Receptor Channel Activity Research Articles

Whole genome methylation sequencing identifies sex‐specific DNA methylation levels in late onset dementia due to Alzheimer’s disease

AbstractBackgroundLate onset dementia due to Alzheimer’s disease (AD) has a sex‐biased incidence with females comprising nearly two thirds of all cases. Females have a more rapid progression in cognitive decline and higher levels of known AD biomarker pathology compared to men. Genetic sequence variation does not account for the sex‐biased incidence of AD, directing attention to the emerging role of epigenetics in AD. Using whole genome methylation sequencing (WGMS), we previously reported 28,038 differentially methylated position (DMPs) within 2,707 genes between persons with and without AD. Here we report sex‐specific DNA methylation levels in AD.MethodWGMS data quantified DNA methylation levels at 25,406,945 CpG loci in 281 blood samples from 109 AD (47 female, 62 male) and 174 cognitively unimpaired (CU) individuals (90 female, 84 male) in the Wisconsin Alzheimer’s Disease Research Center and the Wisconsin Registry for Alzheimer’s Prevention cohorts. Beta‐binomial regression models were developed to test for significant sex‐specific DNA methylation levels between CU and AD accounting for batch effects, estimated white blood cell proportions, BMI, and age. P‐values were corrected and local false discovery rates (lFDRs) were calculated.Result13,293 DMPs were identified in females and 20,719 DMPs were identified in males (Figure 1) when comparing CU and AD (lFDR < 0.05; mean methylation difference > 2.5%). Approximately 2% (280) of the DMPs overlap between sexes with 87 having opposite mean methylation differences. The majority of DMPs annotate to introns (55.9% females; 54.8% males) with most of the remainder intergenic (37.0% females; 37.9% males). Annotation of DMPs to genes identified 1,498 genes that are only differentially methylated in females, and 2,183 genes that are only differentially methylated in males, with 1,365 differentially methylated genes shared between females and males (Figure 2). Gene ontological analyses identified significant enrichment of pathways related to glutamate receptor and ion channel activity in shared gene sets (Figure 3).Conclusion33,732 unique, sex‐specific DMPs in AD were identified with more than 60% annotated to one of 5,067 genes. DMP‐associated genes have sex‐specific DNA methylation levels suggesting that these DMPs may play a role in the biased pathogenesis of AD.

Decrease of KATP channel expression through D3 receptor-mediated GSK3β signaling alleviates levodopa-induced dyskinesia (LID) in Parkinson's disease mouse model

AimsThe standard Parkinson's disease (PD) treatment is L-3,4-dihydroxyphenylalanine (L-DOPA); however, its long-term use may cause L-DOPA-induced dyskinesia (LID). Aberrant activation of medium spiny neurons (MSNs) contributes to LID, and MSN excitability is regulated by dopamine D3 receptor (D3R) and ATP-sensitive potassium (KATP) channel activity. Nevertheless, it remains unclear if D3R and KATP channels may be linked in the context of LID. MethodsWild-type and tyrosine hydroxylase (TH)-specific Kir6.2 knockout mice were injected with 6-hydroxydopamine (6-OHDA) to generate a PD mouse model, then chronically treated with L-DOPA to induce LID. Analyses included immunohistochemical staining, biochemical endpoints, and behavior tests. The mechanisms by which D3R/KATP channels regulate LID in the PD/LID mouse model were probed by treatment with a D3R antagonist, KATP channel opener and glycogen synthase kinase 3β (GSK3β) inhibitor, followed by evaluation of abnormal involuntary movements (AIMs). Key findingsThe D3R antagonist FAUC365 alleviated LID, reducing AIMs and protecting against degeneration of the nigrostriatal pathway, which occurred through a direct interaction between D3Rs and KATP channels. In line with this mechanism, activation of D3R/GSK3β signaling increased KATP channel expression in the striatum of PD/LID mice. Additionally, the KATP channel opener Diz slowed LID progression and preserved nigrostriatal projections. Consistently, mice with TH-specific knockout of Kir6.2 exhibited reduced PD-like symptoms and less severe LID. SignificanceD3Rs act through GSK3β signaling to regulate expression of KATP channels, which may subsequently modulate LID. Inhibition of KATP channels in TH-positive cells is sufficient to reduce AIMs in a mouse model of PD/LID.

