NMDA receptors are tetrameric ligand-gated ion channels comprised of GluN1, GluN2, and GluN3 subunits. Two different GluN2 subunits have been identified in most NMDA receptor-expressing cells, and ...the majority of native receptors are triheteromers containing two GluN1 and two different GluN2. In contrast to diheteromeric NMDA receptors, little is known about the function of triheteromers. We developed a method to provide selective cell-surface expression of recombinant GluN1/GluN2A/GluN2B triheteromers and compared properties of these receptors with those of GluN1/GluN2A and GluN1/GluN2B diheteromers. We show that glutamate deactivation of triheteromers is distinct from those of GluN1/GluN2A and GluN1/GluN2B and reveal modulation of triheteromers by subunit-selective antagonists ifenprodil, CP-101,606, TCN-201, and extracellular Zn2+. Furthermore, kinetic measurements suggest variation in the ifenprodil binding site of triheteromers compared to GluN1/GluN2B diheteromers. This work provides insight into the distinct properties of GluN1/GluN2A/GluN2B triheteromers, which are presumably the most abundant NMDA receptors in the adult forebrain.
•A method to selectively express recombinant GluN1/GluN2A/GluN2B triheteromers•GluN1/GluN2A/GluN2B triheteromers have distinct pharmacological properties•Variation in the ifenprodil binding site of triheteromers compared to GluN1/GluN2B•GluN1/GluN2A/GluN2B triheteromers have distinct glutamate deactivation
Hansen et al. develop a method to study triheteromeric NMDA receptors and provide a comprehensive assessment of properties relevant for synaptic signaling and therapeutic manipulation of the most abundant NMDA receptor subtype in the adult hippocampus and cortex.
NMDA-type glutamate receptors are ligand-gated ion channels that mediate a Ca
-permeable component of excitatory neurotransmission in the central nervous system (CNS). They are expressed throughout ...the CNS and play key physiological roles in synaptic function, such as synaptic plasticity, learning, and memory. NMDA receptors are also implicated in the pathophysiology of several CNS disorders and more recently have been identified as a locus for disease-associated genomic variation. NMDA receptors exist as a diverse array of subtypes formed by variation in assembly of seven subunits (GluN1, GluN2A-D, and GluN3A-B) into tetrameric receptor complexes. These NMDA receptor subtypes show unique structural features that account for their distinct functional and pharmacological properties allowing precise tuning of their physiological roles. Here, we review the relationship between NMDA receptor structure and function with an emphasis on emerging atomic resolution structures, which begin to explain unique features of this receptor.
The mammalian ionotropic glutamate receptor family encodes 18 gene products that coassemble to form ligand-gated ion channels containing an agonist recognition site, a transmembrane ion permeation ...pathway, and gating elements that couple agonist-induced conformational changes to the opening or closing of the permeation pore. Glutamate receptors mediate fast excitatory synaptic transmission in the central nervous system and are localized on neuronal and non-neuronal cells. These receptors regulate a broad spectrum of processes in the brain, spinal cord, retina, and peripheral nervous system. Glutamate receptors are postulated to play important roles in numerous neurological diseases and have attracted intense scrutiny. The description of glutamate receptor structure, including its transmembrane elements, reveals a complex assembly of multiple semiautonomous extracellular domains linked to a pore-forming element with striking resemblance to an inverted potassium channel. In this review we discuss International Union of Basic and Clinical Pharmacology glutamate receptor nomenclature, structure, assembly, accessory subunits, interacting proteins, gene expression and translation, post-translational modifications, agonist and antagonist pharmacology, allosteric modulation, mechanisms of gating and permeation, roles in normal physiological function, as well as the potential therapeutic use of pharmacological agents acting at glutamate receptors.
Key points
Triheteromeric NMDA receptors contain two GluN1 and two distinct GluN2 subunits and mediate excitatory neurotransmission in the CNS.
Triheteromeric GluN1/2B/2D receptors have functional ...properties intermediate to those of diheteromeric GluN1/2B and GluN1/2D receptors.
