Functional expression and ligand identification of homo- and heteromeric Drosophila melanogaster\textit {Drosophila melanogaster} CO2CO_2 receptors in the Xenopus laevis\textit {Xenopus laevis} oocyte system

Carbon dioxide ($CO_2$) is an important olfactory cue in $\textit {Drosophila melanogaster}$ and can elicit both attractive and aversive behaviors. It is detected by gustatory receptors, Gr21a and Gr63a, found in the ab1C neuron in basiconic sensilla on the third antennal segment. Volatile substances that modulate the receptors’ function are of interest for pest control. While several substances block ab1C neurons or mimic the activating effect of carbon dioxide, it is not known if these substances are indeed ligands of the $CO_2$ receptor or might act on other proteins in the receptor neuron. In this study, we used the recombinant $\textit {Xenopus laevis}$ expression system and two-electrode voltage-clamp technology to investigate the receptor function. We found that application of sodium bicarbonate evokes large inward currents in oocytes co-expressing Gr21a and Gr63a. The receptors most likely form hetromultimeric complexes. Homomultimeric receptors of Gr21a or Gr63a are sufficient for receptor functionality, although oocytes gave significantly lower current responses compared to the probable heteromultimeric receptor. We screened for putative blockers of the sodium bicarbonate response and confirmed that some of the substances identified by spike recordings of olfactory receptor neurons, such as 1-hexanol, are also blockers in the $\it Xenopus$ oocyte system. We also identified a new blocking substance, citronellol, which is related to insect repellents. Many substances that activate receptor neurons were inactive in the $\it Xenopus$ oocyte system, indicating that they may not be ligands for the receptor, but may act on other proteins. However, methyl pyruvate and n-hexylamine were found to be activators of the recombinant Gr21a/Gr63a receptor

Identification of Glycyrrhiza\it Glycyrrhiza as the rikkunshito constituent with the highest antagonistic potential on heterologously expressed 5−HT3A5-HT_{3A} 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 $5-HT_{3}$ receptor antagonism, due to the involvement of these receptors in such pathophysiological processes. E.g., setrons, specific $5-HT_{3}$ receptor antagonists are the strongest antiemetics, developed so far. Therefore, the antagonistic effects of the eight rikkunshito constituents at heterologously expressed $5-HT_{3A}$receptors were analyzed using the two-electrode voltage-clamp technique. The results indicate that tinctures from $\textit {Aurantii, Ginseng, Zingiberis, Atractylodis}$ and $\it Glycyrrhiza$ inhibited the $5-HT_{3A}$ receptor response, whereas the tinctures of $\textit {Poria cocos, Jujubae}$ and $\it Pinellia$ exhibited no effect. Surprisingly, the strongest antagonism was found for $\it Glycyrrhiza$, whereas the $\it Zingiberis$ tincture, which is considered to be primarily responsible for the effect of rikkunshito, exhibited the weakest antagonism of $5-HT_{3A}$ receptors. Rikkunshito contains various vanilloids, ginsenosides and flavonoids, a portion of which show an antagonistic effect on $5-HT_{3}$ 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 $\it Glycyrrhiza$ species were found to be the most effective inhibitors of the 5-HT-induced currents in the screening. The flavonoids (-)-liquiritigenin and hesperetin from $\it Aurantii$ 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 $5-HT_{3A}$ receptors. Thus, this study reveals new $5-HT_{3A}$ receptor antagonists with the aid of increasing the comprehension of the complex effects of rikkunshito

