Lack of interaction between nitric oxide and the redox modulatory site of the NMDA receptor

Abstract

1. The inhibitory effects of nitric oxide (NO) on N-methyl-D-aspartate (NMDA) receptor function have been proposed to be mediated via the interaction of this gas with a redox-sensitive thiol moiety on the receptor. Here, we evaluated this suggested mechanism by examining the actions of various NO donors on native neuronal receptors as well as in wild-type and cysteine-mutated recombinant NMDA receptors expressed in Chinese hamster ovary (CHO) cells. 2. The NO donor N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydraxino)ethanamine (NOC-12; 100 microM) produced a rapid and readily reversible inhibition of whole-cell currents induced by NMDA (30 microM) in cultured cortical neurons. The inhibition was apparent at all holding potentials, though a more pronounced block was observed at negative voltages. The effects of NOC-12 disappeared when the donor was allowed to expire. A similar receptor block was observed with another NO-releasing agent, S-nitroso-N-acetylpenicillamine (SNAP; 1 mM). 3. The blocking effects of NO released by SNAP, 3-morpholinosydnonimine (SIN-1; 1 mM), and 3-[2-hydroxy-1-(1-methylethyl)-2-nitrosohydrazino]-1-propanamin e (NOC-5; 100 microM) on currents mediated by recombinant NRI/NR2B receptors were virtually indistinguishable from those observed on native receptors. Furthermore, mutating cysteines 744 and 798 of NR1, which constitute the principal redox modulatory site of the NR1/NR2B receptor configuration, did not affect the inhibition produced by NO. 4. The NR2A subunit may contribute its own redox-sensitive site. However, the effects of NO on NR1/NR2A receptors were very similar to those seen for all other receptor configurations evaluated. Hence, we conclude that NO does not exert its inhibition of NMDA-induced responses via a modification of any of the previously described redox-sensitive sites on the receptor.

Figure 1

Inhibition of NMDA-activated currents by NO in neurons. (A) Representative whole-cell current traces obtained at −60, −30, 0, +15 and +30 mV in rat cortical neurons in culture during activation by NMDA (30 μM) in the absence and presence of freshly-prepared NOC-12 (100 μM). (B) Current-voltage relationship of steady-state NMDA responses in the absence and presence of NOC-12 for the same cell shown in A. (C) Whole-cell current trace during activation by NMDA at −60 mV in the absence and presence of NOC-12 (100 μM) which had been allowed to expire overnight.

Figure 2

Inhibition of NMDA-activated currents by NO in transfected CHO cells does not require the presence of a functional redox site. (A) Representative whole-cell currents obtained at −60 and +30 mV in a CHO cell expressing NR1/NR2B receptors during application of NMDA (30 μM) in the absence and presence of SIN-1 (1 mM). (B) Current-voltage relationship of steady-state NMDA responses in the absence and presence of NOC-12 for the same cell shown in A. (C) Representative whole-cell currents obtained in a CHO cell expressing NR1 (C744A, C798A)/NR2B receptors, which lack a functional redox site, during application of NMDA with or without SIN-1. (D) Current-voltage relationship of steady-state NMDA responses in the absence and presence of NOC-12 for the same cell shown in C.