Cyanide-induced enhancement of NMDA receptor responses in cerebellar granule cells

Peiwen Sun, Purdue University

Abstract

To determine mechanisms by which cyanide causes neurotoxic damage, the effects of cyanide on NMDA receptor-mediated responses were studied in cultured cerebellar granule cells. The NMDA-stimulated increase in cytosolic free calcium ( (Ca$\sp{2+}$) $\sb{\rm i}$) was enhanced by cyanide at doses of 20-100 $\mu$M, which alone had no effect on basal (Ca$\sp{2+}\rbrack \sb{\rm i}$. By contrast, depolarization by KCl (56 mM) resulted in large increases in (Ca$\sp{2+}\rbrack \sb{\rm i}$ which were not altered by cyanide. Cyanide enhancement of NMDA-evoked (Ca$\sp{2+}\rbrack \sb{\rm i}$ elevation was blocked by the NMDA receptor antagonists APV and MK-801 and by extracellular Mg$\sp{2+}$. However, voltage sensitive calcium channel (VSCC) blockers nifedepine and diltiazem did not affect cyanide's action. The cyanide action was also not altered when tetrodotoxin was used to prevent Na$\sp+$ channel-mediated glutamate release. These results suggest that cyanide specifically modulates the NMDA receptor response in neuronal cells. This action of cyanide does not involve VSCC activation or glutamate release. In patch-clamp studies, cyanide increased the amplitude and duration of NMDA-stimulated whole-cell currents. In single-channel recordings, cyanide increased NMDA receptor channel opening probability without affecting channel conductance or mean channel open-time. These studies support that cyanide can interact directly with the NMDA receptor channel complex. Interactions between cyanide and the NMDA receptor agonist recognition and allosteric modulation mechanisms were investigated. Cyanide did not enhance the (Ca$\sp{2+}\rbrack \sb{\rm i}$ elevation evoked by a saturating concentration of NMDA. Enhancement of the NMDA response by cyanide was not affected by glycine, a positive modulator of NMDA receptor activity. The metal ion chelator EDTA did not alter the action of cyanide, indicating that interference with Zn$\sp{2+}$ inhibition of the receptor does not account for the cyanide effect. Modulation of the NMDA receptor redox state using disulfide-reducing agents (dithiothreitol (DTT) and cysteine) significantly enhanced the cyanide potentiation. N-ethylmaleimide (NEM) irreversibly alkylates protein cysteine residues and thus decreases the availability of free sulfhydryl groups. NEM attenuated the cyanide-induced potentiation of the NMDA response. These results indicate that cyanide modulates NMDA receptor responses via a direct interaction with the receptor channel complex, a process that involves free sulfhydryl groups which may make up the redox regulatory site(s) of the receptor.

Degree

Ph.D.

Advisors

Isom, Purdue University.

Subject Area

Pharmacology|Neurology

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