The effect of competitive antagonist chain length on NMDA receptor subunit selectivity

doi:10.1016/j.neuropharm.2004.11.004

Neuropharmacology

Volume 48, Issue 3, March 2005, Pages 354-359

https://doi.org/10.1016/j.neuropharm.2004.11.004 Get rights and content

Abstract

The widely-used N-methyl-d-aspartate (NMDA) receptor antagonists (R)-4-(3-phosphonopropyl) piperazine-2-carboxylic acid ((R)-CPP) and (R)-2-amino-7-phosphonoheptanoate ((R)-AP7) are frequently used as general NMDA receptor antagonists and assumed not to display significant selectivity among NMDA receptor NR2 subunits. However, electrophysiological studies have suggested that certain longer chain N-methyl-d-aspartate (NMDA) receptor competitive antagonists, such as (R)-CPP are ineffective at subpopulations of NMDA receptors in the red nucleus, superior colliculus, and hippocampus. Using recombinant receptors expressed in Xenopus oocytes, we have examined the effect of antagonist chain length on NR2 subunit selectivity. All antagonists displayed the potency order (high to low affinity) of NR2A > NR2B > NR2C > NR2D, however the longer chain antagonists (having 7 instead of 5 bond lengths between acidic groups) displayed much greater subunit selectivity than their short-chain homologues. For example (R)-CPP displayed a 50-fold difference in affinity between NR2A-containing and NR2D-containing NMDA receptors, while the shorter chain homologue 4-(phosphonomethyl) piperazine-2-carboxylic acid (PMPA) displayed only a 5-fold variation in affinity. These results can account for the earlier physiological findings and suggest that longer chain antagonists such as (R)-CPP and (R)-AP7 should not be used as general NMDA receptor antagonists.

Introduction

NMDA receptors mediate synaptic transmission and neural plasticity throughout the CNS; they also contribute to epileptiform activity and neuronal cell death in a number of pathological conditions. Although there are different subtypes of NMDA receptors, their respective roles in synaptic transmission, plasticity and pathology are not yet known. NMDA receptors are a multimeric complex, probably a tetramer (Laube et al., 1998) composed of at least two subunit families, the glycine-binding NR1 subunits (NR1a-h) (Sugihara et al., 1992, Wafford et al., 1995) and the glutamate-binding NR2 subunits (NR2A-D) (Meguro et al., 1992, Monyer et al., 1992, Ishii et al., 1993, Laube et al., 1997). To a limited extent, NMDA receptors may also contain an NR3 subunit (Sucher et al., 1995). The NR2 subunits impart distinct physiological and pharmacological properties on the NR1/NR2 heterodimer (Buller et al., 1994, Laurie and Seeburg, 1994, Monyer et al., 1994).

Despite the varied physiological properties and anatomical distributions of the NR2 subunits, relatively little is known about their different physiological actions in native brain tissue. A major limitation to understanding the individual roles of NMDA receptor subtypes has been the lack of subtype-selective NMDA receptor antagonists. Other than the ifenprodil-like NR2B-selective antagonists (Nikam and Meltzer, 2002, Chazot, 2004), and a recently described NR2A/NR2C-partially selective antagonist (Auberson et al., 2002, Feng et al., 2004), NMDA receptor antagonists are generally considered not to be subtype-selective.

