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.
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