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Proquest Dissertations MOLECULAR CHARACTERISATION OF THE N-METHYL D- ASPARTATE RECEPTOR EXPRESSED IN MAMMALIAN CELLS by Miroslav Cik A thesis submitted for the degree of Doctor of Philosophy of the University of London Department of Pharmaceutical Chemistry School of Pharmacy 29/39 Brunswick Square London, WCIN lAX February 1994 ProQuest Number: U066153 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest U066153 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 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, NMDARl 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 NMDARl-la 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 Mg^^. The percentage cell death was quantified by either Trypan Blue exclusion or the CytoTox 96™ assay system. The combination of AP5 and DKA gave optimal protection with no significant difference between co­ transfected and control samples. Site-directed in vitro mutagenesis was used to generate point mutations of the NMDARl-la 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 Ca^^ permeability of cloned receptors, significantly reduced cell toxicity, thus providing evidence for the role of Ca^^ in NMDA-receptor mediated cytotoxic mechanisms. To Mum, with love and happy memories CONTENTS A bstract...................................................................................................... 2 Contents ...................................................................................................... 4 List of Figures ............................................................................................. 9 List of T ab les ............................................................................................. 11 Abbreviations ............................................................................................. 12 Acknowledgements ....................................................................................15 Chapter 1 General Introduction ............................................................. 16 1.1 Foreword .................................................................................. 17 1.2 Glutamate as a neurotransmitter ............................................. 18 1.3 Molecular pharmacology of glutamate receptors ................... 20 1.3.1 NMDA receptor .......................................................................21 1.3.1.1 The glycine binding site .........................................................21 1.3.1.2 The divalent cation binding sites ........................................... 23 1.3.1.3 The polyamine binding site .................................................... 25 1.3.1.4 The PCP binding site ..............................................................26 1.3.2 Non-NMDA receptors ............................................................. 27 1.3.3 Metabotropic glutamate receptors ...........................................29 1.4 Molecular biology of non-NMDA receptors ...........................30 1.4.1 Cloning of non-NMDA receptors ...........................................30 1.4.2 Properties of expressed non-NMDAreceptors ....................... 35 1.4.3 Distribution of non-NMDA receptors ....................................39 1.4.4 Immunochemical characterization of the non-NMDA glutamate receptors using subunit specific antibodies .... 39 1.5 Molecular biology of NMDA receptors ..................................41 1.5.1 Cloning of NMDA receptors ..................................................41 1.5.2 Properties of expressed NMDA receptors .............................48 1.5.3 Distribution of NMDA receptors ........................................... 52 1.5.4 Identification of functional determinants of the NMDA receptor ...............................................................53 1.6 Glutamate in physiology and pathophysiology ...................... 54 1.6.1 LTP and m em ory .....................................................................54 1.6.2 Glutamate neurotoxicity ............................................................55 1.7 The objectives of this study.....................................................58 Chapter 2 Transient expression of NMDA heteromeric receptors in mammalian cells....................................................................59 2.1 Introduction ............................................................................60 2.1.1 Expression of genes in mammalian c e lls ................................60 2.1.2 Cell lines for expression of cDNAs .........................................61 2.1.3 Functional components of mammalian expression vectors . 62 2.1.3.1 Promoter and enhancer elements ..............................................62 2.1.3.2 mRNA processing signals .......................................................63 2.1.4 Examples of vectors for expression of cDNAs encoding eukaryotic genes in mammalian cells ................................... 63 2.1.5 Methods for introducing DNA into mammalian cells .... 65 2.2 Materials and methods ......................................................... 67 2.2.1 M aterials...................................................................................67 2.2.2 Storage of the E. coli cells ....................................................... 6 8 2.2.3 Preparation of competent E. coli c e lls .....................................6 8 2.2.4 Transformation of competent E. coli c e lls ..............................69 2.2.5 Preparation of plasmid DNA ...................................................69 2.2.5.1 Small-scale preparation of plasmid D N A ................................69 2.2.5.2 Large-scale preparation of plasmid D N A ................................71 2.2.6 Spectrophotometric quantification of plasmid DNA ............. 72 2.2.7 Restriction enzyme digestion ...................................................73 2.2.8 Dephosphorylation of linearized plasmid D N A .......................73 2.2.9 DNA ligation recetions ............................................................ 74 2.2.10 Magic™ DNA purification ....................................................... 74 2.2.11 Preparation of dialysis tubing ..................................................75 2.2.12 Flat bed agarose gel electrophoresis ......................................75 2.2.12.1 The agarose gel ...................................................................... 75 2.2.12.2 The LMP flat bed agarose gel electrophoresis ......................76 2.2.13 Qiaex agarose gel extraction ..................................................77 2.2.14 Preparation of cell culture media ...........................................78 2.2.15 The cell culture conditions ...................................................... 78 2.2.16 Liquid nitrogen storage of HEK 293 c e lls .............................78 2.2.17 Ca^^ Phosphate-mediated transfection of HEK 293 cells . 79 2.2.18 Determination of transfection efficiency of HEK 293 cells 80 2.2.19 Viable cell number determination ...........................................81 2.2.20 CytoTox 96™ non-radioactive cytotoxicity a s sa y ................. 82 2.3 R esu lts.....................................................................................83 2.3.1 Subcloning of the NMDARl-la cDNA into the pCIS expression vector ....................................................................83 2.3.2 Subcloning of the NMDAR2A cDNA into the pCIS expression vector ....................................................................85 2.3.3 Transient expression of the pCISNMDAR1 -1 a recombinant in mammalian cells................................................................. 87 2.3.4 Transient expression of the pCISNMDAR2A recombinant in mammalian cells................................................................
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