Specific pathogenic mutations in the M3 domain of the GluN1 subunit regulate the surface delivery and pharmacological sensitivity of NMDA receptors.

N-methyl-d-aspartate receptors (NMDARs) play an essential role in regulating glutamatergic neurotransmission. Recently, pathogenic missense mutations were identified in genes encoding NMDAR subunits; however, their effect on NMDAR activity is often poorly understood. Here, we examined whether three previously identified pathogenic mutations (M641I, A645S, and Y647S) in the M3 domain of the GluN1 subunit affect the receptor's surface delivery, agonist sensitivity, Mg2+ block, and/or inhibition by the FDA-approved NMDAR blocker memantine. When expressed in HEK293 cells, we found reduced surface expression of GluN1-M641I/GluN2A, GluN1-Y647S/GluN2A, and GluN1-Y647S/GluN2B receptors; other mutation-bearing NMDAR combinations, including GluN1/GluN3A receptors, were expressed at normal surface levels. When expressed in rat hippocampal neurons, we consistently found reduced surface expression of the GluN1-M641I and GluN1-Y647S subunits when compared with wild-type GluN1 subunit. At the functional level, we found that GluN1-M641I/GluN2 and GluN1-A645S/GluN2 receptors expressed in HEK293 cells have wild-type EC50 values for both glutamate and glycine; in contrast, GluN1-Y647S/GluN2 receptors do not produce glutamate-induced currents. In the presence of a physiological concentration of Mg2+, we found that GluN1-M641I/GluN2 receptors have a lower memantine IC50 and slower offset kinetics, whereas GluN1-A645S/GluN2 receptors have a higher memantine IC50 and faster offset kinetics when compared to wild-type receptors. Finally, we found that memantine was the most neuroprotective in hippocampal neurons expressing GluN1-M641I subunits, followed by neurons expressing wild-type GluN1 and then GluN1-A645S subunits in an NMDA-induced excitotoxicity assay. These results indicate that specific pathogenic mutations in the M3 domain of the GluN1 subunit differentially affect the trafficking and functional properties of NMDARs.

GluN2 Subunit-Dependent Redox Modulation of NMDA Receptor Activation by Homocysteine.

Homocysteine (HCY) molecule combines distinct pharmacological properties as an agonist of N-methyl-d-aspartate receptors (NMDARs) and a reducing agent. Whereas NMDAR activation by HCY was elucidated, whether the redox modulation contributes to its action is unclear. Here, using patch-clamp recording and imaging of intracellular Ca2+, we study dithiothreitol (DTT) effects on currents and Ca2+ responses activated by HCY through native NMDARs and recombinant diheteromeric GluN1/2A, GluN1/2B, and GluN1/2C receptors. Within a wide range (1-800 µM) of [HCY]s, the concentration-activation relationships for recombinant NMDARs revealed a biphasicness. The high-affinity component obtained between 1 and 100 µM [HCY]s corresponding to the NMDAR activation was not affected by 1 mM DTT. The low-affinity phase observed at [HCY]s above 200 µM probably originated from thiol-dependent redox modulation of NMDARs. The reduction of NMDAR disulfide bonds by either 1 mM DTT or 1 mM HCY decreased GluN1/2A currents activated by HCY. In contrast, HCY-elicited GluN1/2B currents were enhanced due to the remarkable weakening of GluN1/2B desensitization. In fact, cleaving NMDAR disulfide bonds in neurons reversed the HCY-induced Ca2+ accumulation, making it dependent on GluN2B- rather than GluN2A-containing NMDARs. Thus, estimated concentrations for the HCY redox effects exceed those in the plasma during intermediate hyperhomocysteinemia but may occur during severe hyperhomocysteinemia.

Nicotinic acetylcholine receptors and nicotine addiction: A brief introduction.