Neurosteroid Binding and Actions on GABAA Receptors

Neurosteroids positively modulate GABAA receptor (GABAAR) channel activity by binding to a transmembrane domain intersubunit site. Using photo-affinity labeling and an ELIC-α1GABAAR chimera, we investigated the impact of mutations within the intersubunit site on neurosteroid binding. These mutations reduce neither photolabeling within the intersubunit site nor competitive prevention of labeling by allopregnanolone. Instead, these mutations change the orientation of neurosteroid photolabeling. The data indicate that mutations at Gln242 or Trp246 that eliminate neurosteroid effects do not eliminate neurosteroid binding within the intersubunit site, but significantly alter the preferred orientation of the neurosteroid within the site. The interactions formed by Gln242 and Trp246 within this pocket play a vital role in determining the orientation of the neurosteroid. We also examined how site-specific binding to three identified neurosteroid-binding sites in the α1β3GABAAR contributes to neurosteroid allosteric modulation. We found that the potentiating neurosteroid, allopregnanolone, but not its inhibitory 3β-epimer epi-allopregnanolone, binds to the canonical β3(+)-α1(-) intersubunit site that mediates receptor activation by neurosteroids. In contrast, both allopregnanolone and epi-allopregnanolone bind to intrasubunit sites in the β3 subunit, promoting receptor desensitization and the α1 subunit promoting effects that vary between neurosteroids. Two neurosteroid analogues with diazirine moieties replacing the 3-hydroxyl bind to all three sites, but do not potentiate GABAAR currents. One is a desensitizing agent, whereas the other is devoid of allosteric activity. Collectively, these data show that differential occupancy and efficacy at three discrete neurosteroid-binding sites determine whether a neurosteroid has potentiating, inhibitory, or competitive antagonist activity on GABAAR.

FMRP regulates GABAA receptor channel activity to control signal integration in hippocampal granule cells.

Fragile X syndrome, the most common inherited form of intellectual disability, is caused by loss of fragile X mental retardation protein (FMRP). GABAergic system dysfunction is one of the hallmarks of FXS, yet the underlying mechanisms remain poorly understood. Here, we report that FMRP interacts with GABAA receptor (GABAAR) and modulates its single-channel activity. Specifically, FMRP regulates spontaneous GABAAR opening through modulating its single-channel conductance and open probability in dentate granule cells. FMRP loss reduces spontaneous GABAAR activity underlying tonic inhibition, while N-terminal FMRP fragment (aa 1-297) is sufficient to rapidly normalize tonic inhibition in Fmr1 knockout (KO) granule cells. FMRP-GABAAR interaction is supported by co-immunoprecipitation of FMRP with at least one GABAAR subunit, the α5. Functionally, FMRP-GABAAR interaction ensures accuracy of coincidence detection of granule cells, which is markedly reduced in Fmr1 KOs. Our study reveals a mechanism underlying FMRP regulation of the GABAergic system and information processing in the hippocampus.

Nicotine-induced C-shape movements in planarians are reduced by antinociceptive drugs: Implications for pain in planarian paroxysm etiology?

