Subunit-specific Interactions of Cyanide with the N-Methyl-d-aspartate Receptor

Abstract

Cyanide is a mitochondrial poison that adversely affects cellular respiration by inhibiting the reoxidation of cytochrome a 3 by molecular oxygen, thereby obstructing the electron transport chain and oxidative phosphorylation (1). Cytochrome a 3 and cytochrome a form the cytochrome c oxidase complex, which is the terminal enzyme in the electron transport chain. Transferring electrons to oxygen, cytochrome c oxidase is the cellular respiratory component responsible for the critical need for oxygen. Thus, cyanide intoxication in cells is akin to oxygen starvation. In neuronal systems, cyanide treatment has been a widely used model of hypoxia (2-4), particularly in relation to excitotoxic processes. For example, cyanide can raise extracellular glutamate levels (5, 6), increase glutamate-triggered intracellular Ca 2ϩ elevations in neurons (7,8), and potentiate glutamate toxicity (7, 9). Moreover, specific inhibitors of Nmethyl-D-aspartate (NMDA) 1 receptors can inhibit cyanide-induced Ca 2ϩ influx in neurons (7, 10, 11) as well as neurotoxicity (12-14).Interestingly, a direct interaction between cyanide and the NMDA receptor has recently been demonstrated. Cyanide treatment of cultured rat hippocampal or cerebellar neurons potentiated NMDA-induced physiological responses, including single channel activity in excised outside-out membrane patches (15, 16). However, the precise site of action of cyanide at the receptor remained to be elucidated. In the present investigations we have evaluated the possible interaction of KCN on the NMDA receptor redox modulatory sites (17). Via these sites, disulfide-reducing agents such as dithiothreitol (DTT; see Refs. 17 and 18), dihydrolipoic acid (19), or tris(carboxyethyl)phosphine (20) enhance NMDA receptor-mediated physiological responses, whereas thiol-containing oxidants (21, 22), reactive quinones (23, 24), or oxygen-derived free radicals (25,26) can reverse the effects of reductants or depress native responses. Cyanide has well established properties as a disulfide reducing agent in many preparations (27)(28)(29)(30), and thus an effect on the NMDA thiol-sensitive sites would not be surprising. Nonetheless, the experiments described here demonstrate that cyanide can be used to distinguish between different NMDA receptor subtypes by producing either a potentiation or a depression of the physiological responses mediated by this ligand-gated ion channel. EXPERIMENTAL PROCEDURESTissue Culture-Tissue culture and all common reagents were purchased from Sigma, excluding the following: iron-supplemented bovine calf serum, HyClone Laboratories (Logan, UT), and minimal essential medium, Life Technologies, Inc. Chinese hamster ovary cells (CHO-K1; ATTC CCL61) were grown in Ham's F-12 nutrient medium with 10% fetal bovine serum, and 1 mM glutamine (CHO media) in 50-or 200-ml flasks at 37°C in 5% CO 2 . Cells were passaged at a 1:10 dilution at 80% confluency, approximately every 2 days, no more than 30 times. Cerebral cortices were obtained from E-16 Sprague-Dawley C-D rats and dissocia...

A Preferential Role for Glycolysis in Preventing the Anoxic Depolarization of Rat Hippocampal Area CA1 Pyramidal Cells

“…7), because cyanide also modulates NMDA receptor activation (Patel et al, 1994;Arden et al, 1998). Solution containing iodoacetate and cyanide (1 mM) and lacking O 2 and glucose produced an AD (Fig.…”

“…The potentiating action of cyanide on NMDA receptors containing NR2A subunits (Patel et al, 1994;Arden et al, 1998), together with the fact that the AD is mostly produced by activation of NMDA receptors (Rossi et al, 2000), seems a likely explanation for this slight acceleration.…”

“…There were five aims, as follows: (1) to ascertain the extent to which glycolysis can delay the regenerative decrease in ion gradients that generates the AD, because plasma membrane ion pumps may be preferentially fueled by glycolysis ; (2) to determine the period for which glycogen can sustain glycolysis in the absence of extracellular glucose; (3) to test whether superfused lactate can prevent the AD from occurring when glycolysis is blocked, as expected if most ATP is generated from lactate supplied to neurons from glia; (4) to compare the effect of a commonly used mitochondrial blocker, cyanide, with that of other blockers (rotenone, antimycin), be-cause cyanide can potentiate or inhibit NMDA receptors according to their subunit composition (Patel et al, 1994;Arden et al, 1998); and (5) to examine whether, in addition to preventing oxidative phosphorylation, removal of oxygen had direct effects on membrane currents contributing to the AD, because some K ϩ and Na ϩ currents can sense oxygen directly and are activated or inhibited when O 2 is removed (Jiang and Haddad, 1994;Hammarstrom and Gage, 2000). Our results demonstrate a preferential role for glycolysis in preventing the AD.…”

Cyanide interaction with redox modulatory sites enhances NMDA receptor responses

“…Because NEM pretreatment blocks the cyanide action on NMDA receptor responses, it is concluded that activation of the redox sites is necessary for the manifestation of cyanide's effect. Recently, Arden et al (18) showed that cyanide potentiates NMDA receptor-mediated whole cell currents in rat cortical neurons. As in the present study, this action was explained by cyanide interacting with putative redox-active sites on the receptor subunits by cyanolating cysteine residues to form thiocyanate adducts.…”

“…Because the enhancement persists in cell-free patches, it is likely that the response results at least in part from direct interaction with the receptor. Recently, Arden et al (18) showed that cyanide selectively potentiates the NMDA receptor influx of calcium possibly by interacting directly with the NR2A receptor subunit. The purpose of the present study is to investigate the mechanism by which cyanide enhances NMDA-mediated Ca ‫ם2‬ influx in neuronal cells.…”