The function and pharmacology of γ-aminobutyric acid type A receptors (GABAARs) are of great physiological and clinical importance and have long been thought to be determined by the channel pore–forming subunits. We discovered that Shisa7, a single-passing transmembrane protein, localizes at GABAergic inhibitory synapses and interacts with GABAARs. Shisa7 controls receptor abundance at synapses and speeds up the channel deactivation kinetics. Shisa7 also potently enhances the action of diazepam, a classic benzodiazepine, on GABAARs. Genetic deletion of Shisa7 selectively impairs GABAergic transmission and diminishes the effects of diazepam in mice. Our data indicate that Shisa7 regulates GABAAR trafficking, function, and pharmacology and reveal a previously unknown molecular interaction that modulates benzodiazepine action in the brain.

Highlights d The epilepsy-associated variant GluN2A-S1459G causes NMDAR trafficking defects d GluN2A-S1459G disrupts NMDAR interactions and leads to reduced mEPSC frequency d GluN2A is phosphorylated by CaMKII at S1459, which is regulated by neuronal activity d GluN2A-S1459 phosphorylation dictates binding to PDZ domain-containing proteins

SUMMARY Tonic inhibition mediated by extrasynaptic γ-aminobutyric acid type A receptors (GABA A Rs) critically regulates neuronal excitability and brain function. However, the mechanisms regulating tonic inhibition remain poorly understood. Here, we report that Shisa7 is critical for tonic inhibition regulation in hippocampal neurons. In juvenile Shisa7 knockout (KO) mice, α5-GABA A R-mediated tonic currents are significantly reduced. Mechanistically, Shisa7 is crucial for α5-GABA A R exocytosis. Additionally, Shisa7 regulation of tonic inhibition requires protein kinase A (PKA) that phosphorylates Shisa7 serine 405 (S405). Importantly, tonic inhibition undergoes activity-dependent regulation, and Shisa7 is required for homeostatic potentiation of tonic inhibition. Interestingly, in young adult Shisa7 KOs, basal tonic inhibition in hippocampal neurons is unaltered, largely due to the diminished α5-GABA A R component of tonic inhibition. However, at this stage, tonic inhibition oscillates during the daily sleep/wake cycle, a process requiring Shisa7. Together, these data demonstrate that intricate signaling mechanisms regulate tonic inhibition at different developmental stages and reveal a molecular link between sleep and tonic inhibition.