Redox-dependent synaptic clustering of gephyrin

Abstract Reactive oxygen species (ROS) have been demonstrated to play central functions as signaling molecules thus extending their role beyond oxidative stress in disease and aging. At inhibitory synapses the scaffolding protein gephyrin clusters glycine and GABA type A receptors. Postsynaptic gephyrin clustering is regulated by post-translational modifications such as phosphorylation, S-palmitoylation, and S-nitrosylation, which all critically impact its scaffolding function. Here, we show that oxidation of surface-exposed cysteine residues in gephyrin triggered reversible, synaptic multimerization providing more receptor binding sites and leading to proteolytic protection. Cys419, located at the previously published E-domain SDII interface important for liquid-liquid phase separation, was identified as a critical residue further regulating liquid-liquid phase separation of gephyrin in a redox-dependent manner. We also observed that surface-exposed cysteines are required to maintain fast miniature inhibitory postsynaptic current rise times, supporting the functional relevance of gephyrin redox regulation. Collectively, our findings suggest that cysteines in gephyrin regulate synaptic localization and clustering as redox-switches, thereby establishing a so far undefined link between neuronal and metabolic activity at inhibitory synapses.