The N-methyl-D-aspartate (NMDA) glutamate receptors function as plasma membrane ionic channels and are part of very tightly controlled cellular processes activating neurogenic and inflammatory pathways. A large body of studies supports their functions in both normal physiology and disease states with a focus on the NMDA NR1 subunit (new terminology GluN1 subunit [1]) function in psychiatric and neurologic conditions. NMDA receptors are activated by excitatory amino acid (EAA) agonists, glutamate, aspartate, and NMDA. NMDA receptors in low levels are essential for neuronal development, differentiation, learning, survival, and plasticity ^[2][3][2,3]. NMDA receptors play a critical role in pre- and post-synaptic plasticity, especially learning and memory [4]. However, when glutamate receptor agonists are present in excess, binding to NMDA receptors produces neuronal/CNS/PNS excitotoxicity, pathology, conditions of acute pain ^[5][6][5,6], and ongoing severe, intractable pain ^[7][8][7,8]. Conditional deletion of the GluN1 subunit in the spinal cord dorsal horn reduces injury-induced pain [9]. Beyond this is involvement in anxiety/depression [10], disease states (schizophrenia, Parkinson’s ^[11][12][11,12]), dementia ^[13][14][15][13,14,15], and seizures ^[16][17][16,17].

Earlier studies established that the GluN1 subunit anchor component was necessary for heterodimer formation, cellular trafficking, and nuclear localization that was functionally specific ^[18][19][18,19]. Four subunits assemble the glutamate receptors, and each heterodimer strictly requires the GluN1 subunit to anchor GluN2 or GluN3 subunits, comprising a functional ionic channel. GluN1 subunits bind combinations of GluN2A, 2B, and GluN3A and 3B subunits that form heterodimers to anchor these components on the cellular plasma membrane ^[20][21][22][20,21,22]. The binding composition is noted as two obligatory GluN1 subunits, of which there are now reported at least eight distinct variant subunits and four or two variable subunits from the GluN2 (GluN2A-2D) and GluN3 (GluN3A-3B), respectively, producing 8 × 6 = 48 potential heterodimers.

The GluN1-1a subunits have a nuclear localization sequence (NLS) exclusive of tissue specificity ^[18][23][18,23]. Additional studies have reported the alternative splicing of messenger RNA producing similar proteins that target different tissues, determine its functional fate [24], and have expanded studies to include expression and functioning in non-neuronal tissues. Much of the recent research has focused on characterizing the GluN2 subunits of the GluN1/GluN2 heterodimer. Shifts in the composition of the heterodimer, based on GluN2 or N3 subunits, denote the functional roles of the receptor complex and promote the neuroplasticity of the glutaminergic system throughout the CNS ^[23][24][23,24].

2. Potential Fates of GluN1 Subunit in the Cellular Nucleus

2.1. GluN1 Subunit Signaling Induces Nuclear Translocation

GluN1 subunit occupation in the cell nucleus has been reported since early 2000, and a resurgence of research is providing a better understanding to fill in the gaps. This has included reports of the bipartite NLS contained in the GluN1-1a subunit ^[18][25][18,38], protein isoforms, and sequence cassettes. For example, the GluN1/N2A/B functional tetramer is inserted into the cellular plasma membrane site as an ionic channel. When activated, the heterodimer is phosphorylated at the GluN2 cytoplasmic regions, and the GluN1 subunit transmembrane region protein undergoes intermembrane proteolysis, cleaving the cytoplasmic portion in the cell membrane. The heterodimer internalizes and translocates from the cell’s plasma membrane to the cell’s nuclear membrane ^[18][26][18,35]. This region contains a bipartite NLS with two clusters of short sequences of basic amino acids, mainly lysines (K) and arginines (R), separated by a link of a variable number of amino acids. The two regions of basic amino acids on the protein surface comprise the regions recognized for binding along the nuclear membrane and passing into the nucleus. In the case of GluN1-1a, the NLS sequence regions with basic amino acids in bold type are: KRHK-spacer region—KKKATFRAITSTLASSFKRRR ^[25][38].

