Ethanol has widespread and significant effect on brain function. Acute ethanol causes intoxication with resultant loss of behavioral and motor control that can progress to sedation, coma, and death as lethal blood concentrations are reached. The molecular and cellular alterations induced by ethanol that underlie these effects are beginning to be understood and it is now appreciated that several ligand-gated ion channels activated by brain neurotransmitters are important targets for ethanol's actions in the brain. Of particular interest is the inhibition of the NMDA subtype of the glutamate receptor by ethanol. This receptor gates the flux of calcium ions upon stimulation by the co- agonists glutamate and glycine and is subject to multiple forms of regulation. These include a voltage-sensitive magnesium block, a redox site which can shift the receptor from an agonist to an antagonist preferring state, proton inhibition, and phosphorylation. This complex regulation underscores the fundamental importance of the NMDA receptor in neuronal signaling. The inhibition of the NMDA receptor by ethanol may underlie some of the marked behavioral and cognitive effects associated with intoxication. The goals of this study are to continue our investigation into the mechanisms of action of ethanol on the NMDA receptor at both the biochemical and molecular level. We will continue to use the NMDA-stimulated release of neurotransmitters from adult rat brain slices to examine the multiple regulatory sites of the native NMDA receptor. This assay has been well characterized and is sensitive to all known modulators of the NMD receptor and unlike many test systems it can be used with living adult brain tissue. In separate studies, cDNA clones encoding both rodent and human NMDA recptor subtypes will be used to more carefully determine where ethanol may exert its inhibitory effects on the receptor. For these studies, two approaches will be used. First, experimental cell lines lacking any native ligand-gated or voltage-sensitive calcium channels will be transfected with NMDA receptor subunits to allow for the NMDA-stimulated calcium flux to be monitored by digital calcium imaging and patch-clamp electrophysiology. Secondly, messenger RNA synthesized from the cloned receptors will be expressed in Xenopus oocytes and NMDA-activated currents will be monitored using two-electrode voltage clamp. This system will not only allow for reconstitution of functional multi-subunit NMDA receptor complexes but will also permit an examination on a site by site basis for the molecular site of action of ethanol on the receptor using site- directed mutagenesis. Results from these studies should lead to new insights into the effects of ethanol on neuronal function and may suggest novel approaches in the treatment of disorders resulting from acute and chronic ethanol ingestion.
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