C-shapes are stereotyped movements in planarians that are elicited by diverse stimuli (e.g. acidity, excitatory neurotransmitters, psychostimulants, and pro-convulsants). Muscle contraction and seizure contribute to the expression of C-shape movements, but a causative role for pain is understudied and unclear. Here, using nicotine-induced C-shapes as the endpoint, we tested the efficacy of three classes of antinociceptive compounds – an opioid, NSAID (non-steroidal anti-inflammatory drug), and transient receptor potential ankyrin 1 (TRPA1) channel antagonist. For comparison we also tested effects of a neuromuscular blocker. Nicotine (0.1–10 mM) concentration-dependently increased C-shapes. DAMGO (1–10 µM), a selective µ-opioid agonist, inhibited nicotine (5 mM)-induced C-shapes. Naloxone (0.1–10 µM), an opioid receptor antagonist, prevented the DAMGO (1 µM)-induced reduction of nicotine (5 mM)-evoked C-shapes, suggesting an opioid receptor mechanism. C-shapes induced by nicotine (5 mM) were also reduced by meloxicam (10–100 µM), a NSAID; HC 030,031 (1–10 µM), a TRPA1 antagonist; and pancuronium (10–100 µM), a neuromuscular blocker. Evidence that nicotine-induced C-shapes are reduced by antinociceptive drugs from different classes, and require opioid receptor and TRPA1 channel activation, suggest C-shape etiology involves a pain component.

Antinociceptive activity of standardized extract of Bacopa monnieri in different pain models of zebrafish

Ethnopharmacological relevanceBacopa monnieri L. (Scrophulariaceae) is commonly known as Brahmi and traditionally used as a neuroprotective herbal medicine. Recently, Bacopa monnieri exhibited significant therapeutic activity against animal model of neuropathic pain. However, the therapeutic potential of methanolic extract of Bacopa monnieri in experimental animal model is yet to establish. Aim of the studyThe present study was designed to evaluate the anti-nociceptive potential of standardized methanolic extract of Bacopa monnieri in experimental adult zebrafish (Danio rerio) model of pain. Materials and methodsThe methanolic extract of Bacopa monnieri (BME) was standardized to bacoside-A using chromatographic method. Subsequently, BME (0.75, 1.25 and 5.0 mg/ml) was evaluated for anti-nociceptive activity using adult zebrafish model. ResultsStandardized BME showed antioxidant effect through radical quenching activity in in vitro study. BME at 1.25 mg/ml significantly decreased the nociceptive effect induced by different noxious agents like acetic acid where as BME at 2.5 mg/ml exhibited significant antinociceptive activity against glutamate, formalin, capsaicin, cinnamaldehyde when compared to control and sham group animals. ConclusionBME exerted antinociceptive activity in adult zebrafish. It could be presumed that BME may involve glutamatergic receptor, ASIC and TRP channel activity in its anti-nociceptive effect. BME could be considered as a potential therapeutic option in the management of pain.

Structural basis of ketamine action on human NMDA receptors.

Ketamine is a non-competitive channel blocker of N-methyl-D-aspartate (NMDA) receptors1. A single sub-anaesthetic dose of ketamine produces rapid (within hours) and long-lasting antidepressant effects in patients who are resistant to other antidepressants2,3. Ketamine is a racemic mixture of S- and R-ketamine enantiomers, with S-ketamine isomer being the more active antidepressant4. Here we describe the cryo-electron microscope structures of human GluN1-GluN2A and GluN1-GluN2B NMDA receptors in complex with S-ketamine, glycine and glutamate. Both electron density maps uncovered the binding pocket for S-ketamine in the central vestibule between the channel gate and selectivity filter. Molecular dynamics simulation showed that S-ketamine moves between two distinct locations within the binding pocket. Two amino acids-leucine 642 on GluN2A (homologous to leucine 643 on GluN2B) and asparagine 616 on GluN1-were identified as key residues that form hydrophobic and hydrogen-bond interactions with ketamine, and mutations at these residues reduced the potency of ketamine in blocking NMDA receptor channel activity. These findings show structurally how ketamine binds to and acts on human NMDA receptors, and pave the way for the future development of ketamine-based antidepressants.

