Cik, Miroslav; (1994) Molecular characterisation of the N-methyl-D-aspartate receptor expressed in mammalian cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

L-Glutamate is the major excitatory neurotransmitter in the vertebrate central nervous system, mediating its effects via interaction with glutamate receptors. Several pharmacological subclasses of the glutamate receptor have been identified, including the N-methyl-D-aspartate (NMDA) receptor. Molecular cloning has recently identified five genes encoding the NMDA receptor subunits, NMDAR1 and NMDAR2A-D. In this thesis, work is described in which conditions for the optimal transient expression of homo- and heteromeric NMDA receptors in mammalian cells were established, thus providing a model system for structure-function studies of this important brain protein. Thus, the cDNAs encoding the NMDAR1-1a and NMDAR2A subunits were subcloned into the mammalian expression vector, pCIS. The resultant constructs were transfected alone or in combination in human embryonic kidney (HEK) 293 cells by the calcium phosphate method. Co-transfection studies resulted in cell death, which was prevented by inclusion of NMDA receptor antagonists in the cell culture medium post-transfection. A study was made of the efficacy for the prevention against cell death for a series of different NMDA receptor antagonists, which included DL-2-amino-5 phosphonovalerate (AP5), 5,7-dichlorokynurenic acid (DKA) and Mg2+. The percentage cell death was quantified by either Trypan Blue exclusion or the CytoTox 96TM assay system. The combination of AP5 and DKA gave optimal protection with no significant difference between cotransfected and control samples. Site-directed in vitro mutagenesis was used to generate point mutations of the NMDAR1-1a subunit. Co-transfection studies with the wild-type and mutant NMDA receptor cDNAs were carried out and the effect of the mutations on cell viability quantified. Most notably, it was found that the mutation (N598Q), previously shown to reduce the Ca2+ permeability of cloned receptors, significantly reduced cell toxicity, thus providing evidence for the role of Ca2+ in NMDA-receptor mediated cytotoxic mechanisms.

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