Estimating the Ca2+ Block of NMDA Receptors with Single-Channel Electrophysiology.

In vertebrate central neurons, NMDA receptors are glutamate- and glycine-gated ion channels that allow the passage of Na+ and Ca2+ ions into the cell when these neurotransmitters are simultaneously present. The passage of Ca2+ is critical for initiating the cellular processes underlying various forms of synaptic plasticity. These Ca2+ ions can autoregulate the NMDA receptor signal through multiple distinct mechanisms to reduce the total flux of cations. One such mechanism is the ability of Ca2+ ions to exclude the passage of Na+ ions resulting in a reduced unitary current conductance. In contrast to the well-characterized Mg2+ block, this "channel block" mechanism is voltage-independent. In this chapter, we discuss theoretical and experimental considerations for the study of channel block by Ca2+ using single-channel patch-clamp electrophysiology. We focus on two classic methodologies to quantify the dependence of unitary channel conductance on external concentrations of Ca2+ as the basis for quantifying Ca2+ block.

Estimating the Ca2+ Permeability of NMDA Receptors with Whole-Cell Patch-Clamp Electrophysiology.

In the mammalian central nervous system (CNS), fast excitatory transmission relies primarily on the ionic fluxes generated by ionotropic glutamate receptors (iGluRs). Among iGluRs, NMDA receptors (NMDARs) are unique in their ability to pass large, Ca2+-rich currents. Importantly, their high Ca2+ permeability is essential for normal CNS function and is under physiological control. For this reason, the accurate measurement of NMDA receptor Ca2+ permeability represents a valuable experimental step in evaluating the mechanism by which these receptors contribute to a variety of physiological and pathological conditions. In this chapter, we provide a theoretical and practical overview of the common methods used to estimate the Ca2+ permeability of ion channels as they apply to NMDA receptors. Specifically, we describe the principles and methodology used to calculate relative permeability (PCa/PNa) and fractional permeability (Pf), along with the relationship between these two metrics. With increasing knowledge about the structural dynamics of ion channels and of the ongoing environmental fluctuations in which channels operate in vivo, the ability to quantify the Ca2+ entering cells through specific ion channels remains a tool essential to delineating the molecular mechanisms that support health and cause disease.

Calcium- and calmodulin-dependent inhibition of NMDA receptor currents.

Calcium ions (Ca2+) reduce NMDA receptor currents through several distinct mechanisms. Among these, calmodulin (CaM)-dependent inhibition (CDI) accomplishes rapid, reversible, and incomplete reduction of the NMDA receptor currents in response to elevations in intracellular Ca2+. Quantitative and mechanistic descriptions of CDI of NMDA receptor-mediated signals have been marred by variability originating, in part, from differences in the conditions and metrics used to evaluate this process across laboratories. Recent ratiometric approaches to measure the magnitude and kinetics of NMDA receptor CDI have facilitated rapid insights into this phenomenon. Notably, the kinetics and magnitude of NMDA receptor CDI depend on the degree of saturation of its CaM binding sites, which represent the bona fide calcium sensor for this type of inhibition, the kinetics and magnitude of the Ca2+ signal, which depends on the biophysical properties of the NMDA receptor or of adjacent Ca2+ sources, and on the relative distribution of Ca2+ sources and CaM molecules. Given that all these factors vary widely during development, across cell types, and with physiological and pathological states, it is important to understand how NMDA receptor CDI develops and how it contributes to signaling in the central nervous system. Here, we review briefly these recent advances and highlight remaining questions about the structural and kinetic mechanisms of NMDA receptor CDI. Given that pathologies can arise from several sources, including mutations in the NMDA receptor and in CaM, understanding how CaM responds to intracellular Ca2+ signals to initiate conformational changes in NMDA receptors, and mapping the structural domains responsible will help to envision novel therapeutic strategies to neuropsychiatric diseases, which presently have limited available treatments.

GluD receptors are functional ion channels.

