The mouse neuroblastoma - Chinese hamster brain hybrid cell line NCB-20 has been demonstrated to have delta opioid, sigma, phencyclidine (PCP) and N-methyl-D-aspartate (NMDA) receptors. The NCB-20 cell line is an excellent system in which to characterize NMDA channel is of considerable interest because of its proposed roles in long-term potentiation, developmental modeling, hypoxic damage and epileptiform seizures. Recent evidence suggests that the NMDA channel is functionally and structurally associated with the PCP receptor which mediates the pharmacological effects of PCP< sigma opioids and dioxalanes. The goals of the proposed research ar to characterize the channel properties in this well-defined system and to determine the relationship of the NMDA and PCP receptors on a molecular level. To achieve these goals we propose to characterize NMDA channels and their regulation by PCP receptor ligands in the NCB-20 cell line. Specifically, these studies explore the hypothesis that the pharmacological receptor for phencyclidine is a binding site within the NMDA-activated channels and the pharmacological actions of PCP receptor ligands in NCB-20 cells. Patch clamp methods will be used to characterize single channel properties and to determine in this cell line how PCP regulates NMDA-induced conductances at the single channel level. Receptor binding of PCP and its regulation by NMDA agonists and antagonists will be characterized. The fluorescence-activated cell sorter (FACS) will be used to sort NCB-20 cells labeled with fluorescent PCP derivatives in order to identify cells which overproduce the PCP/NMDA receptor protein(s). Finally, the expression of NMDA and PCP receptors will be examined in Xenopus oocytes after injection of mRNA from NCB-20 cells before and after- size-fractionation. Together, these experiments should provide insight into the molecular and cellular mechanisms of regulation of the NMDA channel and demonstrate the feasibility of using the NCB-20 cell line for cloning of the gene(s) encoding the NMDA receptor.
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