Molecular signaling of synthetic cannabinoids: Comparison of CB1 receptor and TRPV1 channel activation

Recreational use of synthetic cannabinoids (SCs) is associated with desirable euphoric and relaxation effects as well as adverse effects including anxiety, agitation and psychosis. These SC-mediated actions represent a combination of potentiated cannabinoid receptor signaling and “off-target” receptor activity. The goal of this study was to compare the efficacy of various classes of SCs in stimulating CB1 receptors and activating “off-target” transient receptor potential (TRP) channels. Cannabinoid-type 1 (CB1) receptor activity was determined by measuring SC activation of G protein-gated inward rectifier K+ (GIRK) channels using a membrane potential-sensitive fluorescent dye assay. SC opening of vanilloid type-1 (TRPV1) channels was measured by recording intracellular Ca2+ transients. All of the SCs tested activated the GIRK channel with an efficacy of 4-fluoro MDMB-BUTINACA > 5-fluoro MDMB-PICA > MDMB-4en-PINACA ≈ WIN 55,212-2 > AB-FUBINACA > AM1220 ≈ JWH-122 N-(5-chloropentyl) > AM1248 > JWH-018 ≈ XLR-11 ≈ UR-144. The potency of the SCs at the CB1 receptor was 5-fluoro MDMB-PICA ≈ 4-fluoro MDMB-BUTINACA > AB-FUBINACA ≈ MDMB-4en-PINACA > JWH-018 > AM1220 > XLR-11 > JWH-122 N-(5-chloropentyl) > WIN 55,212-2 ≈ UR-144 > AM1248. In contrast, when tested at a SC concentration that produced a maximal effect on the Gi/GIRK channel, only XLR-11, UR-144 and AM1220 caused a significant activation of the TRPV1 channels. The TRPV1 channel/Ca2+ signal measured during application of 10 μM XLR-11 was similar to the signal induced by the endocannabinoid N-arachidonoylethanolamine (AEA). Thus, while various SCs share the ability to stimulate CB1 receptor/Gi signaling, they display limited efficacy in opening TRPV1 channels.

Epicortical Brevetoxin Treatment Promotes Neural Repair and Functional Recovery after Ischemic Stroke

Emerging literature suggests that after a stroke, the peri-infarct region exhibits dynamic changes in excitability. In rodent stroke models, treatments that enhance excitability in the peri-infarct cerebral cortex promote motor recovery. This increase in cortical excitability and plasticity is opposed by increases in tonic GABAergic inhibition in the peri-infarct zone beginning three days after a stroke in a mouse model. Maintenance of a favorable excitatory–inhibitory balance promoting cerebrocortical excitability could potentially improve recovery. Brevetoxin-2 (PbTx-2) is a voltage-gated sodium channel (VGSC) gating modifier that increases intracellular sodium ([Na+]i), upregulates N-methyl-D-aspartate receptor (NMDAR) channel activity and engages downstream calcium (Ca2+) signaling pathways. In immature cerebrocortical neurons, PbTx-2 promoted neuronal structural plasticity by increasing neurite outgrowth, dendritogenesis and synaptogenesis. We hypothesized that PbTx-2 may promote excitability and structural remodeling in the peri-infarct region, leading to improved functional outcomes following a stroke. We tested this hypothesis using epicortical application of PbTx-2 after a photothrombotic stroke in mice. We show that PbTx-2 enhanced the dendritic arborization and synapse density of cortical layer V pyramidal neurons in the peri-infarct cortex. PbTx-2 also produced a robust improvement of motor recovery. These results suggest a novel pharmacologic approach to mimic activity-dependent recovery from stroke.