2.2. GluN1 Subunit Staining in the Cellular Nucleus

The nuclear translocation of the GluN1 subunit after glutamate activation suggests it also plays a direct role in the fast intracellular signaling responses to extracellular glutamate activation. Long-term potentiation (LTP) mediated facilitation requires an active importin nuclear import pathway ^[27][39]. Examination of the function of the GluN1 subunit in nuclear translocation has found the C1 domain binds calmodulin to assist calcium entry into the nucleus ^[19][23][19,23]. This regulates LTP and synaptic plasticity in hippocampal cultures. Glutamate-mediated (LTP-like) overactivation in pain states likely involves calcium entry into the nucleus through ion channels that include GluN1 subunits. The evidence for this appears below. Detection of increased cell nuclear staining of GluN1 subunit in activated cells is easily appreciated in vivo or in vitro. For example, increased cell nuclear staining of the GluN1 subunit was observed with visual microscopic examination of human clonal neuroblastoma cell cultures (SH-SY5Y) activated with glutamate or NMDA for 4 h, and nuclear staining was inhibited by preincubation with active NMDA protein tyrosine kinase (PTK) inhibitors genistein or staurosporin in vitro ^[28][40]. Increased nuclear staining was also easily observed by 2 h after incubation with NMDA and ACPD in human synovial fibroblast cells ^[29][27]. Additionally, staining of the GluN1 subunit on the nuclear rim and nucleoplasm was appreciated in the presence of cycloheximide ^[28][40], demonstrating that nuclear localization is an incitement event for the subunit and not solely the purview of newly synthesized GluN1 subunit. In complementary studies inducing synovial joint capsule inflammation, secondary tactile allodynia in the same hindlimb resulted in the same time span of two hours following excess glutamate released into the joint capsule from activated peripheral nerves in a rat intra-articular K/C arthritis model ^[30][41]. The afferent nerve and spinal glutamate neurons release glutamate into the spinal cord dorsal horn (SCDH) in response to activation or injury ^[31][42]. Models providing information on nociception/pain study either acute pain, where glutamate receptor stimulation is limited timewise, or chronic pain, where the glutamate receptor is overstimulated long-term. Many molecular, epigenetic, and inflammatory events are ongoing, accompanied by prolonged exposure to glutamate and overactivation of glutamate receptors. Continued activation of the glutamate receptors results in excessive intracellular chloride that increases the neuronal membrane potential above the threshold, resulting in the reversal of GABA from inhibitory to excitatory continuous firing, contributing to chronic pain ^[32][33][43,44].

3. Roles and Potential Fates of GluN1 Subunit in The Cellular Nucleus

The regional and functional assignments provided in the GluN1/2 subunit heterodimers have been attributed mostly to the GluN2 subunits, after they are trafficked and delivered to the nucleus via the GluN1 receptor subunit. However, the GluN1 subunit has several indicators of its own potential to influence heterodimer and/or subnuclear organelle assignments. 1. The putative NLS and/or nucleolar localization sequences (NoLS) located in cassette 1 of the GluN1 subunit has two short regions rich in basic amino acids, which comprise the NLS and are potentially involved in nucleolar signaling ^[34][35][66,67]. GluN1 subunits are tightly subjected to multiple levels of regulation, affecting subunit expression, subcellular location, and assembly of functional receptors, and their signaling complexes ^[36][34]. 2. The gene for the GluN1 subunit is expressed in early development in virtually all neurons, and is transcriptionally upregulated during neuronal differentiation. NMDA agonists and EAAs increase cellular GluN1 subunit levels, as demonstrated with widespread cell membrane, intracellular, and nuclear GluN1 subunit staining. GluN1 subunit nuclear translocalization is reported for both human synoviocytes and for rat spinal cord nociceptive neurons at light and EM levels ^[28][37][40,45]. Nuclear translocation of GluN1 is reported for neurons in eye tissue ^[38][39][68,69]. Additionally, GluN1 subunit activation has been reported in models of ischemia, neurogenic, and inflammatory responses, which also stimulate EAA release and increase GluN1 subunit expression. Zhou and Duan have reported that both GluN1 and GluN2 are needed to translocate [23]. 3. There is close coordination in neurons between the assembly of functional heteromeric to tetrameric receptors and the fates of these individual subunits. In addition, two pools of mRNA for the GluN1 subunit have been reported with distinct translational activities. These generate two stores of GluN1 subunits that are differentially assembled with GluN2 subunits to form heterodimers with distinct functions and turnover rates, providing an additional and possibly tissue-specific level of control for protein turnover and trafficking [20]. Nuclear membrane translocation reportedly occurs by endocytotic and de novo mechanisms. Activity dependent clathrin mediated internalization of GluN1 subunit is reported ^[26][35]. Nociceptive neurons are overactivated in pain states with increased GluN1 subunit cellular and nuclear ring immunostaining ^[28][40]. Staining is greatly reduced by tyrosine kinase inhibition. Post-transcriptional mechanisms also contribute to GluN1 subunit regulation in brain development ^[40][41][70,71]. Studies have also reported the importance of post-translational histone modifications in epigenetic transcriptional control of nociceptive pathways ^[42][43][32,72]. 4. Structurally, the GluN1 subunit promoter region is located directly upstream of the transcriptional start site (TSS) and is exceptional for the number of transcriptionally reactive binding regions close to the 3’ site. Transcriptionally active binding regions have been reported for SP-1, NFkB, MEF-2, GC-rich regions, CREB, REI, AP-1, egr-1, and ARC binding regions [5]. The GluN1 promoter allows a spectrum of potential responses, such as activation, de-repression, or suppression of downstream transcribable sequences. 5. GluN1 subunit activation is reported to consequently activate Immediate Early Genes (IEG), e.g., c-fos, zif268, and egr-1 ^[44][45][73,74], and conversely, IEG products are reported to activate GluN subunits. The term IEG describes viral regulatory proteins or cellular proteins generated immediately following stimulation of a resting cell by internal or external stimuli, triggering immediate gene transcription that does not require de novo protein synthesis. IEG products are usually transcription factors, which are DNA-binding protein activators of signaling pathways. They are rapidly and transiently activated to respond to a plethora of cellular and extracellular stimuli, serving as an important cellular first response system. The GluN1 subunit can direct downstream nuclear functioning via nuclear DNA binding sites, immediate early gene products, cytoplasmic input, and environmental signals.