Activation of NMDA receptors (NMDARs) within the CNS represents a major signal for persistent alterations in glutamatergic signaling, such as long-term potentiation (LTP) and long-term depression. NMDARs are composed of a combination of NR1 and NR2 subunits, with distinct NR2 subunits imparting distinct characteristics on the receptor. One particular NR2 subunit, NR2A (NR⑀1), has been proposed to play an integral role in LTP induction in the hippocampus and cortex. Here, we report studies investigating the role of NR2A in LTP induction in the dorsolateral bed nucleus of the stria terminalis (dlBNST). The putative NR2A-specific inhibitor NVP-AAM077 (AAM077) has been used previously to demonstrate the dependence of cortical and hippocampal LTP on NMDARs containing NR2A subunits. We report here the same sensitivity of LTP to pretreatment with AAM077 (0.4 M) in the dlBNST. However, inconsistent with the conclusion that LTP in the dlBNST is NR2A dependent, we see intact LTP in the dlBNST of NR2A knock-out mice. Because we also see blockade of this dlBNST LTP in NR2A knock-out mice after pretreatment with AAM077, we conclude that the antagonist is targeting non-NR2A subunitcontaining receptors. Using a variety of cultured cell types, we find that AAM077 (0.4 M) can attenuate transmission of NR2B subunitcontaining NMDARs when preapplied rather than coapplied with an agonist. Therefore, we conclude that NR2A is not obligatory for the induction of LTP in the dlBNST. Furthermore, our data demonstrate that care must be exercised in the interpretation of data generated with AAM077 when the compound is applied before an agonist.

The N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptor is an important mediator of the behavioral effects of ethanol in the central nervous system. Although ethanol is known to inhibit NMDA receptors by influencing ion-channel gating, its molecular site of action and the mechanism underlying this effect have not been established. We have previously identified a conserved methionine residue in the fourth membrane-associated domain of the NMDA receptor NR2A subunit (Met 823 ) that influences desensitization and gating of the ion channel. Here we report that this residue plays an important role in mediating the effect of ethanol on the NMDA receptor. Ethanol IC 50 values among functional substitution mutants at this position varied over the range ϳ130 -225 mM. There was a weak correlation between ethanol IC 50 and mean open time of NR2A(Met 823 ) mutants that was dependent on inclusion of the value for the tryptophan mutant. In the absence of this value, there was no trend toward a correlation among the remaining mutants. Desensitization appeared to influence the action of ethanol, because ethanol IC 50 of the mutants was correlated with the steadystate to peak current ratio. With the exception of tryptophan, ethanol sensitivity was significantly related to the molecular volume and hydrophobicity of the substituent. The relation between ethanol sensitivity and the molecular volume and hydrophobicity at this position suggests that this residue interacts with or forms part of a site of ethanol action and that the presence of a tryptophan residue in this site disrupts its ability to interact with ethanol.Ethyl alcohol or ethanol, one of the oldest and most widely abused drugs, produces a well known spectrum of behavioral effects primarily through actions on ion channels in the nervous system. The N-methyl-D-aspartate (NMDA) 1 receptor, a subtype of receptor for the major excitatory neurotransmitter glutamate, is thought to be of particular importance in mediating the effects of alcohols in the mammalian brain. Ethanol inhibits NMDA-activated current in central neurons (1, 2) as well as NMDA-evoked Ca 2ϩ flux, cyclic GMP production, and neurotransmitter release (3-6). Studies using in vivo electrophysiological techniques have also reported ethanol inhibition of NMDA receptors at relevant concentrations (7). Ethanol appears to interact with a novel allosteric site, which is independent of the recognition site for the agonist glutamate or coagonist glycine (6, 8 -12), and reduces the mean open time and opening frequency of the channel (13,14).Presumed alcohol binding sites have been identified on a small number of receptors and ion channels, but the location and nature of these sites vary considerably. Sites of alcohol interaction have been reported to be located in the ion-channel lumen of nicotinic acetylcholine receptors from muscle (15) in a cytoplasmic loop of a Drosophila potassium channel (16), in the cytoplasmic C terminus of G-protein-coupled inwardly rectifying potassium channels (17), and in ...

