PSD-95, a principal scaffolding component of the post-synaptic density, is targeted to synapses by palmitoylation where it couples NMDA receptor stimulation to production of nitric oxide (NO) by neuronal nitric oxide synthase (nNOS). Here, we show that PSD-95 is physiologically S-nitrosylated. We identify cysteines 3 and 5, which are palmitoylated, as sites of nitrosylation, suggesting a competition between these two modifications. In support of this hypothesis, physiologically produced NO inhibits PSD-95 palmitoylation in granule cells of the cerebellum, decreasing the number of PSD-95 clusters at synaptic sites. Further, decreased palmitoylation, as seen in heterologous cells treated with 2-bromopalmitate or in ZDHHC8 knockout mice deficient in a PSD-95 palmitoyltransferase, results in increased PSD-95 nitrosylation. These data support a model in which NMDA mediated production of NO regulates targeting of PSD-95 to synapses via mutually competitive cysteine modifications. Thus, differential modification of cysteines may represent a general paradigm in signal transduction.

Serine racemase (SR) generates D-serine, a coagonist with glutamate at NMDA receptors. We show that SR is physiologically S-nitrosylated leading to marked inhibition of enzyme activity. Inhibition involves interactions with the cofactor ATP reflecting juxtaposition of the ATP-binding site and cysteine-113 (C113), the site for physiological S-nitrosylation. NMDA receptor physiologically enhances SR S-nitrosylation by activating neuronal nitricoxide synthase (nNOS) . These findings support a model whereby postsynaptic stimulation of nitric-oxide (NO) formation feeds back to presynaptic cells to S-nitrosylate SR and decrease D-serine availability to postsynaptic NMDA receptors.neuronal nitric-oxide synthase ͉ NMDA receptor ͉ S-nitrosylation G lutamate neurotransmission through NMDA receptors requires a coagonist originally thought to be glycine. Recent studies indicate that in most portions of the brain, D-serine is the physiological coagonist because selective degradation of D-serine but not glycine markedly reduces NMDA transmission (1, 2), whereas retraction of D-serine-producing glia in the hypothalamus of lactating rats also diminishes NMDA transmission (2). D-serine is formed from L-serine by serine racemase (SR), which, like D-serine, is selectively enriched in glia (2, 3), although recent studies indicate some neuronal localization (4). SR, a pyridoxal phosphate-requiring enzyme, also displays an absolute requirement for ATP, which is not hydrolyzed during SR activation (5). SR binds the glutamate receptor interacting protein, which also binds to AMPA subtypes of glutamate receptors with glutamate receptor interacting protein markedly activating SR and providing a means whereby glutamate stimulation of SR-containing cells augments D-serine formation (6).In postsynaptic cells, NMDA signaling is mediated in part by neuronal nitric-oxide synthase (nNOS) because calcium entering through NMDA receptor channels binds to calmodulin associated with nNOS (7,8). Extensive studies have documented a feedback from postsynaptic to presynaptic glutamatergic nerve terminals, which modulates NMDA neurotransmission, especially in long-term potentiation (9). Nitric oxide (NO) may be a retrograde messenger of long-term potentiation (10-12), although the area is controversial (12). Because SR is a component of the NMDA synaptic complex, we wondered whether it is influenced by NO. In the present study, we demonstrate that SR is physiologically S-nitrosylated leading to inhibition of enzyme activity mediated by interactions with ATP. NMDA transmission stimulates SR S-nitrosylation suggesting a feedback mechanism to diminish presynaptic formation of D-serine. ResultsWe demonstrated S-nitrosylation of SR in multiple ways. Incubation of the NO donor S-nitroso-glutathione (GSNO) with SR in vitro leads to robust nitrosylation (Fig. 1A). In HEK293 cells, treatment with the NO donor sodium nitroprusside also provides S-nitrosylation (Fig. 1B). NO produced by nNOS S-nitrosylates SR, as is evident in HEK293 cells transfected with nNOS (Fi...

Synaptic plasticity is mediated by changes in the surface expression of AMPA receptors (AMPARs). Stargazin and related transmembrane AMPAR regulatory proteins have emerged as the principal regulators of AMPAR surface expression. Here, we show in heterologous cells and primary neurons that stargazin is physiologically S-nitrosylated, resulting in increased surface expression. Snitrosylation of stargazin increases binding to the AMPAR subunit GluR1, causing increased surface expression of the AMPAR. NMDAR stimulation, well known to activate neuronal nitric oxide synthase, increases both nitrosylation of stargazin and its binding to AMPAR. Thus, S-nitrosylation of stargazin is a physiologic regulator of AMPAR surface expression.

NMDA receptor activation can elicit synaptic plasticity by augmenting conductance of the AMPA receptor GluA1 subsequent to phosphorylation at S831 by Ca 2+ -dependent kinases. NMDA receptor activation also regulates synaptic plasticity by causing endocytosis of AMPA receptor GluA1. We demonstrate a unique signaling cascade for these processes mediated by NMDA receptor-dependent NO formation and GluA1 S-nitrosylation. Thus, S-nitrosylation of GluA1 at C875 enhances S831 phosphorylation, facilitates the associated AMPA receptor conductance increase, and results in endocytosis by increasing receptor binding to the AP2 protein of the endocytotic machinery.A MPA receptors (AMPARs) are tetramers in their physiologic form, with GluA1 being one of the more important regulatory subunits (1). Synaptic plasticity frequently is mediated by activation of NMDA receptors (NMDARs), which can modulate surface expression and single-channel conductance of AMPARs during the early phase of long-term potentiation (LTP) (1, 2). Phosphorylation of S831 of GluA1 mediates such plasticity, as phosphorylation of this site increases during LTP (3, 4), and mutant mice overexpressing a phosphomimetic S831D mutation display enhanced AMPAR conductance and LTP (5, 6).Mechanisms whereby NMDAR transmission augments AMPAR conductance have been unclear. NMDAR activation leads to calcium entry, which stimulates catalytic activity of neuronal nitric oxide synthase (nNOS) (7,8). NO (nitric oxide) signals by activating guanylate cyclase, and by S-nitrosylating cysteines of many target proteins (9, 10). NO regulates AMPARs by targeting AMPARinteracting proteins that determine receptor surface expression. Thus, physiologic S-nitrosylation of NSF (N-ethylmaleimide sensitive factor) elicits enhanced surface expression of GluA2 (11). Transmembrane AMPAR regulatory proteins are prominent auxiliary proteins for AMPARs, with stargazin being the best characterized (12). NMDAR transmission triggers S-nitrosylation of stargazin, which increases surface expression of GluA1 (13).The surface expression of AMPARs that determines synaptic plasticity is regulated by endocytosis because inhibitors of endocytosis block AMPAR removal from the plasma membrane, resulting in increased surface expression of AMPARs and long term depression (LTD) (14). Although NO has been implicated in regulation of protein endocytosis (15), its role in regulating AMPAR endocytosis is unknown.In the present study, we show that GluA1 is physiologically S-nitrosylated under basal conditions, with increased nitrosylation upon NMDAR stimulation. Moreover, the NMDAR-dependent phosphorylation of GluA1-S831 is regulated by endogenous NO, which acts by S-nitrosylating GluA1-C875. These events regulate single-channel conductance of GluA1, as the increase of such conductance by phosphorylation is markedly slowed in GluA1-C875S mutants. Endocytosis of AMPAR also is regulated by nitrosylation of GluA1-C875, because the C875S mutant of GluA1 reduces AMPAR endocytosis in neuronal cells and decreases binding ...