In this article, we review contemporary evidence that GluD receptors are functional ion channels whose depolarizing currents contribute to their biological functions, akin to all other members of the ionotropic glutamate receptor (iGluR) family.

Complex functional phenotypes of NMDA receptor disease variants.

NMDA receptors have essential roles in the physiology of central excitatory synapses and their dysfunction causes severe neuropsychiatric symptoms. Recently, a series of genetic variants have been identified in patients, however, functional information about these variants is sparse and their role in pathogenesis insufficiently known. Here we investigate the mechanism by which two GluN2A variants may be pathogenic. We use molecular dynamics simulation and single-molecule electrophysiology to examine the contribution of GluN2A subunit-residues, P552 and F652, and their pathogenic substitutions, P552R and F652V, affect receptor functions. We found that P552 and F652 interact during the receptors' normal activity cycle; the interaction stabilizes receptors in open conformations and is required for a normal electrical response. Engineering shorter side-chains at these positions (P552A and/or F652V) caused a loss of interaction energy and produced receptors with severe gating, conductance, and permeability deficits. In contrast, the P552R side chain resulted in stronger interaction and produced a distinct, yet still drastically abnormal electrical response. These results identify the dynamic contact between P552 and F652 as a critical step in the NMDA receptor activation, and show that both increased and reduced communication through this interaction cause dysfunction. Results show that subtle differences in NMDA receptor primary structure can generate complex phenotypic alterations whose binary classification is too simplistic to serve as a therapeutic guide.

Membrane Stretch Gates NMDA Receptors.

NMDARs are ionotropic glutamate receptors widely expressed in the CNS, where they mediate phenomena as diverse as neurotransmission, information processing, synaptogenesis, and cellular toxicity. They function as glutamate-gated Ca2+-permeable channels, which require glycine as coagonist, and can be modulated by many diffusible ligands and cellular cues, including mechanical stimuli. Previously, we found that, in cultured astrocytes, shear stress initiates NMDAR-mediated Ca2+ entry in the absence of added agonists, suggesting that more than being mechanosensitive, NMDARs may be mechanically activated. Here, we used controlled expression of rat recombinant receptors and noninvasive on-cell single-channel current recordings to show that mild membrane stretch can substitute for the neurotransmitter glutamate in gating NMDAR currents. Notably, stretch-activated currents maintained the hallmark features of the glutamate-gated currents, including glycine-requirement, large unitary conductance, high Ca2+ permeability, and voltage-dependent Mg2+ blockade. Further, we found that the stretch-gated current required the receptor's intracellular domain. Our results are consistent with the hypothesis that mechanical forces can gate endogenous NMDAR currents even in the absence of synaptic glutamate release, which has important implications for understanding mechanotransduction and the physiological and pathologic effects of mechanical forces on cells of the CNS.SIGNIFICANCE STATEMENT We show that, in addition to enhancing currents elicited with low agonist concentrations, membrane stretch can gate NMDARs in the absence of the neurotransmitter glutamate. Stretch-gated currents have the principal hallmarks of the glutamate-gated currents, including requirement for glycine, large Na+ conductance, high Ca2+ permeability, and voltage-dependent Mg2+ block. Therefore, results suggest that mechanical forces can initiate cellular processes presently attributed to glutamatergic neurotransmission, such as synaptic plasticity and cytotoxicity. Given the ubiquitous presence of mechanical forces in the CNS, this discovery identifies NMDARs as possibly important mechanotransducers during development and across the lifespan, and during pathologic processes, such as those associated with traumatic brain injuries, shaken infant syndrome, and chronic traumatic encephalopathy.

Homecoming of the estranged GluD channels.

A recent paper by Carrillo and colleagues demonstrates that GluD proteins form bona fide ligand-gated ion channels when their intrinsic flexibility is constrained by interactions with protein partners. Therefore, Delta receptors resemble all other members of the ionotropic glutamate receptor family not only by sequence and structural homology, but also by functional dynamics.

