Molecular Mechanism of L-Pyroglutamic Acid Interaction with the Human Sour Receptor.

Taste is classified into five types, each of which has evolved to play its respective role in mammalian survival. Sour taste is one of the important ways to judge whether food has gone bad, and the sour taste receptor (PKD2L1) is the gene behind it. Here, we investigated whether L-pyroglutamic acid interacts with sour taste receptors through electrophysiology and mutation experiments using Xenopus oocytes. R299 of hPKD2L1 was revealed to be involved in L-pyroglutamic acid binding in a concentration-dependent manner. As a result, it is possible to objectify the change in signal intensity according to the concentration of L-pyroglutamic acid, an active ingredient involved in the taste of kimchi, at the molecular level. Since the taste of other ingredients can also be measured with the method used in this experiment, it is expected that an objective database of taste can be created.

DDX3 Upregulates Hydrogen Peroxide-Induced Melanogenesis in Sk-Mel-2 Human Melanoma Cells.

DDX3 is a DEAD-box RNA helicase with diverse biological functions through multicellular pathways. The objective of this study was to investigate the role of DDX3 in regulating melanogenesis by the exploring signaling pathways involved. Various concentrations of hydrogen peroxide were used to induce melanogenesis in SK-Mel-2 human melanoma cells. Melanin content assays, tyrosinase activity analysis, and Western blot analysis were performed to determine how DDX3 was involved in melanogenesis. Transient transfection was performed to overexpress or silence DDX3 genes. Immunoprecipitation was performed using an antityrosinase antibody. Based on the results of the cell viability test, melanin content, and activity of tyrosinase, a key melanogenesis enzyme, in SK-Mel-2 human melanoma cells, hydrogen peroxide at 0.1 mM was chosen to induce melanogenesis. Treatment with H2O2 notably increased the promoter activity of DDX3. After treatment with hydroperoxide for 4 h, melanin content and tyrosinase activity peaked in DDX3-transfected cells. Overexpression of DDX3 increased melanin content and tyrosinase expression under oxidative stress induced by H2O2. DDX3 co-immunoprecipitated with tyrosinase, a melanogenesis enzyme. The interaction between DDX3 and tyrosinase was strongly increased under oxidative stress. DDX3 could increase melanogenesis under the H2O2-treated condition. Thus, targeting DDX3 could be a novel strategy to develop molecular therapy for skin diseases.

The Application of the Neuroprotective and Potential Antioxidant Effect of Ergotamine Mediated by Targeting N-Methyl-D-Aspartate Receptors.

(1) Background: The N-methyl-D-aspartate receptors (NMDARs) mediate fast excitatory currents leading to depolarization. Postsynaptic NMDARs are ionotropic glutamate receptors that mediate excitatory glutamate or glycine signaling in the CNS and play a primary role in long-term potentiation, which is a major form of use-dependent synaptic plasticity. The overstimulation of NMDARs mediates excessive Ca2+ influx to postsynaptic neurons and facilitates more production of ROS, which induces neuronal apoptosis. (2) Methods: To confirm the induced inward currents by the coapplication of glutamate and ergotamine on NMDARs, a two-electrode voltage clamp (TEVC) was conducted. The ergotamine-mediated inhibitory effects of NR1a/NR2A subunits were explored among four different kinds of recombinant NMDA subunits. In silico docking modeling was performed to confirm the main binding site of ergotamine. (3) Results: The ergotamine-mediated inhibitory effect on the NR1a/NR2A subunits has concentration-dependent, reversible, and voltage-independent properties. The major binding sites were V169 of the NR1a subunit and N466 of the NR2A subunit. (4) Conclusion: Ergotamine effectively inhibited NR1a/NR2A subunit among the subtypes of NMDAR. This inhibition effect can prevent excessive Ca2+ influx, which prevents neuronal death.

Identification and molecular study on the interaction of Schisandrin C with human 5-HT3A receptor.

