The TRPM8 ion channel comprises direct Gq protein-activating capacity.

The transient receptor potential (TRP) family of ion channels comprises receptors that are activated by a vast variety of physical as well as chemical stimuli. TRP channels interact in a complex manner with several intracellular signaling cascades, both up- and downstream of receptor activation. Investigating cascades stimulated downstream of the cold and menthol receptor TRPM8, we found evidence for both, functional and structural interaction of TRPM8 with Gαq. We demonstrated menthol-evoked increase in intracellular Ca(2+) under extracellular Ca(2+)-free conditions, which was blocked by the PLC inhibitors U73122 or edelfosine. This metabotropic Ca(2+) signal could be observed also in cells expressing a channel-dead (i.e. non-conducting) or a chloride-conducting TRPM8 pore mutant. However, this intracellular metabotropic Ca(2+) signal could not be detected in Gαq deficient cells or in the presence of dominant-negative GαqX. Evidence for a close spatial proximity necessary for physical interaction of TRPM8 and Gαq was provided by acceptor bleaching experiments demonstrating FRET between TRPM8-CFP and Gαq-YFP. A Gαq-YFP mobility assay (FRAP) revealed a restricted diffusion of Gαq-YFP under conditions when TRPM8 is immobilized in the plasma membrane. Moreover, a menthol-induced and TRPM8-mediated G protein activation could be demonstrated by FRET experiments monitoring the dissociation of Gαq-YFP from a Gß/Gγ-CFP complex, and by the exchange of radioactive [(35)S]GTPγS for GDP. Our observations lead to a view that extends the operational range of the TRPM8 receptor from its function as a pure ion channel to a molecular switch with additional metabotropic capacity.

Inhibitory odorant signaling in Mammalian olfactory receptor neurons.

Odorants inhibit as well as excite olfactory receptor neurons (ORNs) in many species of animals. Cyclic nucleotide-dependent activation of canonical mammalian ORNs is well established but it is still unclear how odorants inhibit these cells. Here we further implicate phosphoinositide-3-kinase (PI3K), an indispensable element of PI signaling in many cellular processes, in olfactory transduction in rodent ORNs. We show that odorants rapidly and transiently activate PI3K in the olfactory cilia and in the olfactory epithelium in vitro. We implicate known G-protein-coupled isoforms of PI3K and show that they modulate not only the magnitude but also the onset kinetics of the electrophysiological response of ORNs to complex odorants. Finally, we show that the ability of a single odorant to inhibit another can be PI3K dependent. Our collective results provide compelling support for the idea that PI3K-dependent signaling mediates inhibitory odorant input to mammalian ORNs and at least in part contributes to the mixture suppression typically seen in the response of ORNs to complex natural odorants.

PI3Kgamma-dependent signaling in mouse olfactory receptor neurons.

Phosphatidylinositol 3-kinase (PI3K)-dependent signaling couples to receptors for many different ligands in diverse cellular systems. Recent findings suggest that PI3K-dependent signaling also mediates inhibition of odorant responses in rat olfactory receptor neurons (ORNs). Here, we present evidence that murine ORNs show PI3K-dependent calcium responses to odorant stimulation, they express 2 G protein-coupled receptor (GPCR)-activated isoforms of PI3K, PI3Kbeta and PI3Kgamma, and they exhibit odorant-induced PI3K activity. These findings support our use of a transgenic mouse model to begin to investigate the mechanisms underlying PI3K-mediated inhibition of odorant responses in mammalian ORNs. Mice deficient in PI3Kgamma, a class IB PI3K that is activated via GPCRs, lack detectable odorant-induced PI3K activity in their olfactory epithelium and their ORNs are less sensitive to PI3K inhibition. We conclude that odorant-dependent PI3K signaling generalizes to the murine olfactory system and that PI3Kgamma plays a role in mediating inhibition of odorant responses in mammalian ORNs.

Identification of a Novel Gnao-Mediated Alternate Olfactory Signaling Pathway in Murine OSNs.

It is generally agreed that in olfactory sensory neurons (OSNs), the binding of odorant molecules to their specific olfactory receptor (OR) triggers a cAMP-dependent signaling cascade, activating cyclic-nucleotide gated (CNG) channels. However, considerable controversy dating back more than 20 years has surrounded the question of whether alternate signaling plays a role in mammalian olfactory transduction. In this study, we demonstrate a specific alternate signaling pathway in Olfr73-expressing OSNs. Methylisoeugenol (MIEG) and at least one other known weak Olfr73 agonist (Raspberry Ketone) trigger a signaling cascade independent from the canonical pathway, leading to the depolarization of the cell. Interestingly, this pathway is mediated by Gnao activation, leading to Cl(-) efflux; however, the activation of adenylyl cyclase III (ACIII), the recruitment of Ca(2+) from extra-or intracellular stores, and phosphatidylinositol 3-kinase-dependent signaling (PI signaling) are not involved. Furthermore, we demonstrated that our newly identified pathway coexists with the canonical olfactory cAMP pathway in the same OSN and can be triggered by the same OR in a ligand-selective manner. We suggest that this pathway might reflect a mechanism for odor recognition predominantly used in early developmental stages before olfactory cAMP signaling is fully developed. Taken together, our findings support the existence of at least one odor-induced alternate signal transduction pathway in native OSNs mediated by Olfr73 in a ligand-selective manner.