Cross-subunit interactions that stabilize open states mediate gating in NMDA receptors.

NMDA receptors are excitatory channels with critical functions in the physiology of central synapses. Their activation reaction proceeds as a series of kinetically distinguishable, reversible steps, whose structural bases are currently under investigation. Very likely, the earliest steps include glutamate binding to glycine-bound receptors and subsequent constriction of the ligand-binding domain. Later, three short linkers transduce this movement to open the gate by mechanical pulling on transmembrane helices. Here, we used molecular and kinetic simulations and double-mutant cycle analyses to show that a direct chemical interaction between GluN1-I642 (on M3 helix) and GluN2A-L550 (on L1-M1 linker) stabilizes receptors after they have opened and thus represents one of the structural changes that occur late in the activation reaction. This native interaction extends the current decay, and its absence causes deficits in charge transfer by GluN1-I642L, a pathogenic human variant.

Hydroxynorketamine Blocks N-Methyl-d-Aspartate Receptor Currents by Binding to Closed Receptors.

Ketamine, a dissociative anesthetic, is experiencing a clinical resurgence as a fast-acting antidepressant. In the central nervous system, ketamine acts primarily by blocking NMDA receptor currents. Although it is generally safe in a clinical setting, it can be addictive, and several of its derivatives are being investigated as preferable alternatives. 2R,6R-Hydroxynorketamine (HNK), a ketamine metabolite, reproduces some of the therapeutic effects of ketamine and appears to lack abuse liability. Here, we report a systematic investigation of the effects of HNK on macroscopic responses elicited from recombinant NMDA receptors expressed in human embryonic kidney 293 cells. We found that, like ketamine, HNK reduced NMDA receptor currents in a dose-, pH-, and voltage-dependent manner. Relative to ketamine, it had 100-fold-lower potency (46 µM at pH 7.2), 10-fold-slower inhibition onset, slower apparent dissociation rate, weaker voltage dependence, and complete competition by magnesium. Notably, HNK inhibition was fully effective when applied to resting receptors. These results revealed unexpected properties of hydroxynorketamine that warrant its further investigation as a possible therapeutic in pathologies associated with NMDA receptor dysfunction. SIGNIFICANCE STATEMENT: NMDA receptors are excitatory ion channels with fundamental roles in synaptic transmission and plasticity, and their dysfunction associates with severe neuropsychiatric disorders. 2R,6R-Hydroxynorketamine, a metabolite of ketamine, mimics some of the neuroactive properties of ketamine and may lack its abuse liability. Results show that 2R,6R-hydroxynorketamine blocks NMDA receptor currents with low affinity and weak voltage dependence and is effective when applied to resting receptors. These properties highlight its effectiveness to a subset of NMDA receptor responses and recommend it for further investigation.

Ca2+-Dependent Inactivation of GluN2A and GluN2B NMDA Receptors Occurs by a Common Kinetic Mechanism.

N-Methyl-d-aspartate (NMDA) receptors are Ca2+-permeable channels gated by glutamate and glycine that are essential for central excitatory transmission. Ca2+-dependent inactivation (CDI) is a regulatory feedback mechanism that reduces GluN2A-type NMDA receptor responses in an activity-dependent manner. Although CDI is mediated by calmodulin binding to the constitutive GluN1 subunit, prior studies suggest that GluN2B-type receptors are insensitive to CDI. We examined the mechanism of CDI subtype dependence using electrophysiological recordings of recombinant NMDA receptors expressed in HEK-293 cells. In physiological external Ca2+, we observed robust CDI of whole-cell GluN2A currents (0.42 ± 0.05) but no CDI in GluN2B currents (0.08 ± 0.07). In contrast, when Ca2+ was supplied intracellularly, robust CDI occurred for both GluN2A and GluN2B currents (0.75 ± 0.03 and 0.67 ± 0.02, respectively). To examine how the source of Ca2+ affects CDI, we recorded one-channel Na+ currents to quantify the receptor gating mechanism while simultaneously monitoring ionomycin-induced intracellular Ca2+ elevations with fluorometry. We found that CDI of both GluN2A and GluN2B receptors reflects receptor accumulation in long-lived closed (desensitized) states, suggesting that the observed subtype-dependent differences in macroscopic CDI reflect intrinsic differences in equilibrium open probabilities (Po). We tested this hypothesis by measuring substantial macroscopic CDI, in physiologic conditions, for high Po GluN2B receptors (GluN1A652Y/GluN2B). Together, these results show that Ca2+ flux produces activity-dependent inactivation for both GluN2A and GluN2B receptors and that the extent of CDI varies with channel Po. These results are consistent with CDI as an autoinhibitory feedback mechanism against excessive Ca2+ load during high Po activation.

