Cloning, Functional Coexpression, and Pharmacological Characterisation of Human cDNAs Encoding NMDA Receptor NR1 and NR2A Subunits

Abstract

Using expression cloning, and more recently using polymerase chain reaction cloning approaches, a family of rat N‐methyl‐d‐aspartate (NMDA) receptor subunit cDNAs has been described (NR1, NR2A, NR2B, NR2C, and NR2D). Here we report cloning and sequencing of cDNAs encoding isoforms of the human NR1 subunit (NR1a, NR1d, and NR1e) that differ at their C‐terminal end as a result of alternative splicing and also of a cDNA encoding the human NR2A subunit. The deduced amino acid sequences of the human NR1 subunit isoforms differed from the published rat NR1 subunit sequences at only eight positions, all of which were N‐terminal to the alternatively spliced domains. The human NR2A subunit deduced amino acid sequence differed from the published rat NR2A subunit sequence at 81 of the 1,464 amino acids, with most of the substitutions being located in the C‐terminal half of the subunit. The gene for NR2A has been localised to human chromosome 16. We also report the expression and pharmacological characterisation of recombinant human NR1a/NR2A heteromeric receptors in Xenopus oocytes. These receptors had EC50 values of 2.14 and 2.05 μM for glutamate and glycine, respectively, and an IC50 of 46.8 μM for Mg2+. Responses were antagonised by d‐2‐amino‐5‐phosphonovalerate, L‐689,560, pH 6.3, zinc, and MK‐801. No modulatory effect was observed on application of ifenprodil, confirming previous observations with rat NR1 + NR2A recombinant receptors.

Identification of an N-Methyl-d-aspartate Receptor in Isolated Nervous System Mitochondria

“…receptors (34), into mitochondrial membranes of GT1-7 cells using a Bcl-2 leader sequence. This leader sequence has been shown to insert Bcl-2 into both the outer (35) and inner (36) mitochondrial membranes.…”

Molecular pharmacology of human NMDA receptors

“…In other words, there is an apparent lack of data from human NMDA receptors. Relatively few studies have described functional properties of heterologously expressed human NMDA receptors (Bednar et al, 2004; Claiborne et al, 2003; Curtis et al, 2003; Daggett et al, 1998; Feuerbach et al, 2010; Grimwood et al, 1996a; Grimwood et al, 1996b; Hess et al, 1996; Hess et al, 1998; Le Bourdelles et al, 1994; Priestley et al, 1996; Priestley et al, 1995; Steinmetz et al, 2002; Steinmetz et al, 2004; Usala et al, 2003; Varney et al, 1996; Wafford et al, 1995). Although the amino acid sequence is highly conserved between rat and human subunits, some variation exist that may nonetheless confer significant pharmacological and functional differences.…”

“…Only few differences are located in the agonist binding and the transmembrane domains, suggesting conservation of key conformational changes that lead to channel gating in response to agonist binding. More detailed analyses of cDNAs and deduced amino acid sequences for human NMDA receptor subunits are described in the original cloning papers (Collins et al, 1993; Daggett et al, 1998; Foldes et al, 1994a; Foldes et al, 1993, 1994b; Hess et al, 1996; Hess et al, 1998; Karp et al, 1993; Le Bourdelles et al, 1994; Lin et al, 1996; Planells-Cases et al, 1993). …”

“…A number agonists and competitive antagonists have been characterized on human recombinant NMDA receptor subtypes using electrophysiological methods and fluorescence-based measurements of intracellular Ca 2+ (Bednar et al, 2004; Daggett et al, 1998; Feuerbach et al, 2010; Hess et al, 1996; Hess et al, 1998; Le Bourdelles et al, 1994; Priestley et al, 1995; Varney et al, 1996; Wafford et al, 1995). The GluN2B-selective non-competitive antagonist ifenprodil has also been evaluated on human NMDA receptors with some variation in the obtained IC 50 -values, ranging from 110 nM to 2.2 µM at human GluN1/GluN2B (Bednar et al, 2004; Feuerbach et al, 2010; Hess et al, 1998; Varney et al, 1996).…”

Selective vulnerabilities of N-methyl-D-aspartate (NMDA) receptors during brain aging

“…The functional subunits of the NMDA receptor complex have been cloned for rats (Moriyoshi et al, 1991; Monyer et al, 1992; Ishii et al, 1993; Seeburg, 1993), mice (Ikeda et al, 1992; Kutsuwada et al, 1992; Meguro et al, 1992; Yamazaki et al, 1992) and humans (Karp et al, 1993; Le Bourdelles et al, 1994; Zimmer et al, 1995). The International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR) has recently published guidelines for receptor and ion channel classification that aim to standardize the nomenclature used to refer to NMDA receptor subunits (Collingridge et al, 2009).…”