Rieske iron-sulfur protein induces FKBP12.6/RyR2 complex remodeling and subsequent pulmonary hypertension through NF-\u03baB/cyclin D1 pathway

Ca2+ signaling in pulmonary arterial smooth muscle cells (PASMCs) plays an important role in pulmonary hypertension (PH). However, the underlying specific ion channel mechanisms remain largely unknown. Here, we report ryanodine receptor (RyR) channel activity and Ca2+ release both are increased, and association of RyR2 by FK506 binding protein 12.6 (FKBP12.6) is decreased in PASMCs from mice with chronic hypoxia (CH)-induced PH. Smooth muscle cell (SMC)-specific RyR2 knockout (KO) or Rieske iron-sulfur protein (RISP) knockdown inhibits the altered Ca2+ signaling, increased nuclear factor (NF)-κB/cyclin D1 activation and cell proliferation, and CH-induced PH in mice. FKBP12.6 KO or FK506 treatment enhances CH-induced PH, while S107 (a specific stabilizer of RyR2/FKBP12.6 complex) produces an opposite effect. In conclusion, CH causes RISP-dependent ROS generation and FKBP12.6/RyR2 dissociation, leading to PH. RISP inhibition, RyR2/FKBP12.6 complex stabilization and Ca2+ release blockade may be potentially beneficial for the treatment of PH.

Panax Ginseng Inhibits Maximal Electroshock Induced Convulsions in Mice

Epilepsy is a serious common neurological disease. Panax ginseng root has been identified as a potential therapy for many disorders. The present study investigated the possible anticonvulsant activity of Panax ginseng on maximal electroshock (MES) induced seizure in mice. Mice were divided into the following groups: Group A (control group): injected with normal saline then exposed to electric shock. Group B (test group): injected with sodium valproate (300 mg/kg, 600 mg/kg, 900 mg/kg). Then exposed to electric shock. Group C (test group): injected with Panax Ginseng (150, 250, 350 mg/kg) then exposed to electric shock. All animals were examined for motor coordination on rotarod test. We concluded that P. ginseng at moderate and high doses significantly decreased the HLE duration of convulsion in mice, denoting that P. ginseng has anticonvulsant effects. the antiepileptic activity of P. ginseng might be due to its antioxidative actions, anti-inflammatory, its regulating effect on sodium ion channels and/or due to its possible modulation of GABA receptor channel activity for further investigations.

Assays for Modulators of Ryanodine Receptor (RyR)/Ca2+ Release Channel Activity for Drug Discovery for Skeletal Muscle and Heart Diseases.

The ryanodine receptor (RyR) is a Ca2+ release channel that is present in the sarcoplasmic reticulum and endoplasmic reticulum (ER) and that plays a central role in excitation-contraction coupling in skeletal and cardiac muscle. Hyperactivation of RyR by genetic mutations or posttranslational modification can cause various skeletal muscle and arrhythmogenic heart diseases. Inhibitors of RyR are therefore expected to be potential drugs for treatment of such diseases. This article describes assays to evaluate RyR channel activity, including an ER Ca2+ measurement assay that is compatible with high-throughput screening and a [3 H]-ryanodine binding assay that provides a quantitative measure of RyR channel activity as a second screen for compound hits. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: [Ca2+ ]ER assay for ryanodine receptor (RyR) channel activity Support Protocol 1: Determination of dose dependence and isoform selectivity of RyR inhibitors using [Ca2+ ]ER assay Basic Protocol 2: [3 H]-Ryanodine binding assay for RyR channel activity Support Protocol 2: Isolation of microsomes from RyR-expressing HEK293 cells.

Molecular mechanisms of human P2X3 receptor channel activation and modulation by divalent cation bound ATP.

P2X3 receptor channels expressed in sensory neurons are activated by extracellular ATP and serve important roles in nociception and sensory hypersensitization, making them attractive therapeutic targets. Although several P2X3 structures are known, it is unclear how physiologically abundant Ca2+-ATP and Mg2+-ATP activate the receptor, or how divalent cations regulate channel function. We used structural, computational and functional approaches to show that a crucial acidic chamber near the nucleotide-binding pocket in human P2X3 receptors accommodates divalent ions in two distinct modes in the absence and presence of nucleotide. The unusual engagement between the receptor, divalent ion and the γ-phosphate of ATP enables channel activation by ATP-divalent complex, cooperatively stabilizes the nucleotide on the receptor to slow ATP unbinding and recovery from desensitization, a key mechanism for limiting channel activity. These findings reveal how P2X3 receptors recognize and are activated by divalent-bound ATP, aiding future physiological investigations and drug development.