GluN1/2B/2D receptors are more sensitive to channel blockade by ketamine and memantine compared to GluN1/2B receptors in the presence of physiological Mg2+.
GluN2B‐selective antagonists produce robust inhibition of GluN1/2B/2D receptors, and the GluN2B‐selective positive allosteric modulator spermine enhances responses from GluN1/2B/2D but not GluN1/2A/2B receptors.
These insights into the properties of triheteromeric GluN1/2B/2D receptors are necessary to appreciate their physiological roles in neural circuit function and the actions of therapeutic agents targeting NMDA receptors.
Triheteromeric NMDA‐type glutamate receptors that contain two GluN1 and two different GluN2 subunits contribute to excitatory neurotransmission in the adult CNS. In the present study, we report properties of the triheteromeric GluN1/2B/2D NMDA receptor subtype that is expressed in distinct neuronal populations throughout the CNS. We show that neither GluN2B, nor GluN2D dominate the functional properties of GluN1/2B/2D receptors because agonist potencies, open probability and the glutamate deactivation time course of GluN1/2B/2D receptors are intermediate to those of diheteromeric GluN1/2B and GluN1/2D receptors. Furthermore, channel blockade of GluN1/2B/2D by extracellular Mg2+ is intermediate compared to GluN1/2B and GluN1/2D, although GluN1/2B/2D is more sensitive to blockade by ketamine and memantine compared to GluN1/2B in the presence of physiological Mg2+. Subunit‐selective allosteric modulators have distinct activity at GluN1/2B/2D receptors, including GluN2B‐selective antagonists, ifenprodil, EVT‐101 and CP‐101‐606, which inhibit with similar potencies but with different efficacies at GluN1/2B/2D (∼65% inhibition) compared to GluN1/2B (∼95% inhibition). Furthermore, the GluN2B‐selective positive allosteric modulator spermine enhances responses from GluN1/2B/2D but not GluN1/2A/2B receptors. We show that these key features of allosteric modulation of recombinant GluN1/2B/2D receptors are also observed for NMDA receptors in hippocampal interneurons but not CA1 pyramidal cells, which is consistent with the expression of GluN1/2B/2D receptors in interneurons and GluN1/2A/2B receptors in pyramidal cells. Altogether, we uncover previously unknown functional and pharmacological properties of triheteromeric GluN1/2B/2D receptors that can facilitate advances in our understanding of their physiological roles in neural circuit function and therapeutic drug actions.
Key points
Triheteromeric NMDA receptors contain two GluN1 and two distinct GluN2 subunits and mediate excitatory neurotransmission in the CNS.
Triheteromeric GluN1/2B/2D receptors have functional properties intermediate to those of diheteromeric GluN1/2B and GluN1/2D receptors.
GluN1/2B/2D receptors are more sensitive to channel blockade by ketamine and memantine compared to GluN1/2B receptors in the presence of physiological Mg2+.
GluN2B‐selective antagonists produce robust inhibition of GluN1/2B/2D receptors, and the GluN2B‐selective positive allosteric modulator spermine enhances responses from GluN1/2B/2D but not GluN1/2A/2B receptors.
These insights into the properties of triheteromeric GluN1/2B/2D receptors are necessary to appreciate their physiological roles in neural circuit function and the actions of therapeutic agents targeting NMDA receptors.
NMDA-type glutamate receptors are ligand-gated ion channels that mediate a major component of excitatory neurotransmission in the central nervous system (CNS). They are widely distributed at all ...stages of development and are critically involved in normal brain functions, including neuronal development and synaptic plasticity. NMDA receptors are also implicated in the pathophysiology of numerous neurological and psychiatric disorders, such as ischemic stroke, traumatic brain injury, Alzheimer's disease, epilepsy, mood disorders, and schizophrenia. For these reasons, NMDA receptors have been intensively studied in the past several decades to elucidate their physiological roles and to advance them as therapeutic targets. Seven NMDA receptor subunits exist that assemble into a diverse array of tetrameric receptor complexes, which are differently regulated, have distinct regional and developmental expression, and possess a wide range of functional and pharmacological properties. The diversity in subunit composition creates NMDA receptor subtypes with distinct physiological roles across neuronal cell types and brain regions, and enables precise tuning of synaptic transmission. Here, we will review the relationship between NMDA receptor structure and function, the diversity and significance of NMDA receptor subtypes in the CNS, as well as principles and rules by which NMDA receptors operate in the CNS under normal and pathological conditions.