Nicotine is a highly addictive drug found in tobacco that drives its continued use despite the harmful consequences. The initiation of nicotine abuse involves the mesolimbic dopamine system, which contributes to the rewarding sensory stimuli and associative learning processes in the beginning stages of addiction. Nicotine binds to neuronal nicotinic acetylcholine receptors (nAChRs), which come in a diverse collection of subtypes. The nAChRs that contain the α4 and ß2 subunits, often in combination with the α6 subunit, are particularly important for nicotine's ability to increase midbrain dopamine neuron firing rates and phasic burst firing. Chronic nicotine exposure results in numerous neuroadaptations, including the upregulation of particular nAChR subtypes associated with long-term desensitization of the receptors. When nicotine is no longer present, for example during attempts to quit smoking, a withdrawal syndrome develops. The expression of physical withdrawal symptoms depends mainly on the α2, α3, α5, and ß4 nicotinic subunits in the epithalamic habenular complex and its target regions. Thus, nicotine affects diverse neural systems and an array of nAChR subtypes to mediate the overall addiction process. This article is part of the special issue on 'Contemporary Advances in Nicotine Neuropharmacology'.

Structural Basis of Functional Transitions in Mammalian NMDA Receptors.

Excitatory neurotransmission meditated by glutamate receptors including N-methyl-D-aspartate receptors (NMDARs) is pivotal to brain development and function. NMDARs are heterotetramers composed of GluN1 and GluN2 subunits, which bind glycine and glutamate, respectively, to activate their ion channels. Despite importance in brain physiology, the precise mechanisms by which activation and inhibition occur via subunit-specific binding of agonists and antagonists remain largely unknown. Here, we show the detailed patterns of conformational changes and inter-subunit and -domain reorientation leading to agonist-gating and subunit-dependent competitive inhibition by providing multiple structures in distinct ligand states at 4 Å or better. The structures reveal that activation and competitive inhibition by both GluN1 and GluN2 antagonists occur by controlling the tension of the linker between the ligand-binding domain and the transmembrane ion channel of the GluN2 subunit. Our results provide detailed mechanistic insights into NMDAR pharmacology, activation, and inhibition, which are fundamental to the brain physiology.

Anthocyanin Encapsulated by Ferulic Acid-Grafted-Maltodextrin (FA-g-MD) Microcapsules Potentially Improved its Free Radical Scavenging Capabilities Against H2O2-Induced Oxidative Stress.

This study aimed to investigate the antioxidant activity and release behavior of anthocyanin (ANC) loaded within FA-g-MD wall (ANC-FA-g-MD microcapsule) in vitro. The microencapsulation of ANC was prepared by spray drying and displayed a biphasic release profile. The combination of ANC and FA-g-MD (0.0625-1 mg/mL) showed a higher antioxidant activity than that of both individuals. A possible intermolecular interaction between ANC and FA-g-MD was studied by UV-vis spectra. Intracellular reactive oxygen species (ROS), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) test, and protein expression of quinone oxidoreductase 1(NQO1), glutathione reductase (GSR) and γ-glutamate cysteine ligase catalytic subunit (γ-GCLC) were measured through human colon cancer cells (HT-29). After a 24-hour incubation of the HT-29, the combinations (0-60 µg/mL) exhibited a high potential to diminish the ROS level. And the distinct upregulated expressions of GCLC and NQO1 of HT-29 were detected after treatment with combinations compared to those of single ones. These results suggested that the ANC-FA-g-MD microcapsules exerts enhanced antioxidant effect with capability of the modulation of GCLC and NQO1.

Distinctive single-channel properties of α4ß2-nicotinic acetylcholine receptor isoforms.

Central nervous system nicotinic acetylcholine receptors (nAChR) are predominantly of the α4ß2 subtype. Two isoforms exist, with high or low agonist sensitivity (HS-(α4ß2)2ß2- and LS-(α4ß2)2α4-nAChR). Both isoforms exhibit similar macroscopic potency and efficacy values at low acetylcholine (ACh) concentrations, mediated by a common pair of high-affinity α4(+)/(-)ß2 subunit binding interfaces. However LS-(α4ß2)2α4-nAChR also respond to higher concentrations of ACh, acting at a third α4(+)/(-)α4 subunit interface. To probe isoform functional differences further, HS- and LS-α4ß2-nAChR were expressed in Xenopus laevis oocytes and single-channel responses were assessed using cell-attached patch-clamp. In the presence of a low ACh concentration, both isoforms produce low-bursting function. HS-(α4ß2)2ß2-nAChR exhibit a single conductance state, whereas LS-(α4ß2)2α4-nAChR display two distinctive conductance states. A higher ACh concentration did not preferentially recruit either conductance state, but did result in increased LS-(α4ß2)2α4-nAChR bursting and reduced closed times. Introduction of an α4(+)/(-)α4-interface loss-of-function α4W182A mutation abolished these changes, confirming this site's role in mediating LS-(α4ß2)2α4-nAChR responses. Small or large amplitude openings are highly-correlated within individual LS-(α4ß2)2α4-nAChR bursts, suggesting that they arise from distinct intermediate states, each of which is stabilized by α4(+)/(-)α4 site ACh binding. These findings are consistent with α4(+)/(-)α4 subunit interface occupation resulting in allosteric potentiation of agonist actions at α4(+)/(-)ß2 subunit interfaces, rather than independent induction of high conductance channel openings.

