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.

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.

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.

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.

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.

Ethanol stimulates the in vivo axonal movement of neuropeptide dense-core vesicles in Drosophila motor neurons.

Proper neuronal function requires essential biological cargoes to be packaged within membranous vesicles and transported, intracellularly, through the extensive outgrowth of axonal and dendritic fibers. The precise spatiotemporal movement of these cargoes is vital for neuronal survival and, thus, is highly regulated. In this study we test how the axonal movement of a neuropeptide-containing dense-core vesicle (DCV) responds to alcohol stressors. We found that ethanol induces a strong anterograde bias in vesicle movement. Low doses of ethanol stimulate the anterograde movement of neuropeptide-DCV while high doses inhibit bi-directional movement. This process required the presence of functional kinesin-1 motors as reduction in kinesin prevented the ethanol-induced stimulation of the anterograde movement of neuropeptide-DCV. Furthermore, expression of inactive glycogen synthase kinase 3 (GSK-3ß) also prevented ethanol-induced stimulation of neuropeptide-DCV movement, similar to pharmacological inhibition of GSK-3ß with lithium. Conversely, inhibition of PI3K/AKT signaling with wortmannin led to a partial prevention of ethanol-stimulated transport of neuropeptide-DCV. Taken together, we conclude that GSK-3ß signaling mediates the stimulatory effects of ethanol. Therefore, our study provides new insight into the physiological response of the axonal movement of neuropeptide-DCV to exogenous stressors. Cover Image for this Issue: doi: 10.1111/jnc.14165.

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.

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.

Ketamine: An Update on Cellular and Subcellular Mechanisms with Implications for Clinical Practice.

BACKGROUND: Ketamine is one of the oldest hypnotic agents used to provide an anesthetic agent with analgesic properties and minimal suppressive effects on respiration. The ability of ketamine in modulating glutamatergic (N-methyl D-aspartate) pain receptors has made this anesthetic drug a new option for the management of patients with chronic pain syndromes. Further preclinical and clinical findings suggest ketamine may have wide ranging effects on both cognition and development. Recent advances have revealed an unprecedented role for ketamine in the acute management of depression. OBJECTIVES: The purpose of this review is to integrate a number of basic science, preclinical, and clinical studies with the goal of providing insight into the possible signaling events underlying ketamine's biological effects in pain management, depression, cognition and memory, and neurodevelopment. STUDY DESIGN: Narrative literature review. SETTING: Health science library. METHODS: A comprehensive literature search was performed for the following medical subject headings and keywords (ketamine, anesthesia, pain, analgesia, depression, NMDA receptors) on PubMed, Google Scholar, and Medline from 1966 to the present time. The search was then limited to those in the English language. The full text of the relevant articles were printed and reviewed by all authors. RESULTS: We provided a comprehensive review of the literature that explored the pharmacologic aspects of ketamine from its conception as an anesthetic to its evolution as a drug used for treatment of depression and pain. To address the patient response variability observed in clinical studies, we have provided possible patient-specific factors that could contribute to outcome variability. LIMITATIONS: Like any review, this study was limited by publication bias and missing information on negative studies which were denied publication. CONCLUSIONS: Ketamine, an old anesthetic agent with analgesic properties, is currently being considered for treating patients with chronic pain and depression. The complex pharmacological characteristics of ketamine make this medication a multifaceted therapeutic option in these cases. Key Words: Ketamine, anesthetics, pain, depression, pharmacology.

prickle modulates microtubule polarity and axonal transport to ameliorate seizures in flies.

Recent analyses in flies, mice, zebrafish, and humans showed that mutations in prickle orthologs result in epileptic phenotypes, although the mechanism responsible for generating the seizures was unknown. Here, we show that Prickle organizes microtubule polarity and affects their growth dynamics in axons of Drosophila neurons, which in turn influences both anterograde and retrograde vesicle transport. We also show that enhancement of the anterograde transport mechanism is the cause of the seizure phenotype in flies, which can be suppressed by reducing the level of either of two Kinesin motor proteins responsible for anterograde vesicle transport. Additionally, we show that seizure-prone prickle mutant flies have electrophysiological defects similar to other fly mutants used to study seizures, and that merely altering the balance of the two adult prickle isoforms in neurons can predispose flies to seizures. These data reveal a previously unidentified pathway in the pathophysiology of seizure disorders and provide evidence for a more generalized cellular mechanism whereby Prickle mediates polarity by influencing microtubule-mediated transport.

Spatial and temporal characteristics of normal and perturbed vesicle transport.

