The modulation of N‐methyl‐D‐aspartate receptors by redox and alkylating reagents in rat cortical neurones in vitro.

Abstract

SUMMARY1. 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 outsideout patch variants of the patch-clamp technique.2. The disulphide reducing agent dithiothreitol (DTT; 0 1-10 mM) potentiated 10 /iM 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,UM) produced a more rapid reversal of the effects of DTT (th = 0-6 min). The spontaneously oxidized receptor could be further oxidized with DTNB and fully reduced with DTT.5. After receptor oxidation with 500 JtM DTNB, NMDA produced whole-cell responses with an EC50 of 68-4 + 9-4 ,jM, whereas after reduction with 4 mm DTT the EC50 for NMDA was 32'5 + 3-4 /tM. 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 Itm DTT when compared to patches which had been exposed to 500 EM DTNB. Small but significant differences were observed in the single channel MS 1122 L. -H. TANG ANlD E. AIZENMAN conductance for the oxidized (34-6 + I 1 pS) and reduced (37±6 + 1P5 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, 60+007 ms after reduction).7. The site or sites responsible for the redox modulation of the NMDA receptor were alkylated with NV-ethylmaleimide (NEM; 300-500 aM) 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,aM), Mg2+(1 mm), protons (pH 6 8) and glycine (3 JIM) were tested before and after alkylation.We observed that while the blocking effects of pro...

Functional consequences of NR2 subunit composition in single recombinant N -methyl- d -aspartate receptors

“…Sulfhydryl agents altered the frequency of channel opening of NR1͞NR2A, NR1͞NR2B, and NR1͞NR2C receptors in a matter analogous to what is observed in native cortical receptors (32). Interestingly, redox agents affected the open dwell-time of NR1͞NR2A channels, but not of NR1͞NR2B-or NR1͞NR2C-containing receptors, suggesting that the presence of the additional redox site on the NR2A subunit can be detected at the microscopic level.…”

“…The NMDA receptor is modulated by sulfhydryl redox reagents such that reductants can potentiate NMDA-induced responses, whereas oxidants can depress native responses and reverse the effects of reducing agents (31,32). Molecular studies have established the presence of two redox sites on the NMDA receptor, one in the putative extracellular loop between M2 and M3 of NR1 (33) and one in the amino terminus of NR2A (34).…”

“…This observation argues that the analysis of NMDA channel behavior must take into account that the receptor can lie in different redox states, which, in turn, could have pronounced repercussions in what is conceived as ''normal'' function, at least during steady-state measurements. For example, we have observed that Mg 2ϩ and Zn 2ϩ block, as well as current rectification properties, can change after redox treatments (32,35).…”

ASIC1a-Specific Modulation of Acid-Sensing Ion Channels in Mouse Cortical Neurons by Redox Reagents

“…1C). Similarly, bath perfusion of NEM, an alkylating agent known to bind to thiol groups of redox modulatory sites (Creighton, 1984;Tang and Aizenman, 1993), also quickly reversed the persistent potentiation of the ASIC current by DTT, suggesting an involvement of critical sulfhydryl groups in the potentiation of the current by DTT (n ϭ 6; p Ͻ 0.01) (Fig. 1 D).…”

“…An additional finding that NEM, an alkylating agent known to irreversibly alkylate free thiol groups (Creighton, 1984;Tang and Aizenman, 1993), blocked the effects of redox reagents supports an involvement of critical sulfhydryl groups in the effects of redox reagents. The finding that both reducing and oxidizing agents have an effect on the currents suggests that the redox modulatory site(s) on ASICs (or closely associated proteins) exist in a dynamic equilibrium between fully reduced and oxidized states.…”