The N-methyl-D-aspartate (NMDA) receptor has four membrane-associated domains, three of which are membrane-spanning (M1, M3, and M4) and one of which is a re-entrant pore loop (M2 The N-methyl-D-aspartate (NMDA) 1 receptor is a subtype of ionotropic glutamate receptor that plays essential roles in neuronal development, synaptic plasticity, and several types of neurological disorders (1). NMDA receptors are heteromers containing NR1 subunits, which bind the co-agonist glycine (2, 3), and NR2 subunits, which bind the agonist glutamate (4, 5). The agonist-binding domains of all ionotropic glutamate receptor subunits consist of two lobes (S1 and S2) that are formed by the region of the extracellular N-terminal domain preceding the first membrane-associated (M) domain and the loop between the M3 and M4 domain, respectively (5-8), and that together form a clamshell structure that undergoes a conformational change to enclose the ligand upon binding (6, 9, 10).Observations from x-ray crystallographic studies on non-NMDA glutamate receptor constructs suggest that the degree of binding domain closure induced by a particular agonist appears to determine both the degree of receptor activation and the extent of desensitization produced by that agonist (10) and that desensitization in these receptors results from the dissociation of dimers of the ligand-binding domains of adjacent subunits (11). NMDA receptors exhibit apparent desensitization sensitive to glycine or intracellular Ca 2ϩ (1, 12) in addition to true glycine-insensitive desensitization (subsequently referred to simply as desensitization). NMDA receptor desensitization is physiologically relevant, as it can influence the amplitude, duration, and following frequency of NMDA receptormediated synaptic events (13-16). Determinants of NMDA receptor desensitization have been localized to two regions in or near the S1 ligand-binding site in the N-terminal domain, one that shows homology to leucine/isoleucine/valine-binding proteins and one located in the region immediately preceding the M1 domain (17, 18), as well as to a highly conserved motif (YTANLAAF) in the C-terminal portion of the M3 domain preceding the S2 ligand-binding lobe (19). The pre-M1 and M3 domains have been suggested to be involved in transducing the conformational changes induced by agonist binding into those responsible for ion channel gating (17,19), but the nature of the conformational changes and molecular determinants underlying NMDA receptor ion channel gating remain unclear. The YTANLAAF motif in M3 has been shown to play an important role in the regulation of ion channel gating, as a point mutation in this region of the NR1 subunit increases mean open time in receptors formed from coexpression with NR2A subunits (19). Mutations at a tryptophan residue in the M2 domain of the NR1 or NR2A subunit have also been reported to subtly affect mean open time and opening frequency (20). We report here that a residue in the M4 domain of the NR2A subunit exerts a powerful regulatory influence on the desens...

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Recent understanding of the systems that mediate complex disease states, has generated a search for molecules that simultaneously modulate more than one component of a pathologic pathway. Chronic pain syndromes are etiologically connected to functional changes (sensitization) in both peripheral sensory neurons and in the central nervous system (CNS). These functional changes involve modifications of a significant number of components of signal generating, signal transducing and signal propagating pathways. Our analysis of disease-related changes which take place in sensory neurons during sensitization led to the design of a molecule that would simultaneously inhibit peripheral NMDA receptors and voltage sensitive sodium channels. In the current report, we detail the selectivity of N,N-(diphenyl)-4-ureido-5,7-dichloro-2-carboxy-quinoline (DCUKA) for action at NMDA receptors composed of different subunit combinations and voltage sensitive sodium channels having different α subunits. We show that DCUKA is restricted to the periphery after oral administration, and that circulating blood levels are compatible with its necessary concentrations for effects at the peripheral cognate receptors/channels that were assayed in vitro. Our results demonstrate that DCUKA, at concentrations circulating in the blood after oral administration, can modulate systems which are upregulated during peripheral sensitization, and are important for generating and conducting pain information to the CNS. Furthermore, we demonstrate that DCUKA ameliorates the hyperalgesia of chronic pain without affecting normal pain responses in neuropathic and inflammation-induced chronic pain models.

These results suggest that prenatal ethanol treatment reduces [3H]MK-801 binding and that this reduction may be due to a decrease in NR2A subunits.