Effect of 158 herbal remedies on human TRPV1 and the two-pore domain potassium channels KCNK2, 3 and 9.

BACKGROUND AND AIM: Herbal medicines are used to treat a broad number of maladies. However, the pharmacological profile of most remedies is poorly understood. We investigated the effect of herbal remedies from kampo, traditional Chinese medicine (TCM) and other phytotherapies on human two-pore domain potassium channels (KCNK channels; TREK-1, TASK-1 and TASK-3) as well as the human TRPV1 channel. KCNK channels are responsible for the background potassium current of excitable cells, thus essential for the maintenance of the resting membrane potential. Hence, modulators of KCNK channels are of medical significance, e.g. for the treatment of sleep disorders and pain. The transient receptor potential channel TRPV1 is a pain detector for noxious heat. Agonists of this receptor are still used for the treatment of pain in ectopic applications. EXPERIMENTAL PROCEDURE: We evaluated the effect of 158 herbal remedies on these channels in a heterologous expression system (Xenopus laevis oocytes) using the two-electrode voltage-clamp technique with the aim of increasing the comprehension of their pharmacological profile. RESULTS AND CONCLUSION: Some remedies with modulating effects were identified such as Angelica pubescens (radix), which inhibit TASK-1 and TASK-3 channels. Furthermore, the modulatory effects of the most effective remedies on the two TASK family members TASK-1 and TASK-3 correlate positively, reflecting their close relation. For the TRPV1 channel Terminalia chebula and Alchemilla xanthochlora were identified as potentiators. This study identifies a variety of herbal remedies as modulators of human K2P and TRPV1 channels and gives new insights into the pharmacological profile of these herbal remedies.

Kampo Medicine: Evaluation of the Pharmacological Activity of 121 Herbal Drugs on GABAA and 5-HT3A Receptors.

Kampo medicine is a form of Japanese phytotherapy originating from traditional Chinese medicine (TCM). During the last several decades, much attention has been paid to the pharmacological effects of these medical plants and their constituents. However, in many cases, a systematic screening of Kampo remedies to determine pharmacologically relevant targets is still lacking. In this study, a broad screening of Kampo remedies was performed to look for pharmacologically relevant 5-HT3A and GABAA receptor ligands. Several of the Kampo remedies are currently used for symptoms such as nausea, emesis, gastrointestinal motility disorders, anxiety, restlessness, or insomnia. Therefore, the pharmacological effects of 121 herbal drugs from Kampo medicine were analyzed as ethanol tinctures on heterologously expressed 5-HT3A and GABAA receptors, due to the involvement of these receptors in such pathophysiological processes. The tinctures of Lindera aggregata (radix) and Leonurus japonicus (herba) were the most effective inhibitory compounds on the 5-HT3A receptor. Further investigation of known ingredients in these compounds led to the identification of leonurine from Leonurus as a new natural 5-HT3A receptor antagonist. Several potentiating herbs (e.g., Magnolia officinalis (cortex), Syzygium aromaticum (flos), and Panax ginseng (radix)) were also identified for the GABAA receptor, which are all traditionally used for their sedative or anxiolytic effects. A variety of tinctures with antagonistic effects Salvia miltiorrhiza (radix) were also detected. Therefore, this study reveals new insights into the pharmacological action of a broad spectrum of herbal drugs from Kampo, allowing for a better understanding of their physiological effects and clinical applications.

Borneol inhibits TRPA1, a proinflammatory and noxious pain-sensing cation channel.

Borneol, a natural product isolated from several species of Artemisia, Blumea and Kaempferia, has a widespread use in traditional medicine. TRP ion channels are a class of nonselective cation channel proteins involved in a variety of physiological and pathological processes in mammals. TRPA1, a member of TRP family of cation channels, is involved in plethora of processes including noxious-cold, noxious-pain sensations, inflammation and the detection of irritant chemicals. Borneol is chemically related to camphor (a known inhibitor of TRPA1 ion channels); therefore, it is beneficial to investigate the effects of borneol on TRPA1. In the present investigation it was found that borneol inhibits TRPA1 mediated cationic currents in low millimolar range (IC50 0.3mM) in heterologous expression systems like Xenopus oocytes and in neurons cultured from trigeminal ganglia. Effects of nicotine, a known chemical irritant and agonist of TRPA1 are also inhibited by borneol in both systems. It is concluded that borneol, being an inhibitor of TRPA1, could be a safer therapeutic-combination in clinical situations where TRPA1 channelopathies like neuropathic-pain, trigeminal neuralgia or nicotine withdrawal treatments are involved.