Efficient intracellular transport is essential for healthy cellular function and structural integrity, and problems in this pathway can lead to neuronal cell death and disease. To spatially and temporally evaluate how transport defects are initiated, we adapted a primary neuronal culture system from Drosophila larval brains to visualize the movement dynamics of several cargos/organelles along a 90 micron axonal neurite over time. All six vesicles/organelles imaged showed robust bi-directional motility at both day 1 and day 2. Reduction of motor proteins decreased the movement of vesicles/organelles with increased numbers of neurite blocks. Neuronal growth was also perturbed with reduction of motor proteins. Strikingly, we found that all blockages were not fixed, permanent blocks that impeded transport of vesicles as previously thought, but that some blocks were dynamic clusters of vesicles that resolved over time. Taken together, our findings suggest that non-resolving blocks may likely initiate deleterious pathways leading to death and degeneration, while resolving blocks may be benign. Therefore evaluating the spatial and temporal characteristics of vesicle transport has important implications for our understanding of how transport defects can affect other pathways to initiate death and degeneration.

The impact of physical complaints, social environment, and psychological functioning on IBS patients' health perceptions: looking beyond GI symptom severity.

OBJECTIVES: In the absence of a reliable biomarker, clinical decisions for a functional gastrointestinal (GI) disorder like irritable bowel syndrome (IBS) depend on asking patients to appraise and communicate their health status. Self-ratings of health (SRH) have proven a powerful and consistent predictor of health outcomes, but little is known about how they relate to those relevant to IBS (e.g., quality of life (QOL), IBS symptom severity). This study examined what psychosocial factors, if any, predict SRH among a cohort of more severe IBS patients. METHODS: Subjects included 234 Rome III-positive IBS patients (mean age=41 years, female=78%) without comorbid organic GI disease. Subjects were administered a test battery that included the IBS Symptom Severity Scale, Screening for Somatoform Symptoms, IBS Medical Comorbidity Inventory, SF-12 Vitality Scale, Perceived Stress Scale, Beck Depression Inventory, Trait Anxiety Inventory, and Negative Interactions Scale. RESULTS: Partial correlations identified somatization, depression, fatigue, stress, anxiety, and medical comorbidities as variables with the strongest correlations with SRH (r values=0.36-0.41, P values <0.05). IBS symptom severity was weakly associated with SRH (r=0.18, P<0.05). The final regression model explained 41.3% of the variance in SRH scores (F=8.49, P<0.001) with significant predictors including fatigue, medical comorbidities, somatization, and negative social interactions. CONCLUSIONS: SRH are associated with psychological (anxiety, stress, depression), social (negative interactions), and extraintestinal somatic factors (fatigue, somatization, medical comorbidities). The severity of IBS symptoms appears to have a relatively modest role in how IBS patients describe their health in general.

Presenilin influences glycogen synthase kinase-3 ß (GSK-3ß) for kinesin-1 and dynein function during axonal transport.

Within axons, molecular motors transport essential components required for neuronal growth and viability. Although many levels of control and regulation must exist for proper anterograde and retrograde transport of vital proteins, little is known about these mechanisms. We previously showed that presenilin (PS), a gene involved in Alzheimer's disease (AD), influences kinesin-1 and dynein function in vivo. Here, we show that these PS-mediated effects on motor protein function are via a pathway that involves glycogen synthase kinase-3ß (GSK-3ß). PS genetically interacts with GSK-3ß in an activity-dependent manner. Excess of active GSK-3ß perturbed axonal transport by causing axonal blockages, which were enhanced by reduction of kinesin-1 or dynein. These GSK-3ß-mediated axonal defects do not appear to be caused by disruptions or alterations in microtubules (MTs). Excess of non-functional GSK-3ß did not affect axonal transport. Strikingly, GSK-3ß-activity-dependent axonal transport defects were enhanced by reduction of PS. Collectively, our findings suggest that PS and GSK-3ß are required for normal motor protein function. Our observations propose a model, in which PS likely plays a role in regulating GSK-3ß activity during transport. These findings have important implications for our understanding of the complex regulatory machinery that must exist in vivo and how this system is coordinated during the motility of vesicles within axons.

Organically modified silica nanoparticles are biocompatible and can be targeted to neurons in vivo.

The application of nanotechnology in biological research is beginning to have a major impact leading to the development of new types of tools for human health. One focus of nanobiotechnology is the development of nanoparticle-based formulations for use in drug or gene delivery systems. However most of the nano probes currently in use have varying levels of toxicity in cells or whole organisms and therefore are not suitable for in vivo application or long-term use. Here we test the potential of a novel silica based nanoparticle (organically modified silica, ORMOSIL) in living neurons within a whole organism. We show that feeding ORMOSIL nanoparticles to Drosophila has no effect on viability. ORMOSIL nanoparticles penetrate into living brains, neuronal cell bodies and axonal projections. In the neuronal cell body, nanoparticles are present in the cytoplasm, but not in the nucleus. Strikingly, incorporation of ORMOSIL nanoparticles into the brain did not induce aberrant neuronal death or interfered with normal neuronal processes. Our results in Drosophila indicate that these novel silica based nanoparticles are biocompatible and not toxic to whole organisms, and has potential for the development of long-term applications.