Elizabeth A. Corey

Ionotropic Crustacean Olfactory Receptors

The nature of the olfactory receptor in crustaceans, a major group of arthropods, has remained elusive. We report that spiny lobsters, Panulirus argus, express ionotropic receptors (IRs), the insect chemosensory variants of ionotropic glutamate receptors. Unlike insects IRs, which are expressed in a specific subset of olfactory cells, two lobster IR subunits are expressed in most, if not all, lobster olfactory receptor neurons (ORNs), as confirmed by antibody labeling and in situ hybridization. Ligand-specific ORN responses visualized by calcium imaging are consistent with a restricted expression pattern found for other potential subunits, suggesting that cell-specific expression of uncommon IR subunits determines the ligand sensitivity of individual cells. IRs are the only type of olfactory receptor that we have detected in spiny lobster olfactory tissue, suggesting that they likely mediate olfactory signaling. Given long-standing evidence for G protein-mediated signaling in activation of lobster ORNs, this finding raises the interesting specter that IRs act in concert with second messenger-mediated signaling.

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.

Blockade of Insect Odorant Receptor Currents by Amiloride Derivatives

Insect odorant receptors (ORs) function as heteromeric odorant-gated ion channels consisting of a conserved coreceptor, Orco, and an odorant-sensitive tuning subunit. Although some OR modulators have been identified, an extended library of pharmacological tools is currently lacking and would aid in furthering our understanding of insect OR complexes. We now demonstrate that amiloride and several derivatives, which have been extensively used as blockers for various ion channels and transporters, also block odorant-gated currents from 2 OR complexes from the malaria vector mosquito Anopheles gambiae. In addition, both heteromeric and homomeric ORs were susceptible to amiloride blockade when activated by VUAA1, an agonist that targets the Orco channel subunit. Amiloride derivatives therefore represent a valuable class of channel blockers that can be used to investigate the pharmacological and biophysical properties of insect OR function.

Odorant-stimulated phosphoinositide signaling in mammalian olfactory receptor neurons

Recent evidence has revived interest in the idea that phosphoinositides (PIs) may play a role in signal transduction in mammalian olfactory receptor neurons (ORNs). To provide direct evidence that odorants indeed activate PI signaling in ORNs, we used adenoviral vectors carrying two different fluorescently tagged probes, the pleckstrin homology (PH) domains of phospholipase C delta 1 (PLC delta 1) and the general receptor of phosphoinositides (GRP1), to monitor PI activity in the dendritic knobs of ORNs in vivo. Odorants mobilized PI(4,5)P(2)/IP(3) and PI(3,4,5)P(3), the substrates and products of PLC and PI3K. We then measured odorant activation of PLC and PI3K in olfactory ciliary-enriched membranes in vitro using a phospholipid overlay assay and ELISAs. Odorants activated both PLC and