Activity-Dependent Inhibitory Synapse Scaling Is Determined by Gephyrin Phosphorylation and Subsequent Regulation of GABAA Receptor Diffusion.

Synaptic plasticity relies on the rapid changes in neurotransmitter receptor number at postsynaptic sites. Using superresolution photoactivatable localization microscopy imaging and quantum dot-based single-particle tracking in rat hippocampal cultured neurons, we investigated whether the phosphorylation status of the main scaffolding protein gephyrin influenced the organization of the gephyrin scaffold and GABAA receptor (GABAAR) membrane dynamics. We found that gephyrin phosphorylation regulates gephyrin microdomain compaction. Extracellular signal-regulated kinase 1/2 and glycogen synthase kinase 3ß (GSK3ß) signaling alter the gephyrin scaffold mesh differentially. Differences in scaffold organization similarly affected the diffusion of synaptic GABAARs, suggesting reduced gephyrin receptor-binding properties. In the context of synaptic scaling, our results identify a novel role of the GSK3ß signaling pathway in the activity-dependent regulation of extrasynaptic receptor surface trafficking and GSK3ß, protein kinase A, and calcium/calmodulin-dependent protein kinase IIα pathways in facilitating adaptations of synaptic receptors.

Several posttranslational modifications act in concert to regulate gephyrin scaffolding and GABAergic transmission.

GABAA receptors (GABAARs) mediate the majority of fast inhibitory neurotransmission in the brain via synergistic association with the postsynaptic scaffolding protein gephyrin and its interaction partners. However, unlike their counterparts at glutamatergic synapses, gephyrin and its binding partners lack canonical protein interaction motifs; hence, the molecular basis for gephyrin scaffolding has remained unclear. In this study, we identify and characterize two new posttranslational modifications of gephyrin, SUMOylation and acetylation. We demonstrate that crosstalk between SUMOylation, acetylation and phosphorylation pathways regulates gephyrin scaffolding. Pharmacological intervention of SUMO pathway or transgenic expression of SUMOylation-deficient gephyrin variants rescued gephyrin clustering in CA1 or neocortical neurons of Gabra2-null mice, which otherwise lack gephyrin clusters, indicating that gephyrin SUMO modification is an essential determinant for scaffolding at GABAergic synapses. Together, our results demonstrate that concerted modifications on a protein scaffold by evolutionarily conserved yet functionally diverse signalling pathways facilitate GABAergic transmission.