Identification of Glycyrrhiza as the rikkunshito constituent with the highest antagonistic potential on heterologously expressed 5-HT3A receptors due to the action of flavonoids.

The traditional Japanese phytomedicine rikkunshito is traditionally used for the treatment of gastrointestinal motility disorders, cachexia and nausea. These effects indicate 5-HT3 receptor antagonism, due to the involvement of these receptors in such pathophysiological processes. E.g., setrons, specific 5-HT3 receptor antagonists are the strongest antiemetics, developed so far. Therefore, the antagonistic effects of the eight rikkunshito constituents at heterologously expressed 5-HT3Areceptors were analyzed using the two-electrode voltage-clamp technique. The results indicate that tinctures from Aurantii, Ginseng, Zingiberis, Atractylodis and Glycyrrhiza inhibited the 5-HT3A receptor response, whereas the tinctures of Poria cocos, Jujubae and Pinellia exhibited no effect. Surprisingly, the strongest antagonism was found for Glycyrrhiza, whereas the Zingiberis tincture, which is considered to be primarily responsible for the effect of rikkunshito, exhibited the weakest antagonism of 5-HT3A receptors. Rikkunshito contains various vanilloids, ginsenosides and flavonoids, a portion of which show an antagonistic effect on 5-HT3 receptors. A screening of the established ingredients of the active rikkunshito constituents and related substances lead to the identification of new antagonists within the class of flavonoids. The flavonoids (-)-liquiritigenin, glabridin and licochalcone A from Glycyrrhiza species were found to be the most effective inhibitors of the 5-HT-induced currents in the screening. The flavonoids (-)-liquiritigenin and hesperetin from Aurantii inhibited the receptor response in a non-competitive manner, whereas glabridin and licochalcone A exhibited a potential competitive antagonism. Furthermore, licochalcone A acts as a partial antagonist of 5-HT3A receptors. Thus, this study reveals new 5-HT3A receptor antagonists with the aid of increasing the comprehension of the complex effects of rikkunshito.

Partial agonism of taurine at gamma-containing native and recombinant GABAA receptors.

Taurine is a semi-essential sulfonic acid found at high concentrations in plasma and mammalian tissues which regulates osmolarity, ion channel activity and glucose homeostasis. The structural requirements of GABAA-receptors (GABAAR) gated by taurine are not yet known. We determined taurine potency and efficacy relative to GABA at different types of recombinant GABAAR occurring in central histaminergic neurons of the mouse hypothalamic tuberomamillary nucleus (TMN) which controls arousal. At binary α(1/2)ß(1/3) receptors taurine was as efficient as GABA, whereas incorporation of the γ(1/2) subunit reduced taurine efficacy to 60-90% of GABA. The mutation γ(2F77I), which abolishes zolpidem potentiation, significantly reduced taurine efficacy at recombinant and native receptors compared to the wild type controls. As taurine was a full- or super- agonist at recombinant αxß1δ-GABAAR, we generated a chimeric γ(2) subunit carrying the δ subunit motif around F77 (MTVFLH). At α(1/2)ß(1)γ2(MTVFLH) receptors taurine became a super-agonist, similar to δ-containing ternary receptors, but remained a partial agonist at ß3-containing receptors. In conclusion, using site-directed mutagenesis we found structural determinants of taurine's partial agonism at γ-containing GABAA receptors. Our study sheds new light on the ß1 subunit conferring the widest range of taurine-efficacies modifying GABAAR function under (patho)physiological conditions.

2-Aminoethoxydiphenyl borate activates the mechanically gated human KCNK channels KCNK 2 (TREK-1), KCNK 4 (TRAAK), and KCNK 10 (TREK-2).

Two-pore domain K(+) (KCNK, K2P) channels underlie the "leak" (background) potassium conductance in many types of excitable cells. They oppose membrane depolarization and cell excitability. These channels have been reported to be modulated by several physical and chemical stimuli. The compound 2-aminoethoxydiphenyl borate (2-APB) was originally described as an inhibitor of IP3-induced Ca(2+) release but has been shown to act as either a blocker or an activator for several ion channels. Here, we report the effects of this compound on members of the TREK (TWIK related K(+) channels) subfamily of human KCNK channels. We injected Xenopus laevis oocytes with cRNAs (complementary RNAs) encoding several KCNK channels and measured their response using the two-electrode voltage clamp technique. 2-APB was found to be an effective activator for all members of the TREK subfamily (hKCNK2, hKCNK4, and hKCNK10), with the highest efficacy in hKCNK10. We also found that 2-APB was able to activate these channels in cell-excised patches of HEK293 (human embryonic kidney 293) cell transfected with hKCNK4 or hKCNK10, demonstrating direct activation. TREK channels are widely expressed in the central nervous system and peripheral tissues, where they play roles in several key processes. However, little is known regarding their pharmacology; therefore, the identification of a common, stable and inexpensive agonist should aid further investigations of these channels. Additionally, 2-APB has been used to study native receptors in cell systems that endogenously express members of the TREK subfamily (e.g., rat dorsal root ganglia); our results thus warn against the use of 2-APB at high concentrations in these systems.

