THE MODULATION OF N-METHYL-D-ASPARTATE RECEPTORS BY REDOX AND ALKYLATING REAGENTS IN RAT CORTICAL-NEURONS INVITRO

1. The properties of sulfhydryl redox modulation of the N-methyl-D-aspartate (NMDA) receptor have been examined in rat cortical neurones in culture. Electrophysiological measurements were performed with the whole-cell and outside-out patch variants of the patch-clamp technique. 2. The disulphide reducing agent dithiothreitol (DTT; 0.1-10 mm) potentiated 10 mum NMDA-mediated whole-cell currents when applied slowly alone via the superfusate. The initial rate of reduction, as well as the degree of potentiation, was dependent on the concentration of DTT although the process was complicated by the fact that a second, large component appeared at a concentration of 10 mm of this agent. 3. DTT (0.1-10 mm) was also rapidly applied together with the agonist from a perfusion pipette. With this method, the second component was not readily apparent, and the concentration of DTT producing a half-maximal potentiation of the NMDA response was 1.9 +/- 0.3 mm. Two other disulphide reducing agents, ethylene glycol bisthioglycolate and meso-bis(NN-dimethyl)adipamide-2,5-dithiol, also potentiated NMDA responses, but were not as effective as DTT. 4. Following a 4 mm DTT treatment, we observed that the NMDA receptor underwent spontaneous oxidation with a half-time of 1.9 min. In contrast, the sulfhydryl oxidizing agent 5,5'-dithio-bis-(2-nitro-benzoic acid) (DTNB; 500 mum) produced a more rapid reversal of the effects of DTT (t1/2 = 0.6 min). The spontaneously oxidized receptor could be further oxidized with DTNB and fully reduced with DTT. 5. After receptor oxidation with 500 mum DTNB, NMDA produced whole-cell responses with an EC50 of 68.4 +/- 9.4 mum, whereas after reduction with 4 mm DTT the EC50 for NMDA was 32.5 +/- 3.4 mum. In addition, the maximum response after reduction with DTT was substantially increased over that observed after oxidation. 6. Single channel measurements performed on outside-out patches revealed that reduction produced a dramatic increase in the number of NMDA-induced channel openings. We observed a 2.1 +/- 0.2-fold increase in the frequency of openings during reduction with 500 mum DTT when compared to patches which had been exposed to 500 mum DTNB. Small but significant differences were observed in the single channel conductance for the oxidized (34.6 +/- 1.1 pS) and reduced (37.6 +/- 1.5 pS) states of the receptor. In contrast, no significant changes were seen in the arithmetic mean channel open time between the two redox conditions (5-4 +/- 0.3 ms after oxidation, 6.0 +/- 0.7 ms after reduction). 7. The site or sites responsible for the redox modulation of the NMDA receptor were alkylated with N-ethylmaleimide (NEM; 300-500 mum) following reduction with DTT. Alkylation of the receptor produced responses which were permanently potentiated and largely insensitive to oxidation by DTNB. 8. The actions of the NMDA receptor modulatory agents Zn2+ (20 mum), Mg 2+ (1 mm), protons (pH 6.8) and glycine (3 mum) were tested before and after alkylation. We observed that while the blocking effects of protons and potentiating actions of glycine were unaffected by alkylation, Zn 2+ and Mg2+ produced a significantly weaker block of the NMDA whole-cell response. 9. The results presented here provide important information regarding the fundamental properties of the modulation of the NMDA receptor via the redox site and begin to address the molecular mechanism of this form of modulation.