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-asp artate (NMDA) receptor have been examined in rat cortical neurones in culture. Electrophysiological measurements were performed with the who le-cell and outside-out patch variants of the patch-clamp technique. 2 . The disulphide reducing agent dithiothreitol (DTT; 0.1-10 mm) potent iated 10 mum NMDA-mediated whole-cell currents when applied slowly alo ne via the superfusate. The initial rate of reduction, as well as the degree of potentiation, was dependent on the concentration of DTT alth ough the process was complicated by the fact that a second, large comp onent 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 perfu sion pipette. With this method, the second component was not readily a pparent, and the concentration of DTT producing a half-maximal potenti ation of the NMDA response was 1.9 +/- 0.3 mm. Two other disulphide re ducing agents, ethylene glycol bisthioglycolate and meso-bis(NN-dimeth yl)adipamide-2,5-dithiol, also potentiated NMDA responses, but were no t as effective as DTT. 4. Following a 4 mm DTT treatment, we observed that the NMDA receptor underwent spontaneous oxidation with a half-tim e 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 rever sal of the effects of DTT (t1/2 = 0.6 min). The spontaneously oxidized receptor could be further oxidized with DTNB and fully reduced with D TT. 5. After receptor oxidation with 500 mum DTNB, NMDA produced whole -cell responses with an EC50 of 68.4 +/- 9.4 mum, whereas after reduct ion 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 incre ased over that observed after oxidation. 6. Single channel measurement s performed on outside-out patches revealed that reduction produced a dramatic increase in the number of NMDA-induced channel openings. We o bserved a 2.1 +/- 0.2-fold increase in the frequency of openings durin g reduction with 500 mum DTT when compared to patches which had been e xposed to 500 mum DTNB. Small but significant differences were observe d 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 tim e 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 th e redox modulation of the NMDA receptor were alkylated with N-ethylmal eimide (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 obs erved that while the blocking effects of protons and potentiating acti ons of glycine were unaffected by alkylation, Zn 2+ and Mg2+ produced a significantly weaker block of the NMDA whole-cell response. 9. The r esults presented here provide important information regarding the fund amental properties of the modulation of the NMDA receptor via the redo x site and begin to address the molecular mechanism of this form of mo dulation.