Studies on the molecular mechanisms underlying anxiety and convulsion with special reference to the roles for PRIP

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The protein PRIP was isolated from rat brain as an inositol 1,4,5-trisphosphate-binding protein with a domain organization similar to that of phospholipase C-δ1 but lacking PLC activity. In this study, we show that PRIP plays an important role in signaling by the type A receptor for γ-aminobutyric acid(GABA). Yeast two-hybrid screening identified GABARAP(GABA_A receptor-associated protein), which is proposed to contribute to the sorting, targeting, or clustering of GABA_A receptors, and PP1(protein phosphatase 1), as proteins that interact with PRIP. PRIP competitively inhibited the binding of the γ2 subunit of the GABA_A receptor to GABARAP in vitro. Electrophysiological analysis revealed that the modulation of GABA-induced Cl^-current by Zn^<2+> or diazepam, both of which act at GABA_A receptors containing y subunits, is impaired in hippocampal neurons of PRIP knockout mice(PRIP^<-/->mice). Behavioral analysis revealed that, compared with wild-type animals, motor coordination was imp … More aired and the intraperitoneal injection of diazepam induced markedly reduced sedative and anti-anxiety effects in the mutant mice. These results indicate that PRIP is essential for the function of GABA_A receptors, especially in response to the agents acting on a γ2 subunit. Furthermore, we have examined the role for PRIP, which binds and inactivates PP1,in facilitating GABA_A receptor phospho-dependent regulation, using PRIP^<-/-> mice. In wild-type animals, robust phosphorylation and functional modulation of GABA_A receptors containing β3 subunits by cAMP-dependent protein kinase was evident, which was diminished in PRIP^<-/-> mice. PRIP^<-/-> mice exhibited enhanced PP1 activity compared to controls. Furthermore, PRIP was able to interact directly with GABA_A receptor p subunits, and moreover, these proteins were found to be PP1 substrates. Finally, phosphorylation of PRIP on threonine 94 facilitated the dissociation of PP1/PRIP complexes, providing a local mechanism for the activation of PP1. Together, these results suggest an essential role for PRIP in controlling GABAA receptor activity, via regulating subunit phosphorylation and thereby the efficacy of neuronal inhibition mediated by these receptors. Less