Structural insights into GABAA receptor potentiation by Quaalude

Abstract

Methaqualone, a quinazolinone marketed commercially as Quaalude, is a central nervous system depressant that was used clinically as a sedative-hypnotic, then became a notorious recreational drug in the 1960s-80s. Due to its high abuse potential, medical use of methaqualone was eventually prohibited, yet it persists as a globally abused substance. Methaqualone principally targets GABA_A receptors, which are the major inhibitory neurotransmitter-gated ion channels in the brain. The restricted status and limited accessibility of methaqualone have contributed to its pharmacology being understudied. Here, we use cryo-EM to localize the GABA_A receptor binding sites of methaqualone and its more potent derivative, PPTQ, to the same intersubunit transmembrane sites targeted by the general anesthetics propofol and etomidate. Both methaqualone and PPTQ insert more deeply into subunit interfaces than the previously-characterized modulators. Binding of quinazolinones to this site results in widening of the extracellular half of the ion-conducting pore, following a trend among positive allosteric modulators in destabilizing the hydrophobic activation gate in the pore as a mechanism for receptor potentiation. These insights shed light on the underexplored pharmacology of quinazolinones and further elucidate the molecular mechanisms of allosteric GABA_A receptor modulation through transmembrane binding sites.

Fig. 1. Quinazolinones act as positive allosteric modulators through binding to TMD sites in the α1β2γ2 GABA_A receptor.

a Exemplary whole-cell patch-clamp electrophysiology trace showing the PAM and agonist activities displayed by methaqualone (MQ) and PPTQ on the EM receptor at their respective EC₅₀ concentrations; n = 6 recordings from independent cells; GABA EC₁₀ = 4 µM. b and c EM density map showing two methaqualone binding sites per receptor. b Side view of the receptor. c Cross section through the transmembrane domain and ligand chemical structure. d and e Same as b and c for PPTQ.

Fig. 2. Methaqualone binds to the same TMD β/α interface sites as the general anesthetics propofol and etomidate.

a Top view of the receptor TMD with one quinazolinone site boxed. b Comparison of quinazolinones and general anesthetics binding at the β2/α1 binding interface (orange – propofol, turquoise – etomidate, pink – methaqualone, green – PPTQ). c and d detailed view of methaqualone and PPTQ binding sites with residues shown by mutagenesis to affect quinazolinone activity as well as residues identified in our structures to likely interact directly with the ligands.

Fig. 3. Mutagenesis supports importance of deep binding sites for quinazolinones in the TMD β/α interface.

a Concentration-response curve showing GABA-mediated activation of α1T265A, α1L269A and EM α1β2γ2 receptors (EC₅₀: 29 µM for EM; 7 µM for α1T265A and α1L269A). b Whole-cell patch-clamp electrophysiology traces showing activity of methaqualone (MQ) on mutated and EM receptors. c Bar graph showing methaqualone PAM activity on EM and mutated receptors normalized to GABA EC₁₀ = 4 µM for EM, 1 µM for α1T265A and α1L269A. d Whole-cell patch-clamp electrophysiology traces showing activity of PPTQ on mutated and EM receptors. e Bar graph for PPTQ PAM and agonist activities on EM and mutated receptors normalized to GABA EC₁₀. n = recordings from independent cells. Results are shown as a mean response ± S.D.; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 4. The ECD benzodiazepine binding site is a silent site for methaqualone.

a Close-up and b global views of the ECD α1/γ2 interface with methaqualone docked into the observed density. c Flumazenil competition and double mutant experiments, representative patch-clamp electrophysiology trace. d Representative patch-clamp electrophysiology trace for double mutant assay. e Statistical analysis of electrophysiology results comparing methaqualone responses in the EM and the double mutant receptors, the bar graph shows mean responses with standard deviation; p = 0.17, n = 8 recordings from independent cells for EM, n = 11 for Y210A/F77A. f Representative patch-clamp recording from the test of methaqualone potentiation on the EM construct and its N265M mutant. g Statistical analysis of electrophysiology results comparing methaqualone potentiation on the EM construct and the N265M mutant, p = 0.0119, n = 4 recordings from independent cells. h Representative patch-clamp recording on N265M receptor from the competition assay for diazepam. i Statistical analysis of electrophysiology results comparing N265M receptors’ response to diazepam without and with methaqualone, p = 0.0194, n = 4 recordings from independent cells. Results are shown as a mean response ± S.D. p ≤ 0.05 was considered statistically significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 5. Quinazolinone binding widens the pore above the 9′ gate.

a HOLE representation of the channel pore comparing resting and desensitization gates in GABA-only, methaqualone, and PPTQ structures. Pore diameters are indicated. b HOLE plot showing diameter along the pore for different structures (PDB IDs: GABA-6X3Z, etomidate-6X3V, propofol-6X3T, bicuculline-6X3S). c Comparison of the M2 distances between GABA-only (turquoise) and methaqualone (pink) structures. d R269 (19′) position when the pocket is not occupied by a ligand (GABA-only structure, PDB ID: 6X3Z). e R269 (19′) position when the pocket is occupied by methaqualone.