Spatial Coupling Tunes NMDA Receptor Responses via Ca2+ Diffusion.

In the CNS, NMDA receptors generate large and highly regulated Ca2+ signals, which are critical for synaptic development and plasticity. They are highly clustered at postsynaptic sites and along dendritic arbors, but whether this spatial arrangement affects their output is unknown. Synaptic NMDA receptor currents are subject to Ca2+-dependent inactivation (CDI), a type of activity-dependent inhibition that requires intracellular Ca2+ and calmodulin (CaM). We asked whether Ca2+ influx through a single NMDA receptor influences the activity of nearby NMDA receptors, as a possible coupling mechanism. Using cell-attached unitary current recordings from GluN1-2a/GluN2A receptors expressed in human HEK293 cells and from NMDA receptors native to hippocampal neurons from male and female rats, we recorded unitary currents from multichannel patches and used a coupled Markov model to determine the extent of signal coupling (κ). In the absence of extracellular Ca2+, we observed no cooperativity (κ < 0.1), whereas in 1.8 mm external Ca2+, both recombinant and native channels showed substantial negative cooperativity (κ = 0.27). Intracellular Ca2+ chelation or overexpression of a Ca2+-insensitive CaM mutant, reduced coupling, which is consistent with CDI representing the coupling mechanism. In contrast, cooperativity increased substantially (κ = 0.68) when overexpressing the postsynaptic scaffolding protein PSD-95, which increased receptor clustering. Together, these new results demonstrate that NMDA receptor currents are negatively coupled through CDI, and the degree of coupling can be tuned by the distance between receptors. Therefore, channel clustering can influence the activity-dependent reduction in NMDA receptor currents.SIGNIFICANCE STATEMENT At central synapses, NMDA receptors are a major class of excitatory glutamate-gated channels and a source of activity-dependent Ca2+ influx. In turn, fluxed Ca2+ ions bind to calmodulin-primed receptors and reduce further entry, through an autoinhibitory mechanism known as Ca2+ -dependent inactivation (CDI). Here, we show that the diffusion of fluxed Ca2+ between active channels situated within submicroscopic distances amplified receptor inactivation. Thus, calmodulin-mediated gating modulation, an evolutionarily conserved regulatory mechanism, endows synapses with sensitivity to both the temporal sequence and spatial distribution of Ca2+ signals. Perturbations in this mechanism, which coordinates the activity of NMDA receptors within a cluster, may cause signaling alterations that contribute to neuropsychiatric conditions.

Kinetic models for activation and modulation of NMDA receptor subtypes.

NMDA receptors are a diverse family of excitatory channels with critical roles in central synaptic transmission, development, and plasticity. Controlled expression of seven subunits and their combinatorial assembly into tetrameric receptors produces a range of molecularly distinct receptor subtypes. Despite relatively similar atomic structures, each subtype has input-output functions with unique biophysical and pharmacologic profiles. Here, we briefly summarize recent advances in understanding how gating and allosteric modulation are similar or distinct across NMDA receptor isoforms and identify open questions that will focus research in this area going forward.

Resident Calmodulin Primes NMDA Receptors for Ca2+-Dependent Inactivation.

