MOLECULAR STUDIES OF BRAIN NMDA RECEPTORS

Project: Research project

Project Details

Description

The NMDA-type glutamate receptor is implicated in long-term potentiation,
memory formation, brain development, and the neurodegeneration associated
with epilepsy, ischemia, Huntington's chorea, Alzheimer's disease and
AIDS encephalopathy. The proposed research aims to study molecular
diversity in neuronal NMDA receptors. Molecular diversity will be
addressed at the following levels of resolution; amino acid residues,
receptor domains, receptor variants, individual cells, and neural
circuits. Experiments outlined in this proposal are directed at the
development of a detailed molecular/functional/anatomic profile of NMDA
receptor variants. This laboratory has cloned two new NMDA receptor
splice variants from rat brain and has characterized their functional
properties. The receptor variants differ in their agonist affinity,
current amplitudes, and regulation by polyamines, zinc and protein kinase
C(PKC). In one project, we will use site-directed mutagenesis to identify
functionally important amino acid residues in the NR1 receptor protein.
Recombinant NMDA receptor channels will be analyzed in Xenopus oocytes
and human embryonic kidney 293 cells by whole cell recording and by patch
clamp. Studies will focus on the receptor domains involved in polyamine
and zinc potentiation, regulation by PKC and in binding of glycine and
glutamate. Working hypotheses are as follows: 1) Positively charged
residues within the N terminal insert N1 govern zinc and spermine
potentiation, agonist potency, and current amplitude, thereby generating
receptors with altered normal responsiveness and sensitivity to glutamate
pathogenicity; 2) In NR1 receptors lacking the C1 insert, serine residues
within the cytoplasmic loops play a critical role in regulation by PKC;
3) A glycine receptor-like motif within the N terminal domain forms part
of the glycine binding site. 4) A glutamate binding protein (QBP)-like
domain within the N terminal domain of NR1, just preceding TMI, is
involved in the glutamate binding site. In a second project we will
characterize NR1 heteromers with normal, mutationally altered and
chimeric NR2 receptors for comparison in a situ receptors and to
determine structure/activity relationships for this receptor subunit. In
a third project we will define cell-specific and circuit-specific
expression of NMDA receptor splice variants in the hippocampus, a brain
region known to be vulnerable to glutamate toxicity. Individual
hippocampal neurons grown in dissociated culture on coverslips will be
analyzed electrophysiologically at the whole cell and single channel
level for sensitivity to polyamines and to PKC. Subpopulations of NMDA
splice variants and subtypes will be identified by in situ hybridization
using exon-specific and splice junction-specific oligonucleotide probes.
As a future direction, individual neurons will be analyzed for expression
of splice variants by patch clamp methods and by the polymerase chain
reaction. Findings from these studies are expected to aid in the
development of new strategies for intervention in neurodegenerative
disorders.
StatusFinished
Effective start/end date7/1/843/1/11

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $380,478.00
  • National Institutes of Health: $363,983.00
  • National Institutes of Health: $113,950.00
  • National Institutes of Health: $380,985.00
  • National Institutes of Health
  • National Institutes of Health: $489,949.00
  • National Institutes of Health: $363,983.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $356,840.00
  • National Institutes of Health: $363,983.00
  • National Institutes of Health
  • National Institutes of Health: $374,290.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $366,474.00
  • National Institutes of Health
  • National Institutes of Health

ASJC

  • Medicine(all)
  • Neuroscience(all)

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