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NMDA receptor subtypes expressed in mammalian cells: A molecular characterisation

Coleman, Sarah; (1997) NMDA receptor subtypes expressed in mammalian cells: A molecular characterisation. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

The N-methyl-D-aspartate (NMDA) subtypes of the excitatory glutamate receptors of the mammalian brain are fast-acting, ligand-gated ion channels. These receptors have been implicated in long term potentiation, synaptic plasticity and neurodegeneration. The NMDA receptor channel is highly permeable to Ca2+ and NMDA receptor activity can be modulated by several classes of compounds which include neurosteroids, Mg2+, Zn2+, H+ and polyamines. There are five genes encoding NMDA receptor subunits, NR1 and NR2A-2D. These genes are classified according to the amino acid homologies of their respective products. It is believed that native NMDA receptors are composed of an NR1 together with (a) NR2 subunit(s) in unknown ratios. It was the aim of this project to elucidate the subunit complements of native cerebellar NMDA receptors. The NR2C mRNA is abundantly expressed in the cerebellum therefore NR1 and NR2C clones were coexpressed transiently in human embryonic kidney (HEK) 293 cells. The recombinant receptor was characterised by both immunoblotting and 3HMK801 radioligand binding assays and the affinity compared to that of native NMDA receptors expressed in the cerebellum. The NR1/NR2C receptor had an affinity, KD, for 3HMK801 = 345 ± 158 nM compared to 22 ± 9 nM for the wild-type. However, cotransfection with NR1, NR2C and NR2A clones using a defined DNA ratio for transfection yielded a KD = 22 ± 5 nM. A detailed characterisation of the pharmacology of this binding site showed that the correlation coefficient for the affinities of a series of compounds between NR1/NR2A/NR2C and native cerebellar receptors was r = 0.992. Further studies showed that the NR1, NR2A and NR2C ratios used for transfection could influence the pharmacological profile of the recombinant receptor. The radioligand binding studies to the cloned NMDA receptor subtypes suggested that the determinant for the high affinity MK801 binding resided in the NR2A subunit. This has been investigated further by site-directed mutagenesis of the NR2C subunit within the transmembrane, TM2, region in conjunction with 3HMK801 radioligand binding and cell cytotoxicity assays.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: NMDA receptor subtypes expressed in mammalian cells: A molecular characterisation
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; NMDA receptors
URI: https://discovery.ucl.ac.uk/id/eprint/10103617
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