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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 GABAA 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 GABAA 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 GABAA receptor modulation through transmembrane binding sites.

Abstract γ-Aminobutyric acid type A (GABAA) receptors mediate fast inhibitory signaling in the brain and are targets of numerous drugs and endogenous neurosteroids. A subset of neurosteroids are GABAA receptor positive allosteric modulators; one of these, allopregnanolone, is the only drug approved specifically for treating postpartum depression. There is a consensus emerging from structural, physiological and photolabeling studies as to where positive modulators bind, but how they potentiate GABA activation remains unclear. Other neurosteroids are negative modulators of GABAA receptors, but their binding sites remain debated. Here we present structures of a synaptic GABAA receptor bound to allopregnanolone and two inhibitory sulfated neurosteroids. Allopregnanolone binds at the receptor-bilayer interface, in the consensus potentiator site. In contrast, inhibitory neurosteroids bind in the pore. MD simulations and electrophysiology support a mechanism by which allopregnanolone potentiates channel activity and suggest the dominant mechanism for sulfated neurosteroid inhibition is through pore block.

Abstract General anesthetics and neuromuscular blockers are used together during surgery to stabilize patients in an unconscious state. Anesthetics act mainly by potentiating inhibitory ion channels and inhibiting excitatory ion channels, with the net effect of dampening nervous system excitability. Neuromuscular blockers act by antagonizing nicotinic acetylcholine receptors at the motor endplate; these excitatory ligand-gated ion channels are also inhibited by general anesthetics. The mechanisms by which anesthetics and neuromuscular blockers inhibit nicotinic receptors are poorly understood but underlie safe and effective surgeries. Here we took a direct structural approach to define how a commonly used anesthetic and two neuromuscular blockers act on a muscle-type nicotinic receptor. We discover that the intravenous anesthetic etomidate binds at an intrasubunit site in the transmembrane domain and stabilizes a non-conducting, desensitized-like state of the channel. The depolarizing neuromuscular blocker succinylcholine also stabilizes a desensitized channel but does so through binding to the classical neurotransmitter site. Rocuronium binds in this same neurotransmitter site but locks the receptor in a resting, non-conducting state. Together, this study reveals a structural mechanism for how general anesthetics work on excitatory nicotinic receptors and further rationalizes clinical observations in how general anesthetics and neuromuscular blockers interact.

GABAA receptors are important targets for anxiety, sedation and anesthesia. Here, the authors present structures bound by zolpidem (Ambien), the most prescribed hypnotic in the US, and DMCM, a negative modulator, providing insights into receptor modulation.