Heteromeric Neuronal Nicotinic Acetylcholine Receptors with Mutant β Subunits Acquire Sensitivity to α7-Selective Positive Allosteric Modulators.

Homomeric α7 nicotinic acetylcholine receptors (nAChR) have an intrinsically low probability of opening that can be overcome by α7-selective positive allosteric modulators (PAMs), which bind at a site involving the second transmembrane domain (TM2). Mutation of a methionine that is unique to α7 at the 15' position of TM2 to leucine, the residue in most other nAChR subunits, largely eliminates the activity of such PAMs. We tested the effect of the reverse mutation (L15'M) in heteromeric nAChR receptors containing α4 and β2, which are the nAChR subunits that are most abundant in the brain. Receptors containing these mutations were found to be strongly potentiated by the α7 PAM 3a,4,5,9b-tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c]quinoline-8-sulfonamide (TQS) but insensitive to the alternative PAM 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)-urea. The presence of the mutation in the β2 subunit was necessary and sufficient for TQS sensitivity. The primary effect of the mutation in the α4 subunit was to reduce responses to acetylcholine applied alone. Sensitivity to TQS required only a single mutant β subunit, regardless of the position of the mutant β subunit within the pentameric complex. Similar results were obtained when β2L15'M was coexpressed with α2 or α3 and when the L15'M mutation was placed in β4 and coexpressed with α2, α3, or α4. Functional receptors were not observed when β1L15'M subunits were coexpressed with other muscle nAChR subunits. The unique structure-activity relationship of PAMs and the α4β2L15'M receptor compared with α7 and the availability of high-resolution α4β2 structures may provide new insights into the fundamental mechanisms of nAChR allosteric potentiation. SIGNIFICANCE STATEMENT: Heteromeric neuronal nAChRs have a relatively high initial probability of channel activation compared to receptors that are homomers of α7 subunits but are insensitive to PAMs, which greatly increase the open probability of α7 receptors. These features of heteromeric nAChR can be reversed by mutation of a single residue present in all neuronal heteromeric nAChR subunits to the sequence found in α7. Specifically, the mutation of the TM2 15' leucine to methionine in α subunits reduces heteromeric receptor channel activation, while the same mutation in neuronal β subunits allows heteromeric receptors to respond to select α7 PAMs. The results indicate a key role for this residue in the functional differences in the two main classes of neuronal nAChRs.

The molecular determinants of neurosteroid binding in the GABA(A) receptor

Neurosteroids positively modulate GABA-A receptor (GABAAR) channel activity by binding to a transmembrane domain intersubunit site. Understanding the interactions in this site that determine neurosteroid binding and its effect is essential for the design of neurosteroid-based therapeutics. Using photo-affinity labeling and an ELIC-α1GABAAR chimera, we investigated the impact of mutations (Q242L, Q242W and W246L) within the intersubunit site on neurosteroid binding. These mutations, which abolish the thermal stabilizing effect of allopregnanolone on the chimera, reduce neither photolabeling within the intersubunit site nor competitive prevention of labeling by allopregnanolone. Instead, these mutations change the orientation of neurosteroid photolabeling. Molecular docking of allopregnanolone in WT and Q242W receptors confirms that the mutation favors re-orientation of allopregnanolone within the binding pocket. Collectively, the data indicate that mutations at Gln242 or Trp246 that eliminate neurosteroid effects do not eliminate neurosteroid binding within the intersubunit site, but significantly alter the preferred orientation of the neurosteroid within the site. The interactions formed by Gln242 and Trp246 within this pocket play a vital role in determining the orientation of the neurosteroid that may be necessary for its functional effect.

Redox Regulation of Ion Channels and Receptors in Pulmonary Hypertension.

