Several ion channels are thought to be directly modulated by nitric oxide (NO), but the molecular basis of this regulation is unclear. Here we show that the NMDA receptor (NMDAR)-associated ion channel was modulated not only by exogenous NO but also by endogenous NO. Site-directed mutagenesis identified a critical cysteine residue (Cys 399) on the NR2A subunit whose S-nitrosylation (NO+ transfer) under physiological conditions underlies this modulation. In cell systems expressing NMDARs with mutant NR2A subunits in which this single cysteine was replaced by an alanine, the effect of endogenous NO was lost. Thus endogenous S-nitrosylation can regulate ion channel activity.

In cultured cerebrocortical neurons, mild excitotoxic insults or staurosporine result in apoptosis. We show here that N-methyl-D-aspartate (NMDA) receptor-mediated, but not staurosporinemediated, apoptosis is preceded by depolarization of the mitochondrial membrane potential (⌬m) and ATP loss. Both insults, however, release cytochrome c (Cyt c) into the cytoplasm. What prompts mitochondria to release Cyt c and the mechanism of release are as yet unknown. We examined the effect of inhibition of the adenine nucleotide translocator (ANT), a putative component of the mitochondrial permeability transition pore. Inhibition of the mitochondrial ANT with bongkrekic acid (BA) prevented NMDA receptor-mediated apoptosis of cerebrocortical neurons. Concomitantly, BA prevented ⌬ m depolarization, promoted recovery of cellular ATP content, and blocked caspase-3 activation. However, in the presence of BA, Cyt c was still released. Because BA prevented NMDA-induced caspase-3 activation and apoptosis, the presence of Cyt c in the neuronal cytoplasm is not sufficient for the induction of caspase activity or apoptosis. In contrast to these findings, BA was ineffective in preventing staurosporine-induced activation of caspases or apoptosis. Additionally, staurosporineinduced, but not NMDA-induced, apoptosis was associated with activation of caspase-8. These results indicate that, in cerebrocortical cultures, excessive NMDA receptor activation precipitates neuronal apoptosis by means of mitochondrial dysfunction, whereas staurosporine utilizes a distinct pathway. Apoptosis is an important mechanism in both the development and degeneration of the nervous system. Evidence suggests that the loss of neurons in many neurologic disorders occurs by apoptosis (1-4). The overstimulation of glutamate receptors can precipitate the death of neurons by either necrosis or apoptosis depending on the severity of the insult (5, 6). Activation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor, in particular, results in an increase in the intracellular free calcium concentration ([Ca 2ϩ ] i ) to levels above the buffering capacity of neurons (7). This increase in [Ca 2ϩ ] i leads to the activation of toxic events, including oxidative and nitrosative stress (8). Mitochondria, which undergo harmful Ca 2ϩ -loading after NMDA receptor activation (9, 10), also have an important signaling function in apoptosis (11-13).Apoptotic cell death is often mediated by a caspase cascade. Although many stimuli exist, the final phases of apoptosis are executed by a few common effector caspases. Mitochondria appear to provide a link between the initiator caspases and the downstream effector caspases. In nonneuronal cells, mitochondria have been shown to accelerate activation of caspases by releasing proapoptotic molecules, such as cytochrome c (Cyt c) (11, 12) and the apoptosis-inducing factor (13). The release of these molecules can be stimulated by some caspases and by Bid and Bax (14-16), whereas Bcl2 prevents their release (11-13).The mechan...

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