Many physiologic effects of l-glutamate, the major excitatory neurotransmitter in the mammalian central nervous system, are mediated via signaling by ionotropic glutamate receptors (iGluRs). These ...ligand-gated ion channels are critical to brain function and are centrally implicated in numerous psychiatric and neurologic disorders. There are different classes of iGluRs with a variety of receptor subtypes in each class that play distinct roles in neuronal functions. The diversity in iGluR subtypes, with their unique functional properties and physiologic roles, has motivated a large number of studies. Our understanding of receptor subtypes has advanced considerably since the first iGluR subunit gene was cloned in 1989, and the research focus has expanded to encompass facets of biology that have been recently discovered and to exploit experimental paradigms made possible by technological advances. Here, we review insights from more than 3 decades of iGluR studies with an emphasis on the progress that has occurred in the past decade. We cover structure, function, pharmacology, roles in neurophysiology, and therapeutic implications for all classes of receptors assembled from the subunits encoded by the 18 ionotropic glutamate receptor genes. SIGNIFICANCE STATEMENT: Glutamate receptors play important roles in virtually all aspects of brain function and are either involved in mediating some clinical features of neurological disease or represent a therapeutic target for treatment. Therefore, understanding the structure, function, and pharmacology of this class of receptors will advance our understanding of many aspects of brain function at molecular, cellular, and system levels and provide new opportunities to treat patients.
NMDA receptors mediate excitatory synaptic transmission and regulate synaptic plasticity in the central nervous system, but their dysregulation is also implicated in numerous brain disorders. Here, ...we describe GluN2A-selective negative allosteric modulators (NAMs) that inhibit NMDA receptors by stabilizing the apo state of the GluN1 ligand-binding domain (LBD), which is incapable of triggering channel gating. We describe structural determinants of NAM binding in crystal structures of the GluN1/2A LBD heterodimer, and analyses of NAM-bound LBD structures corresponding to active and inhibited receptor states reveal a molecular switch in the modulatory binding site that mediate the allosteric inhibition. NAM binding causes displacement of a valine in GluN2A and the resulting steric effects can be mitigated by the transition from glycine bound to apo state of the GluN1 LBD. This work provides mechanistic insight to allosteric NMDA receptor inhibition, thereby facilitating the development of novel classes NMDA receptor modulators as therapeutic agents.
•GluN2A-selective NMDA receptor antagonists negatively modulate glycine binding•Crystal structures show the modulatory binding site in the GluN1-GluN2A interface•Modulator binding stabilizes the apo state of the GluN1 ligand binding domain•A molecular switch in the modulatory binding site mediates allosteric inhibition
NMDA receptors are ionotropic glutamate receptors that mediate excitatory neurotransmission. Yi at el. show the molecular mechanism for subunit-selective negative allosteric modulators of NMDA receptors that bind the subunit interface between GluN1 and GluN2A ligand-binding domains.
During development of the central nervous system, there is a shift in the subunit composition of NMDA receptors (NMDARs) resulting in a dramatic acceleration of NMDAR-mediated synaptic currents. This ...shift coincides with upregulation of the GluN2A subunit and triheteromeric GluN1/2A/2B receptors with fast deactivation kinetics, whereas expression of diheteromeric GluN1/2B receptors with slower deactivation kinetics is decreased. Here, we show that allosteric interactions occur between the glutamate-binding GluN2 subunits in triheteromeric GluN1/2A/2B NMDARs. This allosterism is dominated by the GluN2A subunit and results in functional properties not predicted by those of diheteromeric GluN1/2A and GluN1/2B NMDARs. These findings suggest that GluN1/2A/2B NMDARs may maintain some signaling properties of the GluN2B subunit while having the kinetic properties of GluN1/2A NMDARs and highlight the complexity in NMDAR signaling created by diversity in subunit composition.