GABAA Receptor Subtypes and the Abuse-Related Effects of Ethanol in Rhesus Monkeys: Experiments with Selective Positive Allosteric Modulators.

BACKGROUND: Previous studies have investigated α1GABAA and α5GABAA receptor mechanisms in the behavioral effects of ethanol (EtOH) in monkeys. However, genetic studies in humans and preclinical studies with mutant mice suggest a role for α2GABAA and/or α3GABAA receptors in the effects of EtOH. The development of novel positive allosteric modulators (PAMs) with functional selectivity (i.e., selective efficacy) at α2GABAA and α3GABAA receptors allows for probing of these subtypes in preclinical models of the discriminative stimulus and reinforcing effects of EtOH in rhesus macaques. METHODS: In discrimination studies, subjects were trained to discriminate EtOH (2 g/kg, intragastrically) from water under a fixed-ratio (FR) schedule of food delivery. In oral self-administration studies, subjects were trained to self-administer EtOH (2% w/v) or sucrose (0.3 to 1% w/v) under an FR schedule of solution availability. RESULTS: In discrimination studies, functionally selective PAMs at α2GABAA and α3GABAA (HZ-166) or α3GABAA (YT-III-31) receptors substituted fully (maximum percentage of EtOH-lever responding ≥80%) for the discriminative stimulus effects of EtOH without altering response rates. Full substitution for EtOH also was engendered by a nonselective PAM (triazolam), an α5GABAA -preferring PAM (QH-ii-066) and a PAM at α2GABAA , α3GABAA , and α5GABAA receptors (L-838417). A partial (MRK-696) or an α1GABAA -preferring (zolpidem) PAM only engendered partial substitution (i.e., ~50 to 60% EtOH-lever responding). In self-administration studies, pretreatments with the functionally selective PAMs at α2GABAA and α3GABAA (XHe-II-053 and HZ-166) or α3GABAA (YT-III-31 and YT-III-271) receptors increased EtOH, but not sucrose, drinking at doses that had few, or no, observable sedative-motor effects. CONCLUSIONS: Our results confirm prior findings regarding the respective roles of α1GABAA and α5GABAA receptors in the discriminative stimulus effects of EtOH and, further, suggest a key facilitatory role for α3GABAA and potentially α2GABAA receptors in several abuse-related effects of EtOH in monkeys. Moreover, they reveal a potential role for these latter subtypes in EtOH's sedative effects.

Development of Cortical Pyramidal Cell and Interneuronal Dendrites: a Role for Kainate Receptor Subunits and NETO1.

During neuronal development, AMPA receptors (AMPARs) and NMDA receptors (NMDARs) are important for neuronal differentiation. Kainate receptors (KARs) are closely related to AMPARs and involved in the regulation of cortical network activity. However, their role for neurite growth and differentiation of cortical neurons is unclear. Here, we used KAR agonists and overexpression of selected KAR subunits and their auxiliary neuropilin and tolloid-like proteins, NETOs, to investigate their influence on dendritic growth and network activity in organotypic cultures of rat visual cortex. Kainate at 500 nM enhanced network activity and promoted development of dendrites in layer II/III pyramidal cells, but not interneurons. GluK2 overexpression promoted dendritic growth in pyramidal cells and interneurons. GluK2 transfectants were highly active and acted as drivers for network activity. GluK1 and NETO1 specifically promoted dendritic growth of interneurons. Our study provides new insights for the roles of KARs and NETOs in the morphological and physiological development of the visual cortex.

Protective Role of NMDAR for Microwave-Induced Synaptic Plasticity Injuries in Primary Hippocampal Neurons.

