Modulation of sleep/wake patterns by gephyrin phosphorylation status

Sleep/wake cycles intricately shape physiological activities including cognitive brain functions, yet the precise molecular orchestrators of sleep remain elusive. Notably, the clinical impact of benzodiazepine drugs underscores the pivotal role of GABAergic neurotransmission in sleep regulation. However, the specific contributions of distinct GABA$_{A}$ receptor subtypes and their principal scaffolding protein, gephyrin, in sleep dynamics remain unclear. The evolving role of synaptic phospho‐proteome alterations at excitatory and inhibitory synapses suggests a potential avenue for modulating gephyrin and, consequently, GABA$_{A}$Rs for sleep through on‐demand kinase recruitment. Our study unveils the distinctive roles of two prevalent GABA$_{A}$ receptor subtypes, α1‐ and α2‐GABA$_{A}$Rs, in influencing sleep duration and electrical sleep activity. Notably, the absence of α1‐GABA$_{A}$Rs emerges as central in sleep regulation, manifesting significant alterations in both non‐rapid eye movement (NREM) and rapid eye movement (REM) sleep during dark or active phases, accompanied by altered electroencephalogram (EEG) patterns across various frequencies. Gephyrin proteomics analysis reveals sleep/wake‐dependent interactions with a repertoire of known and novel kinases. Crucially, we identify the regulation of gephyrin interaction with ERK1/2, and phosphorylations at serines 268 and 270 are dictated by sleep/wake cycles. Employing AAV‐eGFP‐gephyrin or its phospho‐null variant (S268A/S270A), we disrupt sleep either globally or locally to demonstrate gephyrin phosphorylation as a sleep regulator. In summary, our findings support the local cortical sleep hypothesis and we unveil a molecular mechanism operating at GABAergic synapses, providing critical insights into the intricate regulation of sleep.