The widely-used competitive NMDA receptor antagonists, (R)-AP7, (R)-CPP and related structures, are frequently used as general, non-subtype-selective NMDA receptor antagonists. However, at least three physiological studies have shown that (R)-CPP does not block all sub-populations of native NMDA receptors. Monosynaptic responses of sensorimotor cortical synaptic terminals in the red nucleus exhibit NMDA receptors which were blocked by (R)-2-amino-5-phosphonopentanoate ((R)-AP5) and PMPA, but not by the longer chain homologues (R)-AP7 and (R)-CPP (Harris and Davies, 1992). Similarly, neurons in the superior colliculus respond to visual stimuli with enhanced firing that is blocked by (R)-AP5, but not by CPP (Binns et al., 1999). In both the red nucleus and superior colliculus, responses evoked by exogenous agonist (NMDA) application were blocked by all NMDA receptor antagonists tested (Harris and Davies, 1992, Binns et al., 1999). A subtype-selective action of (R)-CPP is also seen at the CA3-CA1 hippocampal synapse where (R)-CPP, but not other NMDA receptor antagonists, selectively inhibits a fast component, but not a slow component, of the NMDA receptor-mediated synaptic response (Lozovaya et al., 2004). As noted in the studies of the red nucleus (Harris and Davies, 1992) and superior colliculus (Binns et al., 1999), the effective blockers have 5 covalent bond lengths between their two acidic groups, whereas the sometimes ineffective blockers, such as (R)-CPP, have 7 bond lengths between their acidic groups.

To evaluate the potential role of chain-length in NMDA receptor subunit selectivity, we evaluated three pairs of competitive, glutamate-binding site NMDA receptor antagonists in which the members of each pair differ only in chain length. The antagonists were tested for activity at recombinant NMDA receptors expressed in Xenopus oocytes using two-electrode voltage-clamp recording.

Section snippets

NR subunit expression in Xenopus oocytes

cDNA encoding the NMDAR1a subunit was a generous gift of Dr. Shigetada Nakanishi (Kyoto, Japan). cDNA encoding the NR2A, NR2C and NR2D were kindly provided by Dr. Peter Seeburg (Heidelburg, Germany) and the NR2B [5′ UTR] cDNA was the generous gift of Drs. Dolan Pritchett and David Lynch (Philadelphia, USA). Plasmids were linearized with Not I (NR1a), EcoR I (NR2A, NR2C and NR2D) or Sal I (NR2B) and transcribed in vitro with T3 (NR2A, NR2C), SP6 (NR2B) or T7 (NR1a, NR2D) RNA polymerase using the

Results

NMDA receptor antagonists that are competitive at the glutamate-binding site were tested for their ability to block glutamate/glycine-evoked currents in Xenopus oocytes injected with NMDA receptor subunit cRNA. The resulting Ki values are shown in Table 1. All compounds displayed the potency order (from high to low) of NR2A > NR2B > NR2C > NR2D. However, within this pattern, there were large variations in relative potency. As shown in Fig. 1, the long-chain antagonist (R)-CPP displayed a wide

Discussion

In each of the pairs of antagonists, the compound having 7 covalent bonds between the two acidic groups displayed significantly greater selectivity among subunits than the corresponding antagonist with 5 covalent bonds between the acidic groups. It is likely, however, that this trend is not just a simple property of chain-length, per se. Comparing the short and long-chain piperazine compounds shows that greater selectivity of the longer-chain antagonists was due to a differential increase in

Acknowledgements

The authors wish to thank Drs. Shigetada Nakanishi, David Lynch, Dolan Pritchett and Peter Seeburg for providing NMDA receptor cDNA constructs. We also thank Dr. Paul Ornstein for providing compounds. This work was supported by NIH grant MH60252.

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The effect of competitive antagonist chain length on NMDA receptor subunit selectivity

Abstract

Introduction

Section snippets

NR subunit expression in Xenopus oocytes

Results

Discussion

Acknowledgements

Bioorg. Med. Chem. Lett.

Eur. J. Pharmacol.

Brain. Res.

J. Biol. Chem.

Neuron

Eur. J. Pharmacol.

Neuron

Biochem. Biophys. Res. Commun.

Brain Res.

Pharmacology of NMDA receptor subtypes

Society for Neuroscience Abstracts

Visual experience alters the molecular profile of NMDA-receptor-mediated sensory transmission

Eur. J. Neurosci.

NR2B and NR2D subunits coassemble in cerebellar Golgi cells to form a distinct NMDA receptor subtype restricted to extrasynaptic sites

J. Neurosci.

The molecular basis of NMDA receptor subtypes: native receptor diversity is predicted by subunit composition

J. Neurosci.