Schisandrin C (Sch C) is one of the main components of Schisandra chinensis (Schisandra). Since the olden times, Schisandra has been used as a traditional herbal medicine in Asia. Recent studies have shown that Schisandra is effective against irritable bowel syndrome (IBS) in an animal model and affects IBS through the 5-HT3A pathway in the IBS rat model. However, there lacks fundamental research on the interaction of specific components of Schisandra with the 5-HT3A receptor for the treatment of IBS. We hypothesized that a component of Schisandra binds to the 5-HT3A receptor and identified Sch C via a screening work using two electrode-voltage clamps (TEVC). Thus, we aimed to elucidate the neuropharmacological actions between Sch C and the 5-HT3A receptor at molecular and cellular levels. Co-treatment of Sch C with 5-HT inhibited I5-HT in a reversible, concentrate-dependent, like-competition, and voltage-independent manner, and IC50 values of Sch C. Besides, the main binding positions of Sch C were identified through 3D modeling and point mutation were V225A and V288Y on 5-HT3A receptor. Thus, we suggest the potential of Sch C in treating IBS in a manner that suppresses excessive neuronal serotonin signaling in the synapse of sensory neurons and enterochromaffin (EC) cells. In conclusion, the results demonstrate the mechanism of interaction between Sch C and 5-HT3A receptor and reveal Sch C as a novel antagonist.

Molecular Regulation of Betulinic Acid on α3ß4 Nicotinic Acetylcholine Receptors.

Betulinic acid (BA) is a major constituent of Zizyphus seeds that have been long used as therapeutic agents for sleep-related issues in Asia. BA is a pentacyclic triterpenoid. It also possesses various anti-cancer and anti-inflammatory effects. Current commercially available sleep aids typically use GABAergic regulation, for which many studies are being actively conducted. However, few studies have focused on acetylcholine receptors that regulate wakefulness. In this study, we utilized BA as an antagonist of α3ß4 nicotinic acetylcholine receptors (α3ß4 nAChRs) known to regulate rapid-eye-movement (REM) sleep and wakefulness. Effects of BA on α3ß4 nAChRs were concentration-dependent, reversible, voltage-independent, and non-competitive. Site-directed mutagenesis and molecular-docking studies confirmed the binding of BA at the molecular level and showed that the α3 subunit L257 and the ß4 subunit I263 residues affected BA binding. These data demonstrate that BA can bind to a binding site different from the site for the receptor's ligand, acetylcholine (ACh). This suggests that BA may be an effective antagonist that is unaffected by large amounts of ACh released during wakefulness and REM sleep. Based on the above experimental results, BA is likely to be a therapeutically useful sleep aid and sedative.

Differential Regulation of Human Serotonin Receptor Type 3A by Chanoclavine and Ergonovine.

Irritable bowel syndrome (IBS) is a chronic disease that causes abdominal pain and an imbalance of defecation patterns due to gastrointestinal dysfunction. The cause of IBS remains unclear, but intestinal-brain axis problems and neurotransmitters have been suggested as factors. In this study, chanoclavine, which has a ring structure similar to 5-hydroxytryptamine (5-HT), showed an interaction with the 5-HT3A receptor to regulate IBS. Although its derivatives are known to be involved in neurotransmitter receptors, the molecular physiological mechanism of the interaction between chanoclavine and the 5-HT3A receptor is unknown. Electrophysiological experiments were conducted using a two-electrode voltage-clamp analysis to observe the inhibitory effects of chanoclavine on Xenopus oocytes in which the h5-HT3A receptor was expressed. The co-application of chanoclavine and 5-HT resulted in concentration-dependent, reversible, voltage-independent, and competitive inhibition. The 5-HT3A response induced by 5-HT was blocked by chanoclavine with half-maximal inhibitory response concentration (IC50) values of 107.2 µM. Docking studies suggested that chanoclavine was positioned close F130 and N138 in the 5-HT3A receptor-binding site. The double mutation of F130A and N138A significantly attenuated the interaction of chanoclavine compared to a single mutation or the wild type. These data suggest that F130 and N138 are important sites for ligand binding and activity. Chanoclavine and ergonovine have different effects. Asparagine, the 130th amino acid sequence of the 5-HT3A receptor, and phenylalanine, the 138th, are important in the role of binding chanoclavine, but ergonovine has no interaction with any amino acid sequence of the 5-HT3A receptor. The results of the electrophysiological studies and of in silico simulation showed that chanoclavine has the potential to inhibit the hypergastric stimulation of the gut by inhibiting the stimulation of signal transduction through 5-HT3A receptor stimulation. These findings suggest chanoclavine as a potential antiemetic agent for excessive gut stimulation and offer insight into the mechanisms of 5-HT3A receptor inhibition.

