The Stimulatory Gαs Protein Is Involved in Olfactory Signal Transduction in Drosophila

Seven-transmembrane receptors typically mediate olfactory signal transduction by coupling to G-proteins. Although insect odorant receptors have seven transmembrane domains like G-protein coupled receptors, they have an inverted membrane topology, constituting a key difference between the olfactory systems of insects and other animals. While heteromeric insect ORs form ligand-activated non-selective cation channels in recombinant expression systems, the evidence for an involvement of cyclic nucleotides and G-proteins in odor reception is inconsistent. We addressed this question in vivo by analyzing the role of G-proteins in olfactory signaling using electrophysiological recordings. We found that Gαs plays a crucial role for odorant induced signal transduction in OR83b expressing olfactory sensory neurons, but not in neurons expressing CO2 responsive proteins GR21a/GR63a. Moreover, signaling of Drosophila ORs involved Gαs also in a heterologous expression system. In agreement with these observations was the finding that elevated levels of cAMP result in increased firing rates, demonstrating the existence of a cAMP dependent excitatory signaling pathway in the sensory neurons. Together, we provide evidence that Gαs plays a role in the OR mediated signaling cascade in Drosophila

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 alpha1beta 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 muM to 42.4+/-2.3 muM. In addition, histamine blocked the spontaneous activity of RNA-edited alpha3beta 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

Deep Sequencing of the Murine Olfactory Receptor Neuron Transcriptome

The ability of animals to sense and differentiate among thousands of odorants relies on a large set of olfactory receptors (OR) and a multitude of accessory proteins within the olfactory epithelium (OE). ORs and related signaling mechanisms have been the subject of intensive studies over the past years, but our knowledge regarding olfactory processing remains limited. The recent development of next generation sequencing (NGS) techniques encouraged us to assess the transcriptome of the murine OE. We analyzed RNA from OEs of female and male adult mice and from fluorescence-activated cell sorting (FACS)-sorted olfactory receptor neurons (ORNs) obtained from transgenic OMP-GFP mice. The Illumina RNA-Seq protocol was utilized to generate up to 86 million reads per transcriptome. In OE samples, nearly all OR and trace amine-associated receptor (TAAR) genes involved in the perception of volatile amines were detectably expressed. Other genes known to participate in olfactory signaling pathways were among the 200 genes with the highest expression levels in the OE. To identify OE-specific genes, we compared olfactory neuron expression profiles with RNA-Seq transcriptome data from different murine tissues. By analyzing different transcript classes, we detected the expression of non-olfactory GPCRs in ORNs and established an expression ranking for GPCRs detected in the OE. We also identified other previously undescribed membrane proteins as potential new players in olfaction. The quantitative and comprehensive transcriptome data provide a virtually complete catalogue of genes expressed in the OE and present a useful tool to uncover candidate genes involved in, for example, olfactory signaling, OR trafficking and recycling, and proliferation

Correlations of expression levels plotted for each detected OR gene.

<p><b>A.</b> Correlation of the OR gene expression patterns between male and female CD1 mice. Only OR genes with detectable expression levels (FPKM>0.1) are shown. The FPKM values are logarithmically presented. The Pearson correlation coefficient of r = 0.83 confirmed the strong correlation of OR gene expression patterns between female and male CD1 mice. The three ORs (Olfr533, Olfr1507 and Olfr309) with the highest expression levels were also the most highly expressed in the RNA-Seq data from both sexes. <b>B.</b> Correlation of OR gene expression patterns between females of strain CD1 and C57BL6. The Pearson correlation coefficient of r = 0.75 confirmed the strong correlation between the expression patterns of OR genes between the different strains; however, these patterns exhibited greater divergence between strains than between the sexes of the CD1 strain. The most highly expressed ORs, Olfr533 and Olfr309, had the same expression ranking, and Olfr1507 was among the ten most highly expressed OR genes in both strains. <b>C.</b> Verification of RNA-Seq results for ORs by real time RT-PCR. Expression levels are relative to mOREG (Olfr73). Error bars represent the SEM.</p

Expression pattern of known genes of the olfactory signal transduction.

<div><p><b>A.</b> Heatmap showing the expression levels of genes known to be involved in olfactory signaling and other genes known to be highly expressed in ORNs as determined by NGS. The FPKM of OR total represents the accumulated gene expression of all ORs and shows that the OR is the most highly expressed gene in the OE. The main components of the signal transduction scheme were among the 200 most highly expressed genes. The FPKM values shown for OMP in sorted ORNs (homozygous) are rough estimations based on the calculation of reads located in the 3’-untranslated regions of OMP and are therefore only valid to a limited extent. Higher FPKM values are indicated by deeper colors.</p> <p><b>B.</b> RT-PCR verification of the highly expressed genes in the OE. Gene expression was normalized to the level of adenylyl cyclase type III (ACIII) RNA. The Investigated genes were RTP1–4, REEP1–4, Stom, Stoml2–3 and transmembrane proteins, including ANO2 (Tmem16b). Error bars represent the SEM.</p></div

Further genes for non-GPCR membrane proteins that were among the 1,000 most highly expressed genes.

<p>Further genes for non-GPCR membrane proteins that were among the 1,000 most highly expressed genes.</p

Expression pattern of chemoreceptor genes.

<p>Heatmap showing the expression levels of the following chemoreceptor classes: TAARs, VNO receptors, GC-D, taste receptors and FPR in olfactory (male and female OE, and FACS-sorted ORNs) and non-olfactory tissue (brain, muscle, liver and testes). Higher FPKM values are indicated by deeper colors. Only genes with a FPKMs >1 are represented in this chart.</p

Summary of RNA-Seq data.

<p>Summary of RNA-Seq data.</p