BACKGROUND/AIMS: The N-methyl-D-aspartic acid receptor (NMDAR) has been extensively studied for its important roles in synaptic plasticity and learning and memory. However, the effects of microwave radiation on the subunit composition and activity of NMDARs and the relationship between NMDARs and microwave-induced synaptic plasticity have not been thoroughly elucidated to date. MATERIALS: In our study, primary hippocampal neurons were used to evaluate the effects of microwave radiation on synaptic plasticity. Structural changes were observed by diolistic (Dil) labeling and scanning electron microscopy (SEM) observation. Functional synaptic plasticity was reflected by the NMDAR currents, which were detected by whole cell patch clamp. We also detected the expression of NMDAR subunits by real-time PCR and Western blot analysis. To clarify the effects of microwave radiation on NMDAR-induced synaptic plasticity, suitable agonists or inhibitors were added to confirm the role of NMDARs on microwave-induced synaptic plasticity. Dil labeling, SEM observation, whole cell patch clamp, real-time PCR and Western blot analysis were used to evaluate changes in synaptic plasticity after treatment with agonists or inhibitors. RESULTS: Our results found that microwave exposure impaired neurite development and decreased mRNA and protein levels and the current density of NMDARs. Due to the decreased expression of NMDAR subunits after microwave exposure, the selective agonist NMDA was added to identify the role of NMDARs on microwave-induced synaptic plasticity injuries. After adding the agonist, the expression of NMDAR subunits recovered to the normal levels. In addition, the microwave-induced structural and functional synaptic plasticity injuries recovered, including the number and length of neurites, the connections between neurons, and the NMDAR current. CONCLUSION: Microwave radiation caused neuronal synaptic plasticity injuries in primary hippocampal neurons, and NMDARs played protective roles on the damage process.

Probing the molecular basis for affinity/potency- and efficacy-based subtype-selectivity exhibited by benzodiazepine-site modulators at GABAA receptors.

The extracellular α(+)/γ2(-) interface in the α1,2,3,5ßγ2 GABAA receptor harbours the allosteric binding site targeted by benzodiazepines and newer generations of subtype-selective modulators. We have probed the molecular determinants for the affinity/potency-based α1-preference exhibited by the hypnotic zolpidem (Ambien®, Stilnox®) and the efficacy-based α3-over-α1 selectivity displayed by the analgesic NS11394. Binding affinities and functional properties of the modulators were characterized at wild-type, concatenated, mutant and chimeric α1,3ß2γ2S receptors expressed in tsA201 cells and Xenopus oocytes by [3H]flumazenil binding and two-electrode voltage clamp electrophysiology. Substitution of Gly201 in α1 with the corresponding Glu in α3 completely eliminated the α1-over-α3 preference exhibited by zolpidem. In contrast, the reverse α3-E225G mutation did not yield corresponding increases in the binding affinity or modulatory potency of zolpidem at α3ß2γ2S, and two additional molecular elements in the extracellular domain of the α-subunit were found also to contribute to its α1-preference. Interestingly, the α1-Gly201/α3-Glu225 residue was also a key determinant of the efficacy-based α3-over-α1 selectivity exhibited by NS11394, and a pronounced correlation existed between the side-chain bulkiness of this residue and the modulatory efficacy of NS11394 at the receptor. The subtype-selectivity determinants identified for zolpidem and NS11394 were found also to apply in different degrees to the α1-preferring modulator indiplon and the α3-over-α1 selective modulator L-838,417, respectively. In conclusion, the molecular origins of subtype-selectivity exhibited by benzodiazepine-site modulators at the α1,2,3,5ßγ2 GABAA receptor seem more complex than previously appreciated, and the importance of the α1-Gly201/α3-Glu225 residue for both potency- and efficacy-based subtype-selective modulation through this site is likely to be rooted in different molecular mechanisms.

Structure-Dependent Activity of Natural GABA(A) Receptor Modulators.

GABA(A) receptors are ligand-gated ion channels consisting of five subunits from eight subfamilies, each assembled in four hydrophobic transmembrane domains. This pentameric structure not only allows different receptor binding sites, but also various types of ligands, such as orthosteric agonists and antagonists, positive and negative allosteric modulators, as well as second-order modulators and non-competitive channel blockers. A fact, that is also displayed by the variety of chemical structures found for both, synthetic as well as nature-derived GABA(A)-receptor modulators. This review covers the literature for natural GABA(A)-receptor modulators until the end of 2017 and discusses their structure-activity relationship.

