally depend on vast fragments of the GABA A R macromolecule, raising a concept of a “diffuse” desensitization gating mechanism rather than its well defined localization. This means that, although the site of the final step of the desensitization transition is located at the constriction of the ion pore (at the level of −2′ residue in the TMD), the preceding structural rearrangements leading to this conformational change would comprise the vast parts of the macromolecule.…”

“…A characteristic feature of pLGICs is that the activation process comprises vast portions of the macromolecules as the ligand binding site (LBS), located at extracellular domain (ECD), is positioned very far (approximately 50 Å) , from the receptor gate, suggesting complexity of gating mechanisms (Figure A–C). Another dimension of GABA A R gating complexity is numerous conformational transitions which can be grouped in three main categories, openings/closings, preactivation, and desensitization, and each of them may be represented by transitions into more than one state (especially for openings/closings and desensitization, typically more than one state are identified). − …”