CHARACTERIZATION OF NEUROBLASTOMA NMDA RECEPTORS

Project: Research project

Project Details

Description

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.
StatusFinished
Effective start/end date7/1/8411/30/93

Funding

  • National Institutes of Health
  • National Institutes of Health

ASJC

  • Medicine(all)
  • Neuroscience(all)
  • Cellular and Molecular Neuroscience
  • Biophysics
  • Physiology
  • Cell Biology
  • Pharmacology

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