Antioxidative and Analgesic Effects of Naringin through Selective Inhibition of Transient Receptor Potential Vanilloid Member 1.

Transient receptor potential vanilloid member 1 (TRPV1) is activated in response to capsaicin, protons, temperature, and free reactive oxygen species (ROS) released from inflammatory molecules after exposure to harmful stimuli. The expression level of TRPV1 is elevated in the dorsal root ganglion, and its activation through capsaicin and ROS mediates neuropathic pain in mice. Its expression is high in peripheral and central nervous systems. Although pain is a response evolved for survival, many studies have been conducted to develop analgesics, but no clear results have been reported. Here, we found that naringin selectively inhibited capsaicin-stimulated inward currents in Xenopus oocytes using a two-electrode voltage clamp. The results of this study showed that naringin has an IC50 value of 33.3 µM on TRPV1. The amino acid residues D471 and N628 of TRPV1 were involved in its binding to naringin. Our study bridged the gap between the pain suppression effect of TRPV1 and the preventive effect of naringin on neuropathic pain and oxidation. Naringin had the same characteristics as a model selective antagonist, which is claimed to be ideal for the development of analgesics targeting TRPV1. Thus, this study suggests the applicability of naringin as a novel analgesic candidate through antioxidative and analgesic effects of naringin.

Molecular Regulation of α3ß4 Nicotinic Acetylcholine Receptors by Lupeol in Cardiovascular System.

Cardiovascular disease (CVD) occurs globally and has a high mortality rate. The highest risk factor for developing CVD is high blood pressure. Currently, natural products are emerging for the treatment of hypertension to avoid the side effects of drugs. Among existing natural products, lupeol is known to be effective against hypertension in animal experiments. However, there exists no study regarding the molecular physiological evidence against the effects of lupeol. Consequently, we investigated the interaction of lupeol with α3ß4 nicotinic acetylcholine receptors (nAChRs). In this study, we performed a two-electrode voltage-clamp technique to investigate the effect of lupeol on the α3ß4 nicotine acetylcholine receptor using the oocytes of Xenopus laevis. Coapplication of acetylcholine and lupeol inhibited the activity of α3ß4 nAChRs in a concentration-dependent, voltage-independent, and reversible manner. We also conducted a mutational experiment to investigate the influence of residues of the α3 and ß4 subunits on lupeol binding with nAChRs. Double mutants of α3ß4 (I37A/N132A), nAChRs significantly attenuated the inhibitory effects of lupeol compared to wild-type α3ß4 nAChRs. A characteristic of α3ß4 nAChRs is their effect on transmission in the cardiac sympathetic ganglion. Overall, it is hypothesized that lupeol lowers hypertension by mediating its effects on α3ß4 nAChRs. The interaction between lupeol and α3ß4 nAChRs provides evidence against its effect on hypertension at the molecular-cell level. In conclusion, the inhibitory effect of lupeol is proposed as a novel therapeutic approach involving the antihypertensive targeting of α3ß4 nAChRs. Furthermore, it is proposed that the molecular basis of the interaction between lupeol and α3ß4 nAChRs would be helpful in cardiac-pharmacology research and therapeutics.

Subunit-specific effects of poricoic acid A on NMDA receptors.

