An elegant study has identified glycogen synthase kinase 3β (GSK3β) as a proline-directed kinase that controls phosphorylation- and proteolytic cleavage-induced turnover of gephyrin (Figure 5A) (Tyagarajan et al., 2011). Using tandem mass spectrometry of gephyrin, the authors identified
Depsipeptide purchase S270 as a residue that is basally phosphorylated in brain tissue. Transfection of cultured neurons with phosphorylation-deficient gephyrinS270A increased the density of gephyrin clusters and the amplitude and frequency of GABAergic mIPSCs, indicating that gephyrin clustering is limited by phosphorylation at S270. However, mutations of S270 had no effect on cluster size. Using kinase-specific inhibitors in in vitro phosphorylation assays the authors identified GSK3β as an important kinase for S270. To address the mechanism by which phosphorylation might increase gephyrin turnover they focused on calpain-1. This Ca2+-dependent cysteine protease was previously shown to cleave gephyrin and to produce a stable C-terminal gephyrin fragment of 48–50 kDa (Kawasaki et al., 1997). Transfection of neurons with the natural calpain-1
inhibitor calpastatin increased the gephyrin cluster density (Tyagarajan et al., 2011). Moreover, this effect was enhanced in the presence of the phosphomimetic mutant gephyrinS270E as a substrate, indicating that calpain-1-mediated degradation of gephyrin is triggered by phosphorylation
of S270. Lastly, the authors showed that S270 phosphostate-dependent clustering of gephyrin is enhanced BMN 673 supplier by chronic treatment of cultured neurons or mice with Li+, a potent inhibitor of GSK3β used as mood-stabilizing agent for the Idoxuridine treatment of bipolar disorder. The findings strongly suggest that Li+-induced enhancement of GABAergic synaptic transmission contributes to the mood-stabilizing effects of Li+ in patients (Tyagarajan and Fritschy, 2010). GSK3β is inhibited as a downstream target of both the canonical Wnt signaling pathway (Inestrosa and Arenas, 2010) and the insulin receptor signaling pathway. Both pathways promote the postsynaptic clustering of GABAARs by additional, gephyrin-independent mechanisms, as detailed further below. Gephyrin forms a stable complex with affinity-purified glycine receptors (Pfeiffer et al., 1982). By contrast, GABAARs in detergent-solubilized membrane extracts do not stably associate with gephyrin (Meyer et al., 1995). Moreover, a major subset of GABAARs comprising α1βγ2 receptors can accumulate and cluster at synapses independently of gephyrin (Kneussel et al., 2001 and Lévi et al., 2004). Nevertheless, in brain gephyrin serves as a reliable postsynaptic marker for all GABAergic synapses (Sassoè-Pognetto et al., 1995, Essrich et al., 1998 and Sassoè-Pognetto and Fritschy, 2000).