Functional and Pharmacological Differences Between RecombinantN-Methyl-d-Aspartate Receptors

Abstract

N-methyl-D-aspartic acid (NMDA) receptors transiently transfected into mammalian HEK-293 cells were characterized with subunit-specific antibodies and electrophysiological recordings. Deactivation time course recorded in response to fast-glutamate pulses were studied in isolated and lifted cells, as well as in outside-out membrane patches excised from cells expressing recombinant NR1 subunits in combination with the NR2A, NR2B, NR2C, or NR2D NMDA receptor subunits. Transfected cells were preidentified by the fluorescence emitted from the coexpressed Aequorea victoria jellyfish Green Lantern protein. Currents generated by NR1/NR2A channels displayed double exponential deactivation time course being faster than that in NR1/NR2B or NR1/NR2C channels. However, a large decay variability was observed within each cotransfection, suggesting that mechanisms additional to subunit composition may also regulate deactivation time course. NR1/NR2D channels displayed slowly deactivating currents. Channel deactivation was fast and comparable among receptors obtained by cotransfecting five distinct spliced variants of the NR1 subunit, each with the NR2A subunit. Additionally, recovery from desensitization was slower for NR1/NR2B than for NR1/NR2A channels. The average deactivation time course of responses to brief L-glutamate applications in cells where NR1/NR2A/NR2B cDNAs were cotransfected at variable ratio was intermediate between those of the NR1/NR2A and NR1/NR2B channels. Although immunocytochemical evidence indicates that the majority of cells are cotransfected by all plasmids in triple transfection, our experimental condition did not allow for a tight control of the expression of NMDA receptor subunits. This produced the result that many cells were characterized by deactivation time course and haloperidol sensitivities of separate NR1/NR2A and NR1/NR2B subunit heteromers. We also speculate on the possible formation of channels resulting from the coassembly in the same receptor of NR1/NR2A/NR2B subunits from a minority of cells that gave responses to brief application of L-glutamate characterized by slow deactivation time course and decreased haloperidol sensitivity.

Alcohol Inhibits NR2B-Containing NMDA Receptors in the Ventral Bed Nucleus of the Stria Terminalis

“…Both the decay time (294747 ms for the NR2A KO, n ¼ 12; 128720 ms for the wild type, n ¼ 10; po0.01; Figure 3b) and the rise time (1171 ms for the NR2A KO, n ¼ 12; 6.670.3 ms for the wild type, n ¼ 10; po0.01; Figure 3c) were significantly slower when compared to wild-type animals. Data from recombinant systems indicate that the kinetics of diheteromeric NR1/NR2A NMDARs are faster than other di-and triheteromeric NMDAR configurations, thus these data are what would be predicted if 'faster' NR2A subunits contribute to the synaptic NMDA-EPSC in the wild-type vBNST glutamate synapses (Vicini et al, 1998;Erreger et al, 2005). Similar observations have been made for synaptic NMDA-EPSCs in cultured cerebellar granule cells from NR2A KO mice (Fu et al, 2005).…”

Zinc reverses glycine-dependent inactivation of NMDARs in cultured rat hippocampal neurons

“…Desensitization characteristics of NMDARs, which depends on the subunits expressed by the neurons, may be remarkably changed from one neuron to another. Accumulating evidence shows that there are subunit-specific differences in the desensitization properties of NMDARs [7,21]. The issue whether this glycinedependent inactivation also appears in NMDAR complex composed of NR1 with other NR2 subunits remains to be elucidated.…”

Enhanced Astrocytic Ca2+Signals Contribute to Neuronal Excitotoxicity after Status Epilepticus

“…The selective attenuation of synaptic NMDA receptors by NVP-AMM077 and attenuation of SICs by ifenprodil show that gliotransmission, which is mediated by SICs, preferentially accesses extrasynaptic NR2B-containing NMDA receptors. Because NR2A-but not NR2C-or NR2D-containing NMDA receptors exhibit rapid kinetics (Vicini et al, 1998), it is likely that the NVP-AAM077-sensitive synaptic NMDA currents are mediated predominantly by NR2A-containing NMDA receptors. Thus, astrocytic, but not synaptic, glutamate preferentially accesses extrasynaptic NR2B subunit-containing NMDA receptors.…”