Chen, Phillip E.; (2000) Identification of amino acid residues of the NR2A subunit that control glutamate potency in recombinant NR1/NR2A NMDA receptors. Doctoral thesis (Ph.D.), University College London.

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Abstract

The NMDA type of glutamate receptor requires both glycine and glutamate to activate it efficiently. The receptor is thought to be an oligomer of two types of subunit, NR1 and NR2. Site-directed mutagenesis has shown that glycine potency is controlled by residues located in two areas on the NR1 subunit, one N-terminal of Ml and the other C-terminal of M3 (termed the S1 and S2 domains respectively). To test the hypothesis that the glutamate site exists on the NR2 subunits, the role of amino acid residues in similar areas on the NR2A subunit were investigated. These areas show homology with the ligand binding domains of bacterial periplasmic amino acid binding proteins. Two mutations caused an increase of glutamate EC50 by at least two orders of magnitude, (H466A and G669A) and one (T671A) by three orders, compared with wild-type. In contrast, glycine EC50s did not differ by more than 2-fold compared with the wild-type. Schild analysis was used to measure the binding of the competitive NMDA receptor antagonist D-(-)-2-amino-5-phosphonopentanoic acid (APV) on the most 'shifted' mutant (T671 A). The slope of the Schild plot for the mutant receptor did not differ greatly from unity, 0.94 ± 0.16 (mean ± S.D.M), which is consistent with APV being a competitive antagonist on the mutant receptor. However the affinity of the receptor for APV (KB = 321 ±30 μM) was 255-fold less than wild-type (KB = 1-26 ± 0.07 μM) (means ± S.D.M). The large reduction of glutamate potency, together with unchanged Hill slope, and no gross reduction in maximum response, suggests that residues contributing to glutamate binding are located on the NR2A subunit. This is supported by the much- reduced affinity for APV in the T671A mutant. Such evidence suggests that this residue is important for the binding of glutamate to NR1/NR2A receptors. In a second project, transgenic founder mice were generated by pronuclear microinjection containing a 8 kb 5'UT fragment from the mouse NR1 gene driving the expression of the tetracycline-sensitive transactivator (tTA). This mouse may be useful for the study of neuronal-specific inducible gene expression in the future.

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