New insights in endogenous modulation of ligand-gated ion channels: histamine is an inverse agonist at strychnine sensitive glycine receptors.

Histamine is involved in many physiological functions in the periphery and is an important neurotransmitter in the brain. It acts on metabotropic H1-H4 receptors mediating vasodilatation, bronchoconstriction and stimulation of gastric acid secretion. In the brain histamine is produced by neurons in the tuberomamillary nucleus (TMN), which controls arousal. Histamine is also a positive modulator of the inhibitory Cys-loop ligand-gated ion channel GABAA. We investigated now its effect on the second member of inhibitory Cys-loop ligand-gated ion channels, the strychnine sensitive glycine receptor. We expressed different human and rat glycine receptor subunits in Xenopus laevis oocytes and characterized the effect of histamine using the two electrode voltage clamp technique. Furthermore we investigated native glycine receptors in hypothalamic neurons using the patch-clamp technique. Histamine inhibited α1ß glycine receptors with an IC50 of 5.2±0.3 mM. In presence of 10 mM histamine the glycine dose-response curve was shifted, increasing the EC50 for glycine from 25.5±1.4 µM to 42.4±2.3 µM. In addition, histamine blocked the spontaneous activity of RNA-edited α3ß glycine receptors. Histamine inhibited glycine receptors expressed in hypothalamic TMN neurons with an IC50 of 4.6±0.3 mM. Our results give strong evidence that histamine is acting on the same binding site as glycine, being an inverse agonist that competitively antagonizes glycine receptors. Thus, we revealed histamine as an endogenous modulator of glycine receptors.

Camphor modulates TRPV3 cation channels activity by interacting with critical pore-region cysteine residues.

TRPV3 ion channels mediate thermo-transduction, nociception, inflammation and dermatitis in mammals. TRPV1-4 proteins have been shown to have conserved cysteine-residues in the pore-forming regions. These residues participate in channel activation via S-nitrosylation of channel proteins. Camphor is a commonly used ligand for TRPV3 channels. Thus the knowledge about the potential binding/interacting site(s) for camphor will help to design effective and potent analgesic compounds. In an overlap-extension PCR method, following primer-pairs were used to mutate conserved cysteine-residues in the pore-region of TRPV3 channels; GATTGAGAATcCTCCAAGGACAAAAAGGAC, TRPV3-C612S-Fw and GTCCTTGGAGgACTTCTCAATCAGTCAGTGAGG, TRPV3-C612S-Rv primers pair. And for TRPV3-C619S: GGACTCcAGTTCCTATGGCCAGC, TRPV3-C619S-Fw and GCTGGCCATAgGAACTGGAGTCC, TRPV3-C619S-Rv respectively. All cDNA constructs were confirmed by DNA-sequencing and used to make cRNAs. Oocytes expressing mTRPV3-C619S and mTRPV3-C612S mutant channels were challenged with 2-APB (1 mM), camphor (10 mM) and dihydrocarveol (10 mM) either at -40 mV or +40 mV holding potentials in voltage-clamp experiments. Responses of both mutants to 2-APB were similar to wild-type mTRPV3. Interestingly, responses to camphor were totally lost in mTRPV3-C619S mutant, while responses to dihydrocarveol remained intact. In contrast mTRPV3-C612S displayed slightly altered (16±2 % reduction) phenotype with respect to camphor sensitivity. It is concluded that pore-region cysteines play critical role in camphor sensitivity of TRPV3 ion channels.

Chemosensory Ca2+ dynamics correlate with diverse behavioral phenotypes in human sperm.