Significance: Pulmonary hypertension (PH) is characterized by elevated vascular resistance due to vasoconstriction and remodeling of the normally low-pressure pulmonary vasculature. Redox stress contributes to the pathophysiology of this disease by altering the regulation and activity of membrane receptors, K+ channels, and intracellular Ca2+ homeostasis. Recent Advances: Antioxidant therapies have had limited success in treating PH, leading to a growing appreciation that reductive stress, in addition to oxidative stress, plays a role in metabolic and cell signaling dysfunction in pulmonary vascular cells. Reactive oxygen species generation from mitochondria and NADPH oxidases has substantial effects on K+ conductance and membrane potential, and both receptor-operated and store-operated Ca2+ entry. Critical Issues: Some specific redox changes resulting from oxidation, S-nitrosylation, and S-glutathionylation are known to modulate membrane receptor and ion channel activity in PH. However, many sites of regulation that have been elucidated in nonpulmonary cell types have not been tested in the pulmonary vasculature, and context-specific molecular mechanisms are lacking. Future Directions: Here, we review what is known about redox regulation of membrane receptors and ion channels in PH. Further investigation of the mechanisms involved is needed to better understand the etiology of PH and develop better targeted treatment strategies.

An inter-dimer allosteric switch controls NMDA receptor activity.

NMDA receptors (NMDARs) are glutamate-gated ion channels that are key mediators of excitatory neurotransmission and synaptic plasticity throughout the central nervous system. They form massive heterotetrameric complexes endowed with unique allosteric capacity provided by eight extracellular clamshell-like domains arranged as two superimposed layers. Despite an increasing number of full-length NMDAR structures, how these domains cooperate in an intact receptor to control its activity remains poorly understood. Here, combining single-molecule and macroscopic electrophysiological recordings, cysteine biochemistry, and in silico analysis, we identify a rolling motion at a yet unexplored interface between the two constitute dimers in the agonist-binding domain (ABD) layer as a key structural determinant in NMDAR activation and allosteric modulation. This rotation acts as a gating switch that tunes channel opening depending on the conformation of the membrane-distal N-terminal domain (NTD) layer. Remarkably, receptors locked in a rolled state display "super-activity" and resistance to NTD-mediated allosteric modulators. Our work unveils how NMDAR domains move in a concerted manner to transduce long-range conformational changes between layers and command receptor channel activity.

Abstract 097: Interaction of miRNA, Endothelial Mineralocorticoid Receptor and Epithelial Sodium Channel in Endothelium Stiffness and Aortic Dysfunction

Excessive endothelial cell (EC) mineralocorticoid receptor (ECMR) and epithelial sodium channel activation in endothelium (EnNaC) increase oxidative stress and inflammation with associated cardiovascular abnormalities. Previous studies implicate elevations in circulating aldosterone (Aldo) in obesity enhance ECMR/EnNaC signaling that contribute to the development of vascular stiffness, in part, by reducing endothelial nitric oxide (NO) synthase (eNOS) activity and NO production. However, the specific role of ECMR/EnNaC signaling and its molecular mechanisms have not been explored. We hypothesized interactions between ECMR, dysregulation of miRNA expression, and EnNaC contribute to obese-/Aldo-induced endothelium dysfunction and aortic stiffness. Six week-old ECMR -/- and wild type littermate mice were fed a mouse chow or Western diet (WD) containing excess fat (46%) and fructose (17.5%) for 16 weeks. The EnNaC alpha subunit KO (EnNaC -/- ) and EnNaC +/+ female mice were administrated Aldo (250 μg/kg/day) via osmotic minipumps for 3 weeks beginning at 25-28 weeks of age. RNA sequencing showed that in ECMR +/+ mice WD induced increased miR-7a, miR-34, and miR-6973a and reduced miR-99, miR-486a, miR-6904, miR-6916, miR-6240 that potentially target EnNaC expression. For EnNaC in vascular function, EnNaC -/- significantly reduced inward sodium currents by patch clamp in isolated mouse ECs. Meanwhile, EnNaC -/- significantly inhibited Aldo-induced endothelium stiffness and endothelium dependent relaxation observed in EnNaC +/+ . Further, chronic Aldo infusion induced aortic endoplasmic reticulum stress, reduced eNOS activation, endothelium permeability, expression of pro-inflammatory cytokines IL1 and IL6, oxidative stress, and aortic collagen 1 deposition and these abnormalities were attenuated in EnNaC -/- mice. These data indicate that interaction of endothelial specific ECMR/miRNA/EnNaC mediates vascular endothelium dysfunction and prompts aortic stiffness.