N-methyl-d-aspartate (NMDA) receptors are glutamate- and glycine-gated channels that flux Na+ and Ca2+ into postsynaptic neurons during synaptic transmission. The resulting intracellular Ca2+ transient is essential to physiological and pathological processes related to synaptic development, plasticity, and apoptosis. It also engages calmodulin (CaM) to reduce subsequent NMDA receptor activity in a process known as Ca2+-dependent inactivation (CDI). Here, we used whole-cell electrophysiology to measure CDI and computational modeling to dissect the sequence of events that underlies it. With these approaches, we estimate that CaM senses NMDA receptor Ca2+ influx at ∼9 nm from the channel pore. Further, when we controlled the frequency of Ca2+ influx through individual channels, we found that a kinetic model where apoCaM associates with channels before their activation best predicts the measured CDI. These results provide, to our knowledge, novel functional evidence for CaM preassociation to NMDA receptors in living cells. This particular mechanism for autoinhibitory feedback reveals strategies and challenges for Ca2+ regulation in neurons during physiological synaptic activity and disease.

Probing the Structural Dynamics of the NMDA Receptor Activation by Coarse-Grained Modeling.

N-Methyl-D-aspartate (NMDA) receptors are glutamate-gated excitatory channels that play essential roles in brain functions. High-resolution structures have been solved for an allosterically inhibited and agonist-bound form of a functional NMDA receptor; however, other key functional states (particularly the active open-channel state) were only resolved at moderate resolutions by cryo-electron microscopy (cryo-EM). To decrypt the mechanism of the NMDA receptor activation, structural modeling is essential to provide presently missing information about structural dynamics. We performed systematic coarse-grained modeling using an elastic network model and related modeling/analysis tools (e.g., normal mode analysis, flexibility and hotspot analysis, cryo-EM flexible fitting, and transition pathway modeling) based on an active-state cryo-EM map. We observed extensive conformational changes that allosterically couple the extracellular regulatory and agonist-binding domains to the pore-forming trans-membrane domain (TMD), and validated these, to our knowledge, new observations against known mutational and functional studies. Our results predict two key modes of collective motions featuring shearing/twisting of the extracellular domains relative to the TMD, reveal subunit-specific flexibility profiles, and identify functional hotspot residues at key domain-domain interfaces. Finally, by examining the conformational transition pathway between the allosterically inhibited form and the active form, we predict a discrete sequence of domain motions, which propagate from the extracellular domains to the TMD. In summary, our results offer rich structural and dynamic information, which is consistent with the literature on structure-function relationships in NMDA receptors, and will guide in-depth studies on the activation dynamics of this important neurotransmitter receptor.

Residues in the GluN1 C-terminal domain control kinetics and pharmacology of GluN1/GluN3A N-methyl-d-aspartate receptors.

N-methyl-d-aspartate (NMDA) receptors assembled from GluN1 and GluN3 subunits are unique in that they form glycine-gated excitatory channels that are insensitive to glutamate and NMDA. Alternative splicing of the GluN1 subunit mRNA results in eight variants with regulated expression patterns and post-translational modifications. Here we investigate the role of residues in the GluN1 C-terminal alternatively spliced cassettes in receptor gating and modulation. We measured whole-cell currents from recombinant GluN1/GluN3A receptors expressed in HEK293 cells that differed in the sequence of their GluN1 C-terminal tail. We found that these residues controlled the level of steady-state activity and the degree to which activity was facilitated by zinc and protons. Further, we found that the phosphorylation status of sites specific to certain variants can also modulate channel activity. Based on these results we suggest that GluN1 C-terminal domain splicing may confer cell-specific and activity-dependent regulation onto the level and pharmacologic sensitivity of GluN1/GluN3A currents.

NMDA receptors: linking physiological output to biophysical operation.