Structural principles of distinct assemblies of the human α4ß2 nicotinic receptor.

Fast chemical communication in the nervous system is mediated by neurotransmitter-gated ion channels. The prototypical member of this class of cell surface receptors is the cation-selective nicotinic acetylcholine receptor. As with most ligand-gated ion channels, nicotinic receptors assemble as oligomers of subunits, usually as hetero-oligomers and often with variable stoichiometries 1 . This intrinsic heterogeneity in protein composition provides fine tunability in channel properties, which is essential to brain function, but frustrates structural and biophysical characterization. The α4ß2 subtype of the nicotinic acetylcholine receptor is the most abundant isoform in the human brain and is the principal target in nicotine addiction. This pentameric ligand-gated ion channel assembles in two stoichiometries of α- and ß-subunits (2α:3ß and 3α:2ß). Both assemblies are functional and have distinct biophysical properties, and an imbalance in the ratio of assemblies is linked to both nicotine addiction2,3 and congenital epilepsy4,5. Here we leverage cryo-electron microscopy to obtain structures of both receptor assemblies from a single sample. Antibody fragments specific to ß2 were used to 'break' symmetry during particle alignment and to obtain high-resolution reconstructions of receptors of both stoichiometries in complex with nicotine. The results reveal principles of subunit assembly and the structural basis of the distinctive biophysical and pharmacological properties of the two different stoichiometries of this receptor.

The downregulation of sweet taste receptor signaling in enteroendocrine L-cells mediates 3-deoxyglucosone-induced attenuation of high glucose-stimulated GLP-1 secretion.

CONTEXT: Sweet taste receptors (STRs) involve in regulating the release of glucose-stimulated glucagon-like peptide-1 (GLP-1). Our in vivo and in vitro studies found that 3-deoxyglucosone (3DG) inhibited glucose-stimulated GLP-1 secretion. OBJECTIVE: This study investigated the role of STRs in 3DG-induced inhibition of high glucose-stimulated GLP-1 secretion. METHODS: STC-1 cells were incubated with lactisole or 3DG for 1 h under 25 mM glucose conditions. Western blotting was used to study the expression of STRs signaling molecules and ELISA was used to analyse GLP-1 and cyclic adenosine monophosphate (cAMP) levels. RESULTS: Lactisole inhibited GLP-1 secretion. Exposure to 25 mM glucose increased the expressions of STRs subunits when compared with 5.6 mM glucose. 3DG decreased GLP-1 secretion and STRs subunits expressions, with affecting other components of STRs pathway, including the downregulation of transient receptor potential cation channel subfamily M member 5 (TRPM5) expression and the reduction of intracellular cAMP levels. CONCLUSION: 3DG attenuates high glucose-stimulated GLP-1 secretion by reducing STR subunit expression and downstream signaling components.

A benzothiadiazine derivative and methylprednisolone are novel and selective activators of transient receptor potential canonical 5 (TRPC5) channels.

The transient receptor potential canonical channel 5 (TRPC5) is a Ca2+-permeable ion channel, which is predominantly expressed in the brain. TRPC5-deficient mice exhibit a reduced innate fear response and impaired motor control. In addition, outgrowth of hippocampal and cerebellar neurons is retarded by TRPC5. However, pharmacological evidence of TRPC5 function on cellular or organismic levels is sparse. Thus, there is still a need for identifying novel and efficient TRPC5 channel modulators. We, therefore, screened compound libraries and identified the glucocorticoid methylprednisolone and N-[3-(adamantan-2-yloxy)propyl]-3-(6-methyl-1,1-dioxo-2H-1λ6,2,4-benzothiadiazin-3-yl)propanamide (BTD) as novel TRPC5 activators. Comparisons with closely related chemical structures from the same libraries indicate important substructures for compound efficacy. Methylprednisolone activates TRPC5 heterologously expressed in HEK293 cells with an EC50 of 12µM, while BTD-induced half-maximal activation is achieved with 5-fold lower concentrations, both in Ca2+ assays (EC50=1.4µM) and in electrophysiological whole cell patch clamp recordings (EC50=1.3 µM). The activation resulting from both compounds is long lasting, reversible and sensitive to clemizole, a recently established TRPC5 inhibitor. No influence of BTD on homotetrameric members of the remaining TRPC family was observed. On the main sensory TRP channels (TRPA1, TRPV1, TRPM3, TRPM8) BTD exerts only minor activity. Furthermore, BTD can activate heteromeric channel complexes consisting of TRPC5 and its closest relatives TRPC1 or TRPC4, suggesting a high selectivity of BTD for channel complexes bearing at least one TRPC5 subunit.

