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Inhibitor-2 (I-2) is a prototypic inhibitor of protein phosphatase-1 (PP1), a major serine-threonine phosphatase that regulates synaptic plasticity and learning and memory. Although I-2 is a potent inhibitor of PP1 in vitro, our previous work has elucidated that, in vivo, I-2 may act as a positive regulator of PP1. Here we show that I-2 and PP1γ, but not PP1α, positively regulate synaptic transmission in hippocampal neurons. Moreover, we demonstrated that I-2 enhanced PP1γ interaction with its major synaptic scaffold, neurabin, by Förster resonance energy transfer (FRET)/Fluorescence lifetime imaging microscopy (FLIM) studies, while having a limited effect on PP1 auto-inhibitory phosphorylation. Furthermore, our study indicates that the effect of I-2 on PP1 activity in vivo is dictated by I-2 threonine-72 phosphorylation. Our work thus demonstrates a molecular mechanism by which I-2 positively regulates PP1 function in synaptic transmission.

Additional file 4: Supplementary Figure 1. Complete acute phase response signaling. Red nodes are upregulated and green nodes are down regulated.

Additional file 3: Supplementary Table 3. A complete list of all proteins identified by SWATH analysis.

Additional file 2: Supplementary Table 2. A complete list of all proteins identified by LTQ Orbitrap Elite Mass Spectrometer. Green highlighted ANOVAs are statistically significant.

Additional file 1: Supplementary Table 1. A demographic table of patient data including gender and major depressive disorder status.