•Allosterism occurs between GluN2 subunits in triheteromeric GluN1/2A/2B NMDARs•These allosteric interactions are asymmetric and dominated by the GluN2A subunit•GluN2A-dominant interactions span multiple domains in the NMDAR•The allosteric interactions endow GluN1/2A/2B with the function of GluN1/2A NMDARs
Sun et al. demonstrate asymmetric inter-GluN2 allosteric interactions within triheteromeric GluN1/2A/2B NMDARs that result in open probability and deactivation kinetics similar to diheteromeric GluN1/2A receptors. This finding highlights the complexity in NMDAR signaling endowed by diversity in subunit composition.
NMDA receptors comprised of different NR2 subunits exhibit strikingly unique biophysical and pharmacological properties. Here, we report that the extracellular amino-terminal domain (ATD) of the NR2 ...subunit controls pharmacological and kinetic properties of recombinant NMDA receptors, such as agonist potency, deactivation time course, open probability (P(OPEN)), and mean open/shut duration. Using ATD deletion mutants of NR2A, NR2B, NR2C, NR2D, and chimeras of NR2A and NR2D with interchanged ATD NR2A-(2D-ATD) and NR2D-(2A-ATD), we show that the ATD contributes to the low glutamate potency of NR2A-containing NMDA receptors and the high glutamate potency of NR2D-containing receptors. The ATD influences the deactivation time courses of NMDA receptors, as removal of the ATD from NR2A slows the deactivation rate, while removal of the ATD from NR2B, NR2C and NR2D accelerates the deactivation rate. Open probability also is influenced by the ATD. Removal of the ATD from NR2A or replacement of the NR2A-ATD with that of NR2D decreases P(OPEN) in single-channel recordings from outside-out patches of HEK 293 cells. In contrast, deletion of the ATD from NR2D or replacement of the NR2D ATD with that of NR2A increases P(OPEN) and mean open duration. These data demonstrate the modular nature of NMDA receptors, and show that the ATD of the different NR2 subunits plays an important role in fine-tuning the functional properties of the individual NMDA receptor subtypes.
Aim
Natriuretic peptides, BNP and ANP increase renal blood flow in experimental animals. The signalling pathway in human kidney vasculature is unknown. It was hypothesized that BNP and ANP cause ...endothelium‐independent relaxation of human intrarenal arteries by vascular natriuretic peptide receptor‐A, but not ‐B and ‐C, which is mimicked by agonists of soluble guanylyl cyclase sGC.
Methods
Human (n = 54, diameter: 665 ± 29 µm 95% CI) and control murine intrarenal arteries (n = 83, diameter 300 ± 6 µm 95% CI) were dissected and used for force recording by four‐channel wire myography. Arterial segments were pre‐contracted, then subjected to increasing concentrations of BNP, ANP, phosphodiesterase 5‐inhibitor sildenafil, sGC‐activator BAY 60‐2770 and ‐stimulator BAY 41‐2272. Endothelial nitric oxide synthase (eNOS) dependence was examined by use of L‐NAME and eNOS knockout respectively. Molecular targets (NPR A‐C, sGC, phosphodiesterase‐5 and neprilysin) were mapped by PCR, immunohistochemistry and RNAscope.
Results
BNP, ANP, sildenafil, sGC‐activation and ‐stimulation caused concentration‐dependent relaxation of human and murine intrarenal arteries. BNP responses were independent of eNOS and were not potentiated by low concentration of phosphodiesterase‐5‐inhibitor, sGC‐stimulator or NPR‐C blocker. PCR showed NPR‐A and C, phosphodiesterase‐5, neprilysin and sGC mRNA in renal arteries. NPR‐A mRNA and protein was observed in vascular smooth muscle and endothelial cells in arteries, podocytes, Bowmans capsule and vasa recta. NPR‐C was observed in tubules, glomeruli and vasculature.
Conclusion
Activation of transmembrane NPR‐A and soluble guanylyl cyclase relax human preglomerular arteries similarly to phosphodiestase‐5 inhibition. The human renal arterial bed relaxes in response to cGMP pathway.
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