Channel opening and gating mechanism in AMPA-subtype glutamate receptors.

AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-subtype ionotropic glutamate receptors mediate fast excitatory neurotransmission throughout the central nervous system. Gated by the neurotransmitter glutamate, AMPA receptors are critical for synaptic strength, and dysregulation of AMPA receptor-mediated signalling is linked to numerous neurological diseases. Here we use cryo-electron microscopy to solve the structures of AMPA receptor-auxiliary subunit complexes in the apo, antagonist- and agonist-bound states and determine the iris-like mechanism of ion channel opening. The ion channel selectivity filter is formed by the extended portions of the re-entrant M2 loops, while the helical portions of M2 contribute to extensive hydrophobic interfaces between AMPA receptor subunits in the ion channel. We show how the permeation pathway changes upon channel opening and identify conformational changes throughout the entire AMPA receptor that accompany activation and desensitization. Our findings provide a framework for understanding gating across the family of ionotropic glutamate receptors and the role of AMPA receptors in excitatory neurotransmission.

α2 Subunit-Containing GABAA Receptor Subtypes Are Upregulated and Contribute to Alcohol-Induced Functional Plasticity in the Rat Hippocampus.

Alcohol (EtOH) intoxication causes changes in the rodent brain γ-aminobutyric acid receptor (GABAAR) subunit composition and function, playing a crucial role in EtOH withdrawal symptoms and dependence. Building evidence indicates that withdrawal from acute EtOH and chronic intermittent EtOH (CIE) results in decreased EtOH-enhanced GABAAR δ subunit-containing extrasynaptic and EtOH-insensitive α1ßγ2 subtype synaptic GABAARs but increased synaptic α4ßγ2 subtype, and increased EtOH sensitivity of GABAAR miniature postsynaptic currents (mIPSCs) correlated with EtOH dependence. Here we demonstrate that after acute EtOH intoxication and CIE, upregulation of hippocampal α4ßγ2 subtypes, as well as increased cell-surface levels of GABAAR α2 and γ1 subunits, along with increased α2ß1γ1 GABAAR pentamers in hippocampal slices using cell-surface cross-linking, followed by Western blot and coimmunoprecipitation. One-dose and two-dose acute EtOH treatments produced temporal plastic changes in EtOH-induced anxiolysis or withdrawal anxiety, and the presence or absence of EtOH-sensitive synaptic currents correlated with cell surface peptide levels of both α4 and γ1(new α2) subunits. CIE increased the abundance of novel mIPSC patterns differing in activation/deactivation kinetics, charge transfer, and sensitivity to EtOH. The different mIPSC patterns in CIE could be correlated with upregulated highly EtOH-sensitive α2ßγ subtypes and EtOH-sensitive α4ßγ2 subtypes. Naïve α4 subunit knockout mice express EtOH-sensitive mIPSCs in hippocampal slices, correlating with upregulated GABAAR α2 (and not α4) subunits. Consistent with α2, ß1, and γ1 subunits genetically linked to alcoholism in humans, our findings indicate that these new α2-containing synaptic GABAARs could mediate the maintained anxiolytic response to EtOH in dependent individuals, rat or human, contributing to elevated EtOH consumption.

Agonist-mediated assembly of the crustacean methyl farnesoate receptor.

The methyl farnesoate receptor (MfR) orchestrates aspects of reproduction and development such as male sex determination in branchiopod crustaceans. Phenotypic endpoints regulated by the receptor have been well-documented, but molecular interactions involved in receptor activation remain elusive. We hypothesized that the MfR subunits, methoprene-tolerant transcription factor (Met) and steroid receptor coactivator (SRC), would be expressed coincident with the timing of sex programming of developing oocytes by methyl farnesoate in daphnids. We also hypothesized that methyl farnesoate activates MfR assembly. Met mRNA was expressed rhythmically during the reproductive cycle, with peak mRNA accumulation just prior period of oocytes programming of sex. Further, we revealed evidence that Met proteins self-associate in the absence of methyl farnesoate, and that the presence of methyl farnesoate stimulates dissociation of Met multimers with subsequent association with SRC. Results demonstrated that the Met subunit is highly dynamic in controlling the action of methyl farnesoate through temporal variation in its expression and availability for receptor assembly.

