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.

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.

Ca2؉ 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. Ca2ϩ ions function as ubiquitous second messengers that control a variety of cellular processes such as differentiation, proliferation, and apoptosis (1). Epithelial cells control their cytosolic Ca 2ϩ level via non-voltage-gated plasma membrane cationic channels and/or depletion of intracellular Ca 2ϩ stores followed by Ca 2ϩ entry via store-operated channels. Several plasma membrane channels have been implicated in modulation of cytosolic Ca 2ϩ in prostate epithelial cells (2-6). Notably, the two highly Ca 2ϩ -selective Ca 2ϩ (re)absorption channels TRPV5 3 and TRPV6 were found to be expressed in prostate cells (7-9). TRPV6 has been proposed to play a role in prostate cancer cell proliferation (9, 10). The biophysical characteristics of recombinantly overexpressed TRPV6 channels have been studied intensively (11). However, no information on channel physiology in native cells is available. Overexpression of TRPV6 leads to constitutively open channels, and so far no activation mechanism has been described (12). We previously reported that activation of PSGR by ␤-ionone induces an increase in cytosolic Ca 2ϩ and a decreased proliferation rate of prostate cancer cells (13). Thus, the aim of the present study was to examine the mechanism of Ca 2ϩ entry into prostate epithelial cells upon stimulation of PSGR, a class A GPCR that was initially identified as a prostate-specific tumor biomarker (14). Here, we identified TRPV6 as an important signaling protein downstream of activated PSGR showing that TRPV6 can function as a receptor-operated channel. We further identified the tyrosine kinase Src as a signaling protein coupling PSGR and TRPV6 independently of G proteins. In this context, we present the first electrophysiological analysis of endogenous TRPV6 channels. EXPERIMENTAL PROCEDURESCell Culture and Transfection-LNCaP cells were maintained in RPMI 1640...

Vertebrates can sense and identify a vast array of chemical cues. The molecular machinery involved in chemodetection and transduction is expressed within the cilia of olfactory sensory neurons. Currently, there is only limited information available on the distribution and density of individual signaling components within the ciliary compartment. Using super-resolution microscopy, we show here that cyclic-nucleotide-gated channels and calcium-activated chloride channels of the anoctamin family are localized to discrete microdomains in the ciliary membrane. In addition to ANO2, a second anoctamin, ANO6, also localizes to ciliary microdomains. This observation, together with the fact that ANO6 and ANO2 co-localize, indicates a role for ANO6 in olfactory signaling. We show that both ANO2 and ANO6 can form heteromultimers and that this heteromerization alters the recombinant channels' physiological properties. Thus, we provide evidence for interaction of ANO2 and ANO6 in olfactory cilia, with possible physiological relevance for olfactory signaling.