Abstract

In recombinant N-methyl-d-aspartate (NMDA) receptors, two redox modulatory sites are thought to exist, one formed by Cys744 and Cys798 on NMDA receptor subunit (NR) 1, and a second one, not yet localized, on NR2A. Reductants increase the open dwell-time and opening frequency of NR1/NR2A channels. In contrast, NR1/NR2B and NR1/NR2C channels exhibit changes only in opening frequency after redox treatments. Here, we evaluated whether the two redox sites act independently of each other, with the NR1 site affecting the opening frequency and the NR2A site altering open dwell-time. Unitary and whole-cell currents mediated by NMDA receptors composed of a cysteine-mutated NR1 subunit, NR1(C744A, C798A) were thus investigated. Dithiothreitol increased the open dwell-time and opening frequency of NR1(C744A, C798A)/NR2A receptors in a manner indistinguishable from that previously seen in wild-type channels. Marginal redox-induced changes in opening frequency of NR1(C744A, C798A)/NR2B receptors were noted. Redox modulation was completely abolished in NR1(C744A, C798A)/NR2C channels. Whole-cell recordings confirmed the single-channel results. Sulfhydryl reagents modulated NR1(C744A, C798A)/NR2A receptors identically to wild-type NR1/NR2A channels, whereas NR1(C744A, C798A)/NR2C receptors were insensitive to redox modulation. The oxidant 5,5′-dithio-bis-(2-nitrobenzoate) attenuated NR1(C744A, C798A)/NR2B receptor-mediated responses in a dithiothreitol-reversible manner. We conclude that cysteines 744 and 798 on the NR1 subunit are not involved in the redox modulation of NR1/NR2A receptors, but are crucial for the modulation of NR1/NR2C-containing receptors. This suggests that the NR2A subunit is necessary and sufficient for the expression of redox sensitivity in NR1/NR2A channels. The slight, but measurable residual redox sensitivity of the mutant NR1(C744A, C798A)/NR2B receptors suggests the existence of an additional redox-sensitive site on NR2B.