Differentially Expressed Genes in Osteomyelitis Induced by Staphylococcus aureus Infection.

Osteomyelitis (OM) is a complicated and serious disease and its underlying molecular signatures of disease initiation and progression remain unclear. Staphylococcus aureus (S. aureus) is the most common causative agent of OM. Previous study of Banchereau et al. has established a link between whole blood transcription profiles and clinical manifestations in patients infected with S. aureus. However, the differentially expressed genes (DEGs) in OM induced by S. aureus infection have not been intensively investigated. In this study, we downloaded the gene expression profile dataset GSE30119 from Gene Expression Omnibus, and performed bioinformatic analysis to identify DEGs in S. aureus infection induced OM from the transcriptional level. The study consisted of 143 whole blood samples, including 44 healthy controls, 42 OM-free, and 57 OM infection patients. A total of 209 S. aureus infection-related genes (SARGs) and 377 OM-related genes (OMRGs) were identified. The SARGs were primarily involved in the immune response by GO functional and pathway enrichment analysis. Several proteins adhere to neutrophil extracellular traps may be critical for the immune response to the process of S. aureus infection. By contrast, the OMRGs differ from the SARGs. The OMRGs were enriched in transmembrane signaling receptor and calcium channel activity, cilium morphogenesis, chromatin silencing, even multicellular organism development. Several key proteins, including PHLPP2 and EGF, were hub nodes in protein–protein interaction network of the OMRGs. In addition, alcoholism, systemic lupus erythematosus and proteoglycans in cancer were the top pathways influenced by the OMRGs associated with OM. Thus, this study has further explored the DEGs and their biological functions associated with S. aureus infection and OM, comparing with the previous study, and may light the further insight into the underlying molecular mechanisms and the potential critical biomarkers in OM development.

The LILI Motif of M3-S2 Linkers Is a Component of the NMDA Receptor Channel Gate.

N-methyl-D-aspartate receptors (NMDARs) mediate excitatory synaptic transmission in the central nervous system, underlie the induction of synaptic plasticity, and their malfunction is associated with human diseases. Native NMDARs are tetramers composed of two obligatory GluN1 subunits and various combinations of GluN2A-D or, more rarely, GluN3A-B subunits. Each subunit consists of an amino-terminal, ligand-binding, transmembrane and carboxyl-terminal domain. The ligand-binding and transmembrane domains are interconnected via polypeptide chains (linkers). Upon glutamate and glycine binding, these receptors undergo a series of conformational changes leading to the opening of the Ca2+-permeable ion channel. Here we report that different deletions and mutations of amino acids in the M3-S2 linkers of the GluN1 and GluN2B subunits lead to constitutively open channels. Irrespective of whether alterations were introduced in the GluN1 or the GluN2B subunit, application of glutamate or glycine promoted receptor channel activity; however, responses induced by the GluN1 agonist glycine were larger, on average, than those induced by glutamate. We observed the most prominent effect when residues GluN1(L657) and GluN2B(I655) were deleted or altered to glycine. In parallel, molecular modeling revealed that two interacting pairs of residues, the LILI motif (GluN1(L657) and GluN2B(I655)), form a functional unit with the TTTT ring (GluN1(T648) and GluN2B(T647)), described earlier to control NMDAR channel gating. These results provide new insight into the structural organization and functional interplay of the LILI and the TTTT ring during the course of NMDAR channel opening and closing.