WNK-dependent phosphorylation of gephyrin tunes GABA A receptors at inhibitory synapses and modulates anxiety behavior

Summary The role of the chloride-sensitive kinase WNK1 and its effector SPAK in the brain remains poorly understood. Here, we identify a regulatory mechanism involving WNK signaling that directly controls the synaptic diffusion and clustering, as well as the membrane stability and endocytosis of inhibitory GABA A receptors (GABA A Rs). We show that activation of WNK signaling stabilizes GABA A Rs at inhibitory synapses, while inhibition enhances receptor internalization. This regulation depends on the phosphorylation state of two previously uncharacterized residues in the central linker region of the gephyrin scaffold protein. Modulating WNK activity alters neuronal activity and the kinetics of GABAergic currents. In vivo , expression of a phospho-mimetic form of gephyrin at WNK-targeted sites produces anxiolytic effects. By orchestrating the recruitment of GABA A Rs at inhibitory synapses, the WNK pathway emerges as a master regulator of GABAergic transmission and establishes chloride as a bona fide second messenger in inhibitory synaptic signaling. Significance Synaptic transmission relies on signaling pathways that control how neurotransmitter receptors move and stabilize at synapses. Many of these pathways use calcium as a second messenger. In contrast, how chloride might regulate synapses has remained unclear. In this study, we identify a chloride-sensitive pathway involving the WNK1 kinase and its partner SPAK, which controls the movement and stability of inhibitory GABA A receptors at synapses. This occurs through the phosphorylation of gephyrin, a key scaffolding protein, at two newly identified sites. Our results show that intracellular chloride can act as a second messenger, reshaping inhibitory synapses and altering behavior in mice, including reducing anxiety-like responses.