Expression profile of ectopic olfactory receptors determined by deep sequencing

Olfactory receptors (ORs) provide the molecular basis for the detection of volatile odorant molecules by olfactory sensory neurons. The OR supergene family encodes G-protein coupled proteins that belong to the seven-transmembrane-domain receptor family. It was initially postulated that ORs are exclusively expressed in the olfactory epithelium. However, recent studies have demonstrated ectopic expression of some ORs in a variety of other tissues. In the present study, we conducted a comprehensive expression analysis of ORs using an extended panel of human tissues. This analysis made use of recent dramatic technical developments of the so-called Next Generation Sequencing (NGS) technique, which encouraged us to use open access data for the first comprehensive RNA-Seq expression analysis of ectopically expressed ORs in multiple human tissues. We analyzed mRNA-Seq data obtained by Illumina sequencing of 16 human tissues available from Illumina Body Map project 2.0 and from an additional study of OR expression in testis. At least some ORs were expressed in all the tissues analyzed. In several tissues, we could detect broadly expressed ORs such as OR2W3 and OR51E1. We also identified ORs that showed exclusive expression in one investigated tissue, such as OR4N4 in testis. For some ORs, the coding exon was found to be part of a transcript of upstream genes. In total, 111 of 400 OR genes were expressed with an FPKM (fragments per kilobase of exon per million fragments mapped) higher than 0.1 in at least one tissue. For several ORs, mRNA expression was verified by RT-PCR. Our results support the idea that ORs are broadly expressed in a variety of tissues and provide the basis for further functional studies

Timberol® inhibits TAAR5-mediated responses to trimethylamine and influences the olfactory threshold in humans

In mice, trace amine-associated receptors (TAARs) are interspersed in the olfactory epithelium and constitute a chemosensory subsystem that is highly specific for detecting volatile amines. Humans possess six putative functional TAAR genes. Human TAAR5 (hTAAR5) is highly expressed in the olfactory mucosa and was shown to be specifically activated by trimethylamine. In this study, we were challenged to uncover an effective blocker substance for trimethylamine-induced hTAAR5 activation. To monitor blocking effects, we recombinantly expressed hTAAR5 and employed a commonly used Cre-luciferase reporter gene assay. Among all tested potential blocker substances, Timberol®, an amber-woody fragrance, is able to inhibit the trimethylamine-induced hTAAR5 activation up to 96%. Moreover, human psychophysical data showed that the presence of Timberol® increases the olfactory detection threshold for the characteristic fishy odor of trimethylamine by almost one order of magnitude. In conclusion, our results show that among tested receptors Timberol® is a specific and potent antagonist for the hTAAR5-mediated response to trimethylamine in a heterologous system. Furthermore, our data concerning the observed shift of the olfactory detection threshold $\textit {in vivo}$ implicate that hTAAR5 or other receptors that may be inhibited by Timberol® could be involved in the high affinity olfactory perception of trimethylamine in humans

Identification of amino acids involved in histamine potentiation of GABAAGABA_{A} receptors

Histamine is a neurotransmitter involved in a number of physiological and neuronal functions. In mammals, such as humans, and rodents, the histaminergic neurons found in the tuberomamillary nucleus project widely throughout the central nervous system. Histamine acts as positive modulator of $GABA_{A}$ receptors $(GABA_{A}Rs)$ and, in high concentrations (10 mM), as negative modulator of the strychnine-sensitive glycine receptor. However, the exact molecular mechanisms by which histamine acts on $GABA_{A}Rs$ are unknown. In our study, we aimed to identify amino acids potentially involved in the modulatory effect of histamine on $GABA_{A}Rs$. We expressed $GABA_{A}Rs$ with 12 different point mutations in Xenopus laevis oocytes and characterized the effect of histamine on GABA-induced currents using the two-electrode voltage clamp technique. Our data demonstrate that the amino acid residues $\beta$2(N265) and $\beta$2(M286), which are important for modulation by propofol, are not involved in the action of histamine. However, we found that histamine modulation is dependent on the amino acid residues $\alpha$1(R120), $\beta$2(Y157), $\beta$2(D163), $\beta$3(V175), and $\beta$3(Q185). We showed that the amino acid residues $\beta$2(Y157) and $\beta$3(Q185) mediate the positive modulatory effect of histamine on GABA-induced currents, whereas $\alpha$1(R120) and $\beta$2(D163) form a potential histamine interaction site in $GABA_{A}Rs$

RNA-Seq analysis of human trigeminal and dorsal root ganglia with a focus on chemoreceptors