BACKGROUND: N-methyl-D-aspartate (NMDA) receptor is a tetrameric protein complex composed of glycine-linked NR1 subunits and glutamate-linked NR2 subunits. There are four NR2 subunits (A-D) that differ in development, anatomy, and function profiles. They play various roles in normal and neuropathologic conditions. Specific agonists, antagonists, and modulators of subunits for selective NMDA receptors may be precious mediational tools and potent agents for treating diseases. The objective of this study was to determine the effect of poricoic acid A on NMDA receptor known to mediate excitatory synaptic transmission factors and cause changes in synaptic strength. Inhibitory effect of poricoic acid A on NR1a combined with NR2A, NR2B, NR2C, or NR2D receptor was evaluated. METHODS: Glutamate-mediated currents for each NR1a and NR2 subunits were investigated using two-electrode voltage-clamp techniques. Molecular modeling and molecular dynamics simulation studies were carried out with Autodock Tools. Poricoic acid A and NMDA receptor protein complex were examined with Ligplot and Pymol docking program. Ligplot shows binding activity at the protein and the ligand. RESULTS: The inhibitory effect of poricoic acid A on glutamate-induced inward current in a concentration-dependent manner that was reversible. Half inhibitory concentrations of glutamate on NR1a/NR2A, NR1a/NR2B, NR1a/NR2C, and NR1a/NR2D receptors were 9.6 ± 1.2, 5.7 ± 0.4, 46.1 ± 21.5, and 21.5 ± 8.2 µM, respectively. This corresponded to the order of inhibitory effect of oocyte expressing NR1a and NR2s subunit of NR1a/NR2B > NR1a/NR2A > NR1a/NR2C > NR1a/NR2D. CONCLUSIONS: Taken together, these results indicate that poricoic acid A can modulate the expression of NMDA receptor. In addition, the regulation of excitatory ligand-gating ion channel by poricoic acid A may have pharmaceutical functions on excitatory synaptic transmission of neuronal system.

Molecular basis involved in the blocking effect of antidepressant metergoline on C-type inactivation of Kv1.4 channel.

Voltage-gated potassium channels (VGKCs) are transmembrane ion channels specific for potassium. Currently there are nine kinds of VGKCs. Kv1.4 is one of shaker-related potassium channels. It is a representative alpha subunit of potassium channels that can inactivate A type-currents, leading to N pattern inactivation. Inactivation of Kv channels plays an important role in shaping electrical signaling properties of neuronal and muscular cells. The shape of N pattern inactivation can be modified by removing the N-terminal (NT) domain which results in non-inactivated currents and C pattern inactivation. In a previous work, we have reported the regulatory effect of metergoline on Kv1.4 and Nav1.2 channel activity. In the present study, we constructed a mutant of deleted 61 residues from NT of Kv1.4 channels (Kv1.4 Δ2-61) and found that it induced an outward peak and steady-state currents We also studied the modulation effect of metergoline on the activity of this Kv1.4 Δ2-61 mutant channel without having the N-terminal quick inactivation domain. Our results revealed that treatment with metergoline inhibited NT deleted Kv1.4 mutant channel activity in a concentration-dependent manner which was reversible. Interestingly, metergoline treatment induced little effects on the outward peak current in the deleted Kv1.4 mutant channel. However, metergoline treatment conspicuously inhibited steady state currents of Kv1.4 Δ2-61 channels with acceleration current mode. The acceleration of steady-state current of deleted Kv1.4 mutant channel occurred in a concentration-dependent manner. This means that metergoline can accelerate C pattern inactivation of Kv1.4 Δ2-61 channel by acting as an open state dependent channel blocker. We also performed site-directed mutations in V561A and K532Y, also known as C-type inactivation sites. V561A, K532Y, and V561A + K532Y substitution mutants significantly attenuated the acceleration effect of metergoline on C pattern inactivation of hKv1.4 channel currents. In docking modeling study, predicted binding residues for metergoline were analyzed for six amino acids. Among them, the K532 residue known as the C-type inactivation site was analyzed to be a major site of action. Then various mutants were constructed. K532 substitution mutant significantly abolished the effect of metergoline on Kv1.4 currents among various mutants whereas other changes had slight inhibitory effects. Furthermore, we found that metergoline had specificity for Kv1.4, but not for Kv1.5 currents. In addition, the A type current in rat neuronal cell was inhibited and accelerated of inactivation. This result further shows that metergoline might interact with Lys532 residue and then accelerate C pattern inactivation of Kv1.4 channels with channel type specificity. Taken together, these results demonstrate the molecular basis involved in the effect of metergoline, an ergot alkaloid, on human Kv1.4 channel, providing a novel interaction ligand.

Molecular Determinants of α3ß4 Nicotinic Acetylcholine Receptors Inhibition by Triterpenoids.

