Reduction of NMDA receptors with dithiothreitol increases [3H]-MK-801 binding and NMDA-induced Ca2+ fluxes.

1. We have investigated the modulation of N-methyl-D-aspartate (NMDA) receptor activation by the sulphydryl redox reagents dithiothreitol (DTT) and 5,5-dithio-bis-2-nitrobenzoic acid (DTNB). 2. Increases in [3H]-MK-801 binding produced by glutamate, glycine and spermidine were enhanced by DTT (2mM) and diminished by DTNB (0.5 mM). 3. The inhibition of [3H]-MK-801 binding by CGS 19755 and 7-chlorokynurenate was not altered by 2 mM DTT. However, the potency of the competitive polyamine antagonist, arcaine, was decreased by DTT. 4. NMDA-induced Ca2+ fluxes into primary cultures of rat forebrain neurones were enhanced by DTT in a DTNB-reversible fashion. In addition to augmenting the magnitude of NMDA-induced increase in intracellular free Ca2+, 10 mM DTT also prolonged the duration of the Ca2+ signal. However, DTT had no effect on the increase in Ca2+ produced by depolarizing neurones with 50 mM KCl. 5. These studies show that the reduction of disulphide bonds on the NMDA receptor complex by DTT increases activation. The precise site of these groups remains unclear but they are unlikely to form an integral part of the glutamate, glycine or polyamine binding domains. The enhancement of the activation of the NMDA receptor by DTT is associated with increased Ca2+ fluxes. The possible pathophysiological consequences of receptor reduction are discussed.