Enhanced ethanol inhibition of recombinant N-methyl-D-aspartate receptors by magnesium: role of NR3A subunits.

N-methyl-D-aspartate receptors are heteromeric ion channels activated by glutamate and related excitatory amino acids (reviewed by Dingledine et al., 1999). They are expressed throughout the brain and are particularly enriched on dendritic spines, specialized neuronal compartments housing structural and functional elements involved in excitatory neurotransmission. NMDA receptors are particularly important in regulating neuronal plasticity, the ability of synapses to undergo reversible changes in signaling efficacy thought to be important in mediating many forms of learning and memory. While changes in synaptic efficacy are likely to be important in the behavioral adaptation during normal experience-dependent learning, alterations in NMDA receptor activity by drugs or disease may be counter-productive and could contribute to the persistence of maladaptive behaviors that characterizes the addictive state (Hyman and Malenka, 2001). In recent years, a growing body of evidence has suggested that changes in glutamate-mediated signaling may be especially important in mediating some of the actions of ethanol (Chandler, 2003). Both native and recombinant NMDA receptors are reversibly inhibited by acute application of ethanol (Lovinger et al., 1989; Mirshahi and Woodward, 1995) while long-term exposure to ethanol induces marked changes in the synaptic localization and function of NMDA receptor subunits (Carpenter-Hyland et al., 2004). These results suggest the acute ethanol sensitivity of NMDA receptors may be an important factor involved in the development of alcohol dependence. The magnitude of ethanol's inhibition of NMDA receptors is influenced by several factors including subunit composition (Jin and Woodward, 2006), interaction with cytoskeletal elements (Anders et al., 2000) and the developmental status of the brain (Lovinger, 1995).In general, NMDA receptors in adolescent brains appear more sensitive to inhibition by ethanol (Swartzwelder et al., 1995) and in some brain areas this appears to be correlated with the level of expression of NR2B subunits that are highest during early periods of development (Lovinger, 1995; but see Popp et al., 1999). One factor that has received only modest attention in the search for conditions that regulate ethanol sensitivity of NMDA receptors is the divalent cation magnesium. Most in vitro studies that measure NMDA receptor function routinely use magnesium-free solutions to avoid the well-known voltage-dependent block of receptor current by this ion. However, in some studies, the ethanol inhibition of NMDA-mediated responses was reported to be significantly enhanced when experiments were conducted with physiological (~1 mM) levels of magnesium (Chandler et al., 1994; Martin et al., 1991; Morrisett et al., 1991). In those studies, the subunit makeup of the neuronal NMDA receptors was not known. Both NR2A and NR2B containing receptors show a high sensitivity to magnesium block while those containing NR2C or NR2D are less affected (Monyer et al., 1994; Stern et al., 1994). Magnesium block of NMDA receptors is also influenced by the presence of the modulatory NR3 subunit that shows both regional and developmentally regulated profiles of expression. In this study, we tested whether the ethanol inhibition of recombinant NMDA receptors is also enhanced by extracellular magnesium and whether this effect is regulated by the NR3A subunit.