In a previous work, we reported the regulatory role of the triterpenoids on 5-hydroxytryptamine (5-HT)3A receptors activity in Xenopus laevis oocytes (Eur. J. Pharmacol., 615, 2009, Lee et al.). In the present report, we studied the modulation of triterpenoids on the activity of the human nicotinic acetylcholine receptor type α3ß4. Two-electrode voltage clamp experiments were used to test acetylcholine mediated inward current (IACh). Treatment with triterpenoids (dehydroeburicoic acid, 6α-hydroxypolyporenic acid C and pachymic acid) inhibited IACh in a concentration dependent and reversible manner. The IC50 values for pachymic acid, dehydroeburicoic acid, and 6α-hydroxypolyporenic acid C were 14.9, 37.7, and 20.9 µM, respectively. The inhibitory regulation of IACh by each triterpenoid showed in a non-competitive manner on the activity of α3ß4 nicotinic acetylcholine receptors. These results show that triterpenoids (pachymic acid, dehydroeburicoic acid, 6α-hydroxypolyporenic acid C) can be used as agents to modulate the activity of nicotinic acetylcholine receptor type α3ß4. Furthermore, molecular docking studies of 6α-hydroxypolyporenic acid C on α3ß4 nicotinic acetylcholine receptors in silico showed that this molecule interacted predominantly with residues at cavities in the α3 subunit and ß4 subunit. This docking assays indicated four potential binding sites for this ligand in the extracellular region at sensor domain of α3ß4 nicotinic acetylcholine receptors. In point mutagenesis of those whose alanine substitution, 6α-hydroxypolyporenic acid C potency decreased on W25A of α3 subunit or N109A of ß4 subunit in both mutants. The double mutation of W25A of α3 subunit and N109A of ß4 subunit was significantly attenuated inhibitory effects by 6α-hydroxypolyporenic acid C. All taken together, this study revealed that molecular basis of α3ß4 nicotinic acetylcholine receptors by triterpenoids and provides a novel potent interaction ligand.

A Molecular Basis for the Inhibition of Transient Receptor Potential Vanilloid Type 1 by Gomisin A.

Transient receptor potential (TRP) channel has critical actions as conditional sensors in primary afferent neurons. We studied the regulatory action of gomisin A on TRPV1 channel current in this report. Schisandra chinensis contains bioactive compounds such as the gomisin derivatives and their related compounds. Coapplication with gomisin A inhibited the capsaicin-mediated inward peak current. This inhibitory effect of gomisin A on capsaicin-induced inward current showed concentration-dependence and was reversible. The half maximal inhibitory concentration of gomisin A was 62.7 ± 8.4 µM. In addition, this inhibition occurred in a noncompetition regulation mode and voltage insensitive manner. Furthermore, molecular docking studies of gomisin A on TRPV1 showed that it interacted predominantly with residues at cavities in the segments 1 and 2 of each subunit. Four potential binding sites for this ligand in the extracellular region at sensor domain of TRPV1 channel were identified. Point mutagenesis studies were undertaken, and gomisin A potency decreased for both the Y453A and N467A mutants. The double mutation of Y453 and N467 significantly attenuated inhibitory effects by gomisin A. In summary, this study revealed the molecular basis for the interaction between TRPV1 and gomisin A and provides a novel potent interaction ligand.

Regulation of Human Kv1.4 Channel Activity by the Antidepressant Metergoline.

Metergoline is an ergot-derived psychoactive drug that is a ligand for various serotonin and dopamine receptors. Little is known about the effect of metergoline on different types of receptors and ion channels. Potassium channels are the most diverse group of ion channels. Kv1.4, a shaker family K channel alpha subunit, is one of a family of voltage gated K channels that mediates transient and rapid inactivating A-type currents and N-type inactivation. We demonstrated previously that metergoline inhibited the activity of neuronal voltage-dependent Na(+) channels in Xenopus laevis oocytes (Acta Pharmacol. Sin., 35, 2014, Lee et al.). In this study, we sought to elucidate the regulatory effects underlying metergoline-induced human Kv1.4 channel inhibition. We used the two electrode voltage-clamp (TEVC) technique to investigate the effect of metergoline on human Kv1.4 channel currents in Xenopus laevis oocytes expressing human Kv1.4 alpha subunits. Interestingly, metergoline treatment also induced inhibition of peak currents in human Kv1.4 channels in a concentration-dependent manner. The IC50 of peak currents of hKv1.4 currents was 3.6±0.6 µM. These results indicate that metergoline might regulate the human Kv1.4 channel activity that is expressed in X. laevis oocytes. Further, this regulation of potassium currents by metergoline might be one of the pharmacological actions of metergoline-mediated psychoactivity.