, 2002). Addition of NMDA to cultured neurons triggers AMPA receptor endocytosis (Beattie et al., 2000). Treatment with 2-BP does not further decrease GluR2 surface clusters in neurons exposed to NMDA (Figures 4C and 4D), suggesting that palmitoylated targets including PSD-95 play a role in both basal and NMDA-dependent AMPA receptor surface clustering. These experiments establish that NMDAR-mediated neurotransmission regulates PSD-95 clustering.

The reduction of PSD-95 clustering in response to glutamatergic transmission involves NO. The regulation of PSD-95 clustering by CHIR-99021 price NO reflects its influence upon palmitoylation of PSD-95. Nitrosylation is increasingly appreciated as a major posttranslational modification of proteins

(Cho et al., 2009, Hess et al., 2005 and Whalen et al., 2007). Besides nitrosylation and palmitoylation, cysteines of proteins are physiologically modified by oxidative mechanisms involving hydrogen peroxide and other agents, as well as by glutathionylation, sulfhydration, and the formation of disulfide bonds. With the exception of disulfides (Takahashi et al., 2007), thus far there has been no evidence for physiologic regulation of nitrosylation by these other posttranslational modifications of cysteines. We asked whether palmitoylation might normally Alectinib manufacturer modulate levels of nitrosylated PSD-95. In HEK-nNOS cells nitrosylation of PSD-95-1-433 is demonstrable and is lost with mutation of C3 and C5 (Figure 6A). Nitrosylation of PSD-95-1-433 is substantially augmented by treatment with 2-BP, indicating that endogenous palmitoylation reduces levels of PSD-95 nitrosylation. This reciprocity of nitrosylation and palmitoylation is selective. Thus, β-tubulin is known to be nitrosylated (Jaffrey

et al., 2001) but has not been conclusively shown to be palmitoylated in vivo. Nitrosylation of tubulin is unaffected by 2-BP. To assess whether endogenous palmitoylation of PSD-95 physiologically because regulates its nitrosylation in intact animals, we examined brains of mice with targeted deletion of the palmitoyl acyltransferase enzyme ZDHHC8, which has been demonstrated to be a physiologic PAT for PSD-95 (Mukai et al., 2008) (Figure 6B). Levels of nitrosylation of PSD-95 are increased 3-fold in the ZDHHC8 knockout brains. By contrast, nitrosylation of GAPDH, which is not physiologically palmitoylated, is unaltered in the mutant mice. The proportionally larger increase in nitrosylation of PSD-95 compared to palmitoylation may be due to higher levels of palmitoylated PSD-95 in unstimulated brains with low basal levels of NO. A small change in palmitoylation may therefore result in a larger percentage change in nitrosylation of PSD-95. Neither palmitoylation (Hayashi et al., 2009) nor nitrosylation (Choi et al., 2000) of NR2A, which occurs on distinct cysteines, is affected in the mutant mice (Figure 6C). Thus, nitrosylation of PSD-95 is physiologically modulated by palmitoylation.