Generation and Characterisation of Stable Cell Lines Expressing Recombinant Human N‐Methyl‐d‐Aspartate Receptor Subtypes

Abstract

Transfection of mouse L(tk‐) cells with human N‐methyl‐d‐aspartate (NMDA) receptor subunit cDNAs under the control of a dexamethasone‐inducible promoter has been used to generate two stable cell lines expressing NR1a/NR2A receptors and a stable cell line expressing NR1a/NR2B receptors. The cell lines have been characterised by northern and western blot analyses, and the pharmacology of the recombinant receptors determined by radioligand binding techniques. Pharmacological differences were identified between the two NMDA receptor subtypes. The glutamate site antagonist d,l‐(ε)‐2‐[3H]amino‐4‐propyl‐5‐phosphono‐3‐pentanoic acid ([3H]CGP 39653) had high affinity for NR1a/NR2A receptors (KD = 3.93 nM) but did not bind to NR1a/NR2B receptors. Glycine site agonists showed a 2.6–5.4‐fold higher affinity for NR1a/NR2B receptors. Data from radioligand binding studies indicated that one of the cell lines, NR1a/NR2A‐I, expressed a stoichiometric excess of the NR1a subunit, which may exist as homomeric assemblies. This observation has implications when interpreting data from pharmacological analysis of recombinant receptors, as well as understanding the assembly and control of expression of native NMDA receptors.

Dopamine receptor contribution to the action of PCP, LSD and ketamine psychotomimetics

“…A similar situation exists for ketamine, where the psychotomimetic concentration in the serum is of the order of 100-500 nM, 8 a value that matches the ketamine action (100-500 nM) at D2 High (Figure 3, top), but not that at the NMDA receptor, which has a ketamine K i of 3100 nM for striatal tissue (Figure 3, bottom), 760-2270 nM for brain membranes 33,34 and 3150 nM 34 for cloned NMDA receptors ( Table 1).…”

“…32 This is precisely the active range of phencyclidine that directly acts on D2 High (Figure 1) and stimulates D2 (Figure 2). In contrast, the NMDA receptor has phencyclidine K i values of 97 nM 34 and 124 nM 35 on rat brain membranes, and 196 nM 35 on the human cloned NMDA receptor (Table 1).…”

Characterisation of N‐Methyl‐D‐Aspartate Receptor‐Specific [3H]Ifenprodil Binding to Recombinant Human NR1a/NR2B Receptors Compared with Native Receptors in Rodent Brain Membranes

“…Rat cortex/hippocampus membranes and cell membranes transfected with human NMDA receptor subunits were prepared as previously reported (Grimwood et al, 1992(Grimwood et al, , 1996a. NR1a, NR1a/NR2A, and NR1a/NR2B receptors were stably expressed in L(tkϪ) cell lines (Grimwood et al, 1996a), and NR2B and NR1b/NR2B receptors were transiently transfected in HEK 293 cells as described previously (Grimwood et al, 1995).…”

“…NR1a, NR1a/NR2A, and NR1a/NR2B receptors were stably expressed in L(tkϪ) cell lines (Grimwood et al, 1996a), and NR2B and NR1b/NR2B receptors were transiently transfected in HEK 293 cells as described previously (Grimwood et al, 1995).…”

“…] reflects that this cell line was selected for its high level of expression of NR2B subunits (see Grimwood et al, 1996a). The affinity value (K D ϭ 33.5 nM; Table 1) obtained for [ 3 H]ifenprodil binding to NR1a/NR2B cell membranes was similar to that observed for both rat cortex/hippocampus membranes (24.8 nM; Table 1) and human cortex membranes [cortical region BA22, 17.3 nM; cortical region BA6, 21.0 nM (Grimwood et al, 1999)], suggesting that the higher ratio of NR2B subunits per receptor in the stably transfected NR1a/NR2B cell line compared with native NMDA receptors did not affect the affinity of [ 3 H]ifenprodil.…”