The chemosensory capacity of the somatosensory system relies on the appropriate expression of chemoreceptors, which detect chemical stimuli and transduce sensory information into cellular signals. Knowledge of the complete repertoire of the chemoreceptors expressed in human sensory ganglia is lacking. This study employed the next-generation sequencing technique (RNA-Seq) to conduct the first expression analysis of human trigeminal ganglia (TG) and dorsal root ganglia (DRG). We analyzed the data with a focus on G-protein coupled receptors (GPCRs) and ion channels, which are (potentially) involved in chemosensation by somatosensory neurons in the human TG and DRG. For years, transient receptor potential (TRP) channels have been considered the main group of receptors for chemosensation in the trigeminal system. Interestingly, we could show that sensory ganglia also express a panel of different olfactory receptors (ORs) with putative chemosensory function. To characterize OR expression in more detail, we performed microarray, semi-quantitative RT-PCR experiments, and immunohistochemical staining. Additionally, we analyzed the expression data to identify further known or putative classes of chemoreceptors in the human TG and DRG. Our results give an overview of the major classes of chemoreceptors expressed in the human TG and DRG and provide the basis for a broader understanding of the reception of chemical cues

Odorant-dependent generation of nitric oxide in mammalian olfactory sensory neurons

The gaseous signalling molecule nitric oxide (NO) is involved in various physiological processes including regulation of blood pressure, immunocytotoxicity and neurotransmission. In the mammalian olfactory bulb (OB), NO plays a role in the formation of olfactory memory evoked by pheromones as well as conventional odorants. While NO generated by the neuronal isoform of NO synthase (nNOS) regulates neurogenesis in the olfactory epithelium, NO has not been implicated in olfactory signal transduction. We now show the expression and function of the endothelial isoform of NO synthase (eNOS) in mature olfactory sensory neurons (OSNs) of adult mice. Using NO-sensitive micro electrodes, we show that stimulation liberates NO from isolated wild-type OSNs, but not from OSNs of eNOS deficient mice. Integrated electrophysiological recordings (electro-olfactograms or EOGs) from the olfactory epithelium of these mice show that NO plays a significant role in modulating adaptation. Evidence for the presence of eNOS in mature mammalian OSNs and its involvement in odorant adaptation implicates NO as an important new element involved in olfactory signal transduction. As a diffusible messenger, NO could also have additional functions related to cross adaptation, regeneration, and maintenance of MOE homeostasis

Human trace amine-associated receptor TAAR5 can be activated by trimethylamine

In addition to the canonical olfactory receptors, TAARs were currently suggested to be a second class of chemosensory receptors in the olfactory epithelium of vertebrates. In contrast to several deorphanized murine TAARs, agonists for the intact human TAAR genes 2, 5, 6, 8 and 9 that are potentially expressed in the human olfactory epithelium have not been determined so far. Moreover, the physiological relevance of TAARs still remains elusive. We present the first successful functional expression of a human TAAR and agonists of human TAAR5. We performed a ligand screening using recombinantly expressed human TAAR5 in HANA3A cells and $\textit {Xenopus laevis}$ oocytes. In order to measure receptor activity, we used a cAMP-dependent reporter gene assay and two-electrode voltage clamp technique. As a result, human TAAR5 can be activated in a concentration-dependent manner by trimethylamine and with less efficacy by dimethylethylamine. It could neither be activated by any other of the tested single amines with a related chemical structure (42 in total), nor by any of the tested odorant mixtures. The hypothesis that Single Nucleotide Polymorphisms (SNP) within the reading frame of an olfactory receptor gene can cause a specific anosmia, formed the basis for clarifying the question, if anosmia for trimethylamine is caused by a SNP in a TAAR coding sequence. All functional human TAAR gene reading frames of subjects with specific anosmia for trimethylamine were amplified and products analyzed regarding SNP distribution. We demonstrated that the observed specific anosmia for trimethylamine is not correlated with a SNP in the coding sequence of one of the putatively functional human TAAR genes

Trigeminal ganglion neurons of mice show intracellular chloride accumulation and chloride-dependent amplification of capsaicin-induced responses

