The aim of this proposed research project is to elucidate how protein kinase C (PKC) modulates ethanol (EtOH) inhibition of the N-methyl-D-aspartate receptor (NMDAR). The NMDAR is believed to be involved in neuronal processes that mediate learning and memory, excitotoxicity and cell death, as well as many neural and behavioral changes attributed to the abuse of EtOH. Our preliminary results suggest PKC may be involved in EtOH inhibition of NMDARs expressed in primary cultured cerebellar granule cells (CGC)s 6-14 days in vitro (DIV). Using both whole-cell and perforated patch-clamp electrophysiological techniques we have observed significant attenuation of EtOH inhibition of NMDA-induced steady-state currents by an eight minute exposure to 100 nM calphostin C, a PKC inhibitor. This reversal of inhibition was significant for three concentrations of EtOH: 10, 50 and 100 mM. Immunoblot results indicate that PKCs alpha, betaI, betaII, epsilon, gamma, delta, mu, theta, lambda/iota and zeta are contained in our cultures between 6-14 DIV. These preliminary data support the hypothesis that acute EtOH exposure activates one (or more) of these PKC isoforms. Activation results in a translocation of the enzyme to the location of its substrate where a phosphorylation-mediated event occurs. This phosphorylation-mediated event modulates EtOH inhibition of native NMDARs. This hypothesis will be tested in the following experiments. CGCs will be exposed to EtOH and activation of PKC will be confirmed by measuring increases in enzyme activity from the cytosol to the membrane by kinase activity assays. Immunoblot analysis and PKC isoform-specific antibodies will be used to identify which isoforms have translocated to the membrane fraction. To determine if EtOH-mediated activation of PKC results in the phosphorylation of the NMDAR NRI subunit, immunoprecipitation studies will be performed with an NR1 antibody followed by immunoblotting with a phosphorylation site-specific antibody that recognizes PKC-mediated phosphorylation of the NRI. The translocation, kinase activity and phosphorylation experiments will be conducted under various physiological conditions to determine if PKC is directly or indirectly activated by EtOH. Electrophysiological experiments will establish that our observed calphostin C attenuation of EtOH inhibition of NMDARs is mediated by PKC, by assessing the EtOH sensitivity of NMDARs after exposure to PKC activators and catalytically active PKC in the presence and absence of PKC inhibitors. PKC isoform-specific inhibitors will identify which PKC isoform is modulating EtOH inhibition of NMDAR function. Additional electrophysiological experiments will examine the role of PKC with other intracellular signaling cascades and how the convergence of these cascades alters the EtOH sensitivity of the NMDAR. Overall, these studies will provide insights into the molecular mechanisms by which acute EtOH exposure alters NMDAR function. Thus, facilitating the future design of novel therapeutic strategies to be used in the treatment of alcoholism and alcohol abuse.