In the female reproductive tract, mammalian sperm undergo a regulated sequence of prefusion changes that "prime" sperm for fertilization. Among the least understood of these complex processes are the molecular mechanisms that underlie sperm guidance by environmental chemical cues. A "hard-wired" Ca(2+) signaling strategy that orchestrates specific motility patterns according to given functional requirements is an emerging concept for regulation of sperm swimming behavior. The molecular players involved, the spatiotemporal characteristics of such motility-associated Ca(2+) dynamics, and the relation between a distinct Ca(2+) signaling pattern and a behavioral sperm phenotype, however, remain largely unclear. Here, we report the functional characterization of two human sperm chemoreceptors. Using complementary molecular, physiological, and behavioral approaches, we comparatively describe sperm Ca(2+) responses to specific agonists of these novel receptors and bourgeonal, a known sperm chemoattractant. We further show that individual receptor activation induces specific Ca(2+) signaling patterns with unique spatiotemporal dynamics. These distinct Ca(2+) dynamics are correlated to a set of stimulus-specific stereotyped behavioral responses that could play vital roles during various stages of prefusion sperm-egg chemical communication.

G protein-coupled receptor signaling via Src kinase induces endogenous human transient receptor potential vanilloid type 6 (TRPV6) channel activation.

Ca(2+) homeostasis plays a critical role in a variety of cellular processes. We showed previously that stimulation of the prostate-specific G protein-coupled receptor (PSGR) enhances cytosolic Ca(2+) and inhibits proliferation of prostate cells. Here, we analyzed the signaling mechanisms underlying the PSGR-mediated Ca(2+) increase. Using complementary molecular, biochemical, electrophysiological, and live-cell imaging techniques, we found that endogenous Ca(2+)-selective transient receptor potential vanilloid type 6 (TRPV6) channels are critically involved in the PSGR-induced Ca(2+) signal. Biophysical characterization of the current activated by PSGR stimulation revealed characteristic properties of TRPV6. The molecular identity of the involved channel was confirmed using RNA interference targeting TrpV6. TRPV6-mediated Ca(2+) influx depended on Src kinase activity. Src kinase activation occurred independently of G protein activation, presumably by direct interaction with PSGR. Taken together, we report that endogenous TRPV6 channels are activated downstream of a G protein-coupled receptor and present the first physiological characterization of these channels in situ.

Inhibition of TRPV1 for the treatment of sensitive skin.

During the past years, the topic sensitive skin became one of the most important fields in dermatology. The tremendous interest is based on several studies showing that about 50% of the population declares to have sensitive skin. The human thermoreceptor hTRPV1 was previously identified to contribute to this skin condition while facilitating neurogenic inflammation leading to hyperalgesia. Furthermore, skin sensitivity towards capsaicin, a natural activator of TRPV1, was shown to correlate with sensitive skin. In a screening campaign based on recombinant HEK293-cells stably transfected with hTRPV1, the selective antagonist trans-4-tert-butylcyclohexanol was identified. This antagonist is able to inhibit capsaicin-induced hTRPV1 activation with an IC(50) value of 34 ± 5 µm tested in HEK293-cells as well as in electrophysiological recordings performed in oocytes expressing hTRPV1. Strikingly, in a clinical study with 30 women using topical treatment with o/w emulsions containing 31.6 ppm capsaicin, we were able to show that 0.4% of this inhibitor significantly reduces capsaicin-induced burning (P < 0.0001) in vivo. Thus trans-4-tert-butylcyclohexanol has the potential as a novel bioactive for the treatment of sensitive skin.

Fragrant dioxane derivatives identify beta1-subunit-containing GABAA receptors.

Nineteen GABA(A) receptor (GABA(A)R) subunits are known in mammals with only a restricted number of functionally identified native combinations. The physiological role of beta1-subunit-containing GABA(A)Rs is unknown. Here we report the discovery of a new structural class of GABA(A)R positive modulators with unique beta1-subunit selectivity: fragrant dioxane derivatives (FDD). At heterologously expressed alpha1betaxgamma2L (x-for 1,2,3) GABA(A)R FDD were 6 times more potent at beta1- versus beta2- and beta3-containing receptors. Serine at position 265 was essential for the high sensitivity of the beta1-subunit to FDD and the beta1N286W mutation nearly abolished modulation; vice versa the mutation beta3N265S shifted FDD sensitivity toward the beta1-type. In posterior hypothalamic neurons controlling wakefulness GABA-mediated whole-cell responses and GABAergic synaptic currents were highly sensitive to FDD, in contrast to beta1-negative cerebellar Purkinje neurons. Immunostaining for the beta1-subunit and the potency of FDD to modulate GABA responses in cultured hypothalamic neurons was drastically diminished by beta1-siRNA treatment. In conclusion, with the help of FDDs we reveal a functional expression of beta1-containing GABA(A)Rs in the hypothalamus, offering a new tool for studies on the functional diversity of native GABA(A)Rs.

Subunit-specific P2X-receptor expression defines chemosensory properties of trigeminal neurons.