Intracellular $Cl^{−}$ concentrations $([Cl^{−}]_{i}$) of sensory neurons regulate signal transmission and signal amplification. In dorsal root ganglion (DRG) and olfactory sensory neurons (OSNs), $Cl^{−}$ is accumulated by the $Na^{+}-K^{+}-2Cl^{−}$ cotransporter 1 (NKCC1), resulting in a $[Cl^{−}]_{i}$ above electrochemical equilibrium and a depolarizing $Cl^{−}$ efflux upon $Cl^{−}$ channel opening. Here, we investigate the $[Cl^{−}]_{i}$ and function of $Cl^{−}$ in primary sensory neurons of trigeminal ganglia (TG) of wild type (WT) and $NKCC1^{−/−}$ mice using pharmacological and imaging approaches, patch-clamping, as well as behavioral testing. The $[Cl^{−}]_{i}$ of WT TG neurons indicated active NKCC1-dependent $Cl^{−}$ accumulation. Gamma-aminobutyric acid $(GABA)_{A}$ receptor activation induced a reduction of $[Cl^{−}]_{i}$ as well as $Ca^{2+}$ transients in a corresponding fraction of TG neurons. $Ca^{2+}$ transients were sensitive to inhibition of NKCC1 and voltage-gated $Ca^{2+}$ channels (VGCCs). $Ca^{2+}$ responses induced by capsaicin, a prototypical stimulus of transient receptor potential vanilloid subfamily member-1 (TRPV1) were diminished in $NKCC1^{−/−}$ TG neurons, but elevated under conditions of a lowered $[Cl^{−}]_{o}$ suggesting a $Cl^{−}$-dependent amplification of capsaicin-induced responses. Using next generation sequencing (NGS), we found expression of different $Ca^{2+}$-activated $Cl^{−}$ channels (CaCCs) in TGs of mice. Pharmacological inhibition of CaCCs reduced the amplitude of capsaicin-induced responses of TG neurons in $Ca^{2+}$ imaging and electrophysiological recordings. In a behavioral paradigm, $NKCC1^{−/−}$ mice showed less avoidance of the aversive stimulus capsaicin. In summary, our results strongly argue for a $Ca^{2+}$-activated $Cl^{−}$-dependent signal amplification mechanism in TG neurons that requires intracellular $Cl^{−}$ accumulation by NKCC1 and the activation of CaCCs

Comprehensive RNA-seq expression analysis of sensory ganglia with a focus on ion channels and GPCRs in trigeminal ganglia

The specific functions of sensory systems depend on the tissue-specific expression of genes that code for molecular sensor proteins that are necessary for stimulus detection and membrane signaling. Using the Next Generation Sequencing technique (RNA-Seq), we analyzed the complete transcriptome of the trigeminal ganglia (TG) and dorsal root ganglia (DRG) of adult mice. Focusing on genes with an expression level higher than 1 FPKM (fragments per kilobase of transcript per million mapped reads), we detected the expression of 12984 genes in the TG and 13195 in the DRG. To analyze the specific gene expression patterns of the peripheral neuronal tissues, we compared their gene expression profiles with that of the liver, brain, olfactory epithelium, and skeletal muscle. The transcriptome data of the TG and DRG were scanned for virtually all known G-protein-coupled receptors (GPCRs) as well as for ion channels. The expression profile was ranked with regard to the level and specificity for the TG. In total, we detected 106 non-olfactory GPCRs and 33 ion channels that had not been previously described as expressed in the TG. To validate the RNA-Seq data, $\textit {in situ}$ hybridization experiments were performed for several of the newly detected transcripts. To identify differences in expression profiles between the sensory ganglia, the RNA-Seq data of the TG and DRG were compared. Among the differentially expressed genes (> 1 FPKM), 65 and 117 were expressed at least 10-fold higher in the TG and DRG, respectively. Our transcriptome analysis allows a comprehensive overview of all ion channels and G protein-coupled receptors that are expressed in trigeminal ganglia and provides additional approaches for the investigation of trigeminal sensing as well as for the physiological and pathophysiological mechanisms of pain