GABAA (γ-aminobutyric acid, type A) receptors are targeted by numerous small-molecule drugs, many of which were discovered before their target was known. Using single particle cryo-electron microscopy (cryo-EM), the first structure of a GABAA receptor in a physiological conformation, reconstituted into a lipid bilayer, has been solved, as have five structures of the same receptor in complex with various drugs. These structures provide insights into the mechanisms of action of GABAA receptor-targeting molecules and could provide the basis for future structure-based drug discovery.
The GABAA receptor modulates phasic and tonal neuronal inhibition. Well-known drugs that target this receptor include the benzodiazepines, which are positive allosteric modulators with sedative, anxiolytic, hypnotic and anticonvulsant properties, but an unknown mechanism of action. Previous work also established that classic benzodiazepines require the presence of specific GABAA receptor subunits, for unclear reasons.
Masiulis and colleagues solved cryo-EM structures of the GABAA receptor to resolutions of 3.0–3.7 Å in complexes with the channel blocker picrotoxin (PTX), PTX plus the endogenous ligand γ-aminobutyric acid (GABA), the antagonist bicuculline (BCC), diazepam (a benzodiazepine) plus GABA, and alprazolam (another benzodiazepine) plus GABA. From their PTX work, the authors concluded that this tool compound binds to the open conformation of the channel pore, after which it unexpectedly induces pore closure. GABA binds to this stabilized conformation with lower affinity, thereby explaining how PTX acts as a competitive antagonist of GABA without binding to either of the orthosteric pockets.
Next, the researchers showed that binding of GABA to the orthosteric sites in the GABAA receptor induces rotation of the extracellular domain, which is reduced in the presence of PTX. Similarly, BCC, which binds to the GABA orthosteric sites, prevents rotation of the extracellular domain and maintains the pore in the closed conformation.
The benzodiazepines bind at an allosteric site between the α and γ subunits. Interestingly, classic benzodiazepines can only modulate GABAA receptors that contain α1, α2, α3 or α5 subunits. In α4 and α6 subunits, an arginine residue replaces the histidine that interacts with diazepam and alprazolam, leading to steric clashes. Of note, a second binding site for diazepam (but not alprazolam) was observed at intersubunit interfaces within the transmembrane domain and is likely responsible for its anaesthetic properties.
The structures presented in these articles could be used to design GABAA receptor modulators with particular properties, which could have increased efficacy and safety, and also highlight the emerging opportunities for applying cryo-EM in drug discovery.