NMDA receptors are preeminent neurotransmitter-gated channels in the CNS, which respond to glutamate in a manner that integrates multiple external and internal cues. They belong to the ionotropic glutamate receptor family and fulfil unique and crucial roles in neuronal development and function. These roles depend on characteristic response kinetics, which reflect the operation of the receptors. Here, we review biologically salient features of the NMDA receptor signal and its mechanistic origins. Knowledge of distinctive NMDA receptor biophysical properties, their structural determinants and physiological roles is necessary to understand the physiological and neurotoxic actions of glutamate and to design effective therapeutics.

Reprogramming Postnatal Human Epidermal Keratinocytes Toward Functional Neural Crest Fates.

During development, neural crest (NC) cells are induced by signaling events at the neural plate border of all vertebrate embryos. Initially arising within the central nervous system, NC cells subsequently undergo an epithelial to mesenchymal transition to migrate into the periphery, where they differentiate into diverse cell types. Here we provide evidence that postnatal human epidermal keratinocytes (KC), in response to fibroblast growth factor 2 and insulin like growth factor 1 signals, can be reprogrammed toward a NC fate. Genome-wide transcriptome analyses show that keratinocyte-derived NC cells are similar to those derived from human embryonic stem cells. Moreover, they give rise in vitro and in vivo to NC derivatives such as peripheral neurons, melanocytes, Schwann cells and mesenchymal cells (osteocytes, chondrocytes, adipocytes, and smooth muscle cells). By demonstrating that human keratin-14+ KC can form NC cells, even from clones of single cells, our results have important implications in stem cell biology and regenerative medicine. Stem Cells 2017;35:1402-1415.

Mechanical stress activates NMDA receptors in the absence of agonists.

While studying the physiological response of primary rat astrocytes to fluid shear stress in a model of traumatic brain injury (TBI), we found that shear stress induced Ca2+ entry. The influx was inhibited by MK-801, a specific pore blocker of N-Methyl-D-aspartic acid receptor (NMDAR) channels, and this occurred in the absence of agonists. Other NMDA open channel blockers ketamine and memantine showed a similar effect. The competitive glutamate antagonists AP5 and GluN2B-selective inhibitor ifenprodil reduced NMDA-activated currents, but had no effect on the mechanically induced Ca2+ influx. Extracellular Mg2+ at 2 mM did not significantly affect the shear induced Ca2+ influx, but at 10 mM it produced significant inhibition. Patch clamp experiments showed mechanical activation of NMDAR and inhibition by MK-801. The mechanical sensitivity of NMDARs may play a role in the normal physiology of fluid flow in the glymphatic system and it has obvious relevance to TBI.

Protons Potentiate GluN1/GluN3A Currents by Attenuating Their Desensitisation.

N-methyl-D-aspartate (NMDA) receptors are glutamate- and glycine-gated channels composed of two GluN1 and two GluN2 or/and GluN3 subunits. GluN3A expression is developmentally regulated, and changes in this normal pattern of expression, which occur in several brain disorders, alter synaptic maturation and function by unknown mechanisms. Uniquely within the NMDA receptor family, GluN1/GluN3 receptors produce glycine-gated deeply desensitising currents that are insensitive to glutamate and NMDA; these currents remain poorly characterised and their cellular functions are unknown. Here, we show that extracellular acidification strongly potentiated glycine-gated currents from recombinant GluN1/GluN3A receptors, with half-maximal effect in the physiologic pH range. This was largely due to slower current desensitisation and faster current recovery from desensitisation, and was mediated by residues facing the heterodimer interface of the ligand-binding domain. Consistent with the observed changes in desensitisation kinetics, acidic shifts increased the GluN1/GluN3A equilibrium current and depolarized the membrane in a glycine concentration-dependent manner. These results reveal novel modulatory mechanisms for GluN1/GluN3A receptors that further differentiate them from the canonical glutamatergic GluN1/GluN2 receptors and provide a new and potent pharmacologic tool to assist the detection, identification, and the further study of GluN1/GluN3A currents in native preparations.