The facial innervation pattern of trigeminal nerve fibres comprises the innervation of the nasal epithelium, where free trigeminal nerve endings contribute to detection and discrimination of chemical stimuli including odourants. The signal transduction mechanisms in sensory nerve endings underlying perception of chemical stimuli remain widely uncovered. Here, we characterized trigeminal ATP-activated P2X receptors in cultured rat trigeminal neurons and investigated their role in chemoperception. We identified a new subpopulation of neurons lacking typical nociceptive characteristics and expressing homomeric P2X(2) receptors. Using a certain group of chemicals known as trigeminal stimuli we found no direct activation of trigeminal neurons, but a modulation of P2X(2) receptor mediated currents. In contrast, P2X(3) receptor mediated currents of nociceptive trigeminal neurons remained unaffected by the tested chemicals. Therefore, we assume a functional role for the newly identified subpopulation in chemodetection of certain trigeminal stimuli.

Characterization of recombinant and native Ih-channels from Apis mellifera.

Recently, a novel class of genes coding for Ih-channels has been identified in several vertebrates and invertebrates. We isolated a cDNA (AMIH) encoding a putative member of these ion channels from Apis mellifera heads by means of polymerase chain reaction and homology screening. High similarity (88% identical amino acids) to the putative Drosophila melanogaster Ih-channel suggests that the Apis cDNA codes for a hyperpolarization-activated and cyclic nucleotide-gated channel. Functional expression of recombinant AMIH in HEK293 cells gave unitary currents that were preferentially selective for potassium over sodium ions and were activated by hyperpolarizing voltage steps. Cyclic nucleotides shifted the voltage activation curve to more positive membrane potentials. The current kinetics, activation by hyperpolarizing voltage steps and modulatory influence of cyclic nucleotides properties closely resemble those of mammalian Ih-channels. RT-PCR analysis showed pronounced mRNA expression in the antennae, head and body of Apis mellifera. Investigation of hyperpolarization-activated currents in olfactory receptor neurons (ORNs) in a primary cell culture of Apis mellifera antennal cells revealed activation properties similar to the heterologously expressed Ih-channel. By in-situ hybridization and immunohistochemistry, expression of AMIH was seen in olfactory receptor neurons of the bee antennae. We conclude that AMIH is the ion channel responsible for the hyperpolarization-activated currents in olfactory receptor neurons of bee.

Overexpression of a hyperpolarization-activated cation current (Ih) channel gene modifies the firing activity of identified motor neurons in a small neural network.

The hyperpolarization-activated cation current (Ih) is widely distributed in excitable cells. Ih plays important roles in regulation of cellular excitability, rhythmic activity, and synaptic function. We previously showed that, in pyloric dilator (PD) neurons of the stomatogastric ganglion (STG) of spiny lobsters, Ih can be endogenously upregulated to compensate for artificial overexpression of the Shal transient potassium channel; this maintains normal firing properties of the neuron despite large increases in potassium current. To further explore the function of Ih in the pyloric network, we injected cRNA of PAIH, a lobster gene that encodes Ih, into rhythmically active PD neurons. Overexpression of PAIH produced a fourfold increase in Ih, although with somewhat different biophysical properties than the endogenous current. Compared with the endogenous Ih, the voltage for half-maximal activation of the PAIH-evoked current was depolarized by 10 mV, and its activation kinetics were significantly faster. This increase in Ih did not affect the expression of IA or other outward currents. Instead, it significantly altered the firing properties of the PD neurons. Increased Ih depolarized the minimum membrane potential of the cell, reduced the oscillation amplitude, decreased the time to the first spike, and increased the duty cycle and number of action potentials per burst. We used both dynamic-clamp experiments, injecting the modeled PAIH currents into PD cells in a functioning STG, and a theoretical model of a two-cell network to demonstrate that the increased Ih was sufficient to cause the observed changes in the PD activity.

Two cDNAs coding for histamine-gated ion channels in D. melanogaster.

Histamine, a neurotransmitter and neuroregulatory compound in diverse species, serves as the neurotransmitter of photoreceptors in insects and other arthropods by directly activating a chloride channel. By systematic expression screening of novel putative ligand-gated anion channels, we identified two cDNAs (DM-HisCl-alpha 1 and-alpha 2) coding for putative histamine-gated chloride channels by functional expression in Xenopus laevis oocytes. DM-HisCl-alpha 1 mRNA localizes in the lamina region of the Drosophila eye, supporting the idea that DM-HisCl-alpha 1 may be a neurotransmitter receptor for histamine in the visual system.