A role for the redox site in the modulation of the NMDA receptor by light

Abstract

Light has been shown to modulate NMDA receptor function. In this study, we have performed experiments aimed at elucidating the putative site of action of light within the receptor structure. Whole-cell recordings were performed in Chinese hamster ovary cells expressing various combinations of NMDA receptor subunits. Although there was no apparent difference in the actions of light between wild-type NR1-NR2A and NR1-NR2B subunit configurations, the light enhancement of NMDA-induced currents was either completely abolished or substantially diminished in the redox site mutants NR1a (C744A, C798A)-NR2B and NR1a (C744A, C798A)-NR2A. Further studies demonstrated that chemical reduction of NR1a-NR2B NMDA receptors decreased its sensitivity to light. In addition, sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES), used to irreversibly bind free sulfhydryl groups and inactivate the redox site, abolished the effects of light on wild-type receptors. In contrast, no free sulfhydryls were available for MTSES following light stimulation, suggesting that light itself could not reduce the redox modulatory site. Our results suggest that a functionally intact, oxidized redox site is necessary for light-induced potentiation. Hence, light and redox modulation of the NMDA receptor may share a common intramolecular pathway for altering the function of this ion channel.

Figure 1. The redox site on the NR1 subunit of the NMDA receptor is required for light sensitivity

A, whole-cell NMDA (10 μm)-induced currents from receptors NMDA receptors transiently expressed in CHO cells (membrane potential: −60 mV in this and all subsequent figures). Sample traces represent currents before (grey trace) and shortly after (5 s; black trace) a brief light pulse (2 s, > 280 nm). Similar effects were seen in 5-12 cells for each subunit configuration. Scale bars: 100 pA and 1 s. B, peak response amplitudes for responses such as those seen in A were measured and a post-flash to pre-flash (I*/I) ratio was calculated (* P < 0.05, significantly different from wild-type control, Student's two tailed t test; n = 5–12 cells). C, top, NMDA-elicited responses from NR1a-NR2B receptors both before and after a brief flash of light (light bulb; 2 s, > 280 nm) Scale bars: 500 pA and 500 ms. C, bottom, NMDA-mediated responses from NR1a-NR2B receptors before and after 8 min of 5 mm DTT incubation. Chemically reduced receptors were then exposed to brief flash (2 s, > 280 nm) followed by chemical oxidation with 500 μm DTNB (2 min). Scale bars: 100 pA and 1 s. D, I*/I ratios demonstrating a significant decrease in light sensitivity following chemical reduction (* P < 0.05, significantly different from ‘-DTT’ control, Student's two tailed t test, n = 5–7 cells).

Figure 2. MTSES inactivates redox modulation following chemical reduction but not light-induced potentiation

A, top, sample trace showing whole-cell NR1a-NR2B receptor-mediated responses (10 μm NMDA) following potentiation with DTT, oxidation with DTNB, and a brief pulse of light (light bulb; 2 s, > 280 nm). A, bottom, redox sites were inactivated by exposure to 10 mm MTSES (+MTSES) following chemical reduction (DTT). Receptors were then oxidized (500 μm DTNB) and exposed to light (2 s, > 280 nm). Scale bars: 500 pA and 500 ms. B, top, quantification of the amount of oxidation following exposure to DTT in the presence and absence of 10 mm MTSES (% oxidation was calculated as (I_DTT/I_DTNB) × 100; * P < 0.05, significantly different from - MTSES control, Student's two tailed t test, n = 5–6 cells). B, bottom, I*/I ratio of receptor responses after incubation with DTT in the presence and absence of MTS compound (* P < 0.05, significantly different from - MTSES control, Student's two tailed t test, n = 5–6 cells). C, sample traces of NMDA-elicited responses both before and after a light flash (light bulb; 2 s, > 280 nm). Cells were then exposed to control external solution, 500 μm DTNB, or MTS reagents followed by 500 μm DTNB. Scale bars: 250 pA and 500 ms. D, quantification of the amount of decay 3 min after a brief flash of light, in the presence of control external solution, DTNB or MTS reagents followed by DTNB (P > 0.05, no significant difference between control external and the DTNB and MTS-DTNB-treated groups, a one-way ANOVA, n = 4–6 cells).