Influence of the intracellular GluN2 C-terminal domain on NMDA receptor function

Excitatory neurotransmission mediated by N−methyl−D−aspartate receptors (NMDARs) is fundamental to learning and memory and, when impaired, causes certain neurological disorders. NMDARs are heterotetrameric complexes composed of two GluN1 [NR1] and two GluN2(A−D) [NR2(A−D)] subunits. The GluN2 subunit is responsible for subunit−specific channel activity and gating kinetics including activation (rise time), peak open probability (peak Po) and deactivation (decay time). The peak Po of recombinant NMDARs was recently described to be controlled by the extracellular GluN2 N−terminal domain (NTD). The cytoplasmic GluN2 C−terminal domain (CTD) could also be involved, because the Po of synaptic NMDARs is reduced in mice expressing C−terminally truncated GluN2 subunits. Here, we examined the role of the GluN2 cytoplasmic tail for NMDAR channel activity and gating in HEK−293 cells. C−terminal truncation of GluN2A, GluN2B or GluN2C did not change the subunit−specific rise time but accelerated the decay time of glutamate−activated currents. Furthermore, the peak Po was reduced by about 50%for GluN2A and GluN2B but not for GluN2C. These results indicated that the CTD of GluN2 has a modulating role in NMDAR gating even in the absence of interacting synaptic proteins. Reduction of peak Po and deactivation kinetics following GluN2 C−terminal truncation were reversed by re−introducing a CTD from a different GluN2 subunit. Thus, the CTDs of GluN2 subunits behave as constitutive structural elements required for normal functioning of NMDARs but are not involved in determining the subunit−specific gating properties of NMDAR

Lack of NMDAR subtype selectivity for hippocampal LTP

NMDA receptor (NMDAR) 2A (NR2A)- and NR2B-type NMDARs coexist in synapses of CA1 pyramidal cells. Recent studies using pharmacological blockade of NMDAR subtypes proposed that the NR2A type is responsible for inducing long-term potentiation (LTP), whereas the NR2B type induces long-term depression (LTD). This contrasts with the finding in genetically modified mice that NR2B-type NMDARs induce LTP when NR2A signaling is absent or impaired, although compensatory mechanisms might have contributed to this result. We therefore assessed the contribution of the two NMDAR subtypes to LTP in mouse hippocampal slices by different induction protocols and in the presence of NMDAR antagonists, including the NR2A-type blocker NVP-AAM077, for which an optimal concentration for subtype selectivity was determined on recombinant and native NMDARs. Partial blockade of NMDA EPSCs by 40%, either by preferentially antagonizing NR2A- or NR2B-type NMDARs or by the nonselective antagonist D-AP-5, did not impair LTP, demonstrating that hippocampal LTP induction can be generated by either NMDAR subtype

Presynaptic {alpha}2-GABAA receptors in primary afferent depolarization and spinal pain control

Spinal dorsal horn GABA(A) receptors are found both postsynaptically on central neurons and presynaptically on axons and/or terminals of primary sensory neurons, where they mediate primary afferent depolarization (PAD) and presynaptic inhibition. Both phenomena have been studied extensively on a cellular level, but their role in sensory processing in vivo has remained elusive, due to inherent difficulties to selectively interfere with presynaptic receptors. Here, we address the contribution of a major subpopulation of GABA(A) receptors (those containing the α2 subunit) to spinal pain control in mice lacking α2-GABA(A) receptors specifically in primary nociceptors (sns-α2(-/-) mice). sns-α2(-/-) mice exhibited GABA(A) receptor currents and dorsal root potentials of normal amplitude in vitro, and normal response thresholds to thermal and mechanical stimulation in vivo, and developed normal inflammatory and neuropathic pain sensitization. However, the positive allosteric GABA(A) receptor modulator diazepam (DZP) had almost completely lost its potentiating effect on PAD and presynaptic inhibition in vitro and a major part of its spinal antihyperalgesic action against inflammatory hyperalgesia in vivo. Our results thus show that part of the antihyperalgesic action of spinally applied DZP occurs through facilitated activation of GABA(A) receptors residing on primary nociceptors