IGF-1-Involved Negative Feedback of NR2B NMDA Subunits Protects Cultured Hippocampal Neurons Against NMDA-Induced Excitotoxicity.

Insulin-like growth factor 1 (IGF-1) is a multifunctional protein involved in neuronal polarity and axonal guidance. In our previous study, it was discovered that IGF-1 alleviated 50-µM NMDA-induced excitotoxicity against neuronal autophagy via depression of NR2B p-Ser1303 activation. However, it was found that NMDA at a higher dose did not cause neuronal autophagy. And, the performance of IGF-1 under severe excitotoxicity still needs to be clarified. In this study, we observed that IGF-1 can salvage the hippocampal neurons in an autophagy-independent manner after 150-µM NMDA exposure using thiazolyl blue tetrazolium bromide (MTT), lactate dehydrogenase (LDH), Western blot assay, and transmission electron microscopy. In addition, over-activation of post-synaptic NMDARs was found with the whole-cell patch clamp recording method. In order to explore whether there is a positive feedback way for post-synaptic NMDARs and the different pathway caused by 150 µM NMDA, the phosphorylation level of Fyn and the phosphorylation site of NR2B were investigated. It was observed that NR2B p-Tyr1472 was increased by the activation of Fyn after 150-µM NMDA exposure. When the neutralizing antibody against NR2B p-Ser1303 was added into the medium, both the activations of Fyn and NR2B p-Tyr1472 were blocked, suggesting NR2B p-Ser1303 may be the initial step of NMDA-induced excitotoxicity. In addition, since IGF-1 can block the initial step of NR2B activation, its effect is concluded to continue with the development of excitotoxicity. Overall, this study strongly indicates that the relationship between different phosphorylation sites of NR2B should be laid more emphasis on, which may be a vital target for the NR2B-involved excitotoxicity.

The pan-Kv7 (KCNQ) Channel Opener Retigabine Inhibits Striatal Excitability by Direct Action on Striatal Neurons In Vivo.

Central Kv7 (KCNQ) channels are voltage-dependent potassium channels composed of different combinations of four Kv7 subunits, being differently expressed in the brain. Notably, striatal dopaminergic neurotransmission is strongly suppressed by systemic administration of the pan-Kv7 channel opener retigabine. The effect of retigabine likely involves the inhibition of the activity in mesencephalic dopaminergic neurons projecting to the striatum, but whether Kv7 channels expressed in the striatum may also play a role is not resolved. We therefore assessed the effect of intrastriatal retigabine administration on striatal neuronal excitability in the rat determined by c-Fos immunoreactivity, a marker of neuronal activation. When retigabine was applied locally in the striatum, this resulted in a marked reduction in the number of c-Fos-positive neurons after a strong excitatory striatal stimulus induced by acute systemic haloperidol administration in the rat. The relative mRNA levels of Kv7 subunits in the rat striatum were found to be Kv7.2 = Kv7.3 = Kv7.5 > >Kv7.4. These data suggest that intrastriatal Kv7 channels play a direct role in regulating striatal excitability in vivo.

Nicotinamide is an endogenous agonist for a C. elegans TRPV OSM-9 and OCR-4 channel.

TRPV ion channels are directly activated by sensory stimuli and participate in thermo-, mechano- and chemo-sensation. They are also hypothesized to respond to endogenous agonists that would modulate sensory responses. Here, we show that the nicotinamide (NAM) form of vitamin B3 is an agonist of a Caenorhabditis elegans TRPV channel. Using heterologous expression in Xenopus oocytes, we demonstrate that NAM is a soluble agonist for a channel consisting of the well-studied OSM-9 TRPV subunit and relatively uncharacterized OCR-4 TRPV subunit as well as the orthologous Drosophila Nan-Iav TRPV channel, and we examine stoichiometry of subunit assembly. Finally, we show that behaviours mediated by these C. elegans and Drosophila channels are responsive to NAM, suggesting conservation of activity of this soluble endogenous metabolite on TRPV activity. Our results in combination with the role of NAM in NAD+ metabolism suggest an intriguing link between metabolic regulation and TRPV channel activity.