A critical role of the N-methyl-D-aspartate (NMDA) receptor subunit (NR) 2A in the expression of redox sensitivity of NR1/NR2A recombinant NMDA receptors

In recombinant N-methyl-D-aspartate (NMDA) receptors, two redox modulatory sites are thought to exist, one formed by Cys744 and Cys798 on NMDA recepto r 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 f requency after redox treatments. Here, we evaluated whether the two redox s ites act independently of each other, with the NR1 site affecting the openi ng 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 increase d the open dwell-time and opening frequency of NR1(C744A, C798A)/NR2A recep tors in a manner indistinguishable from that previously seen in wild-type c hannels. 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 si ngle-channel results. Sulfhydryl reagents modulated NR1(C744A, C798A)/NR2A receptors identically to wild-type NR1/NR2A channels, whereas NR1(C744A, C7 98A)/ NR2C receptors were insensitive to redox modulation. The oxidant 5,5' -dithio-bis-(2-nitrobenzoate) attenuated NR1(C744A, C798A)/NR2B receptor-me diated responses in a dithiothreitol-reversible manner. We conclude that cy steines 744 and 798 on the NR1 subunit are not involved in the redox modula tion 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. T he slight, but measurable residual redox sensitivity of the mutant NR1(C744 A, C798A)/NR2B receptors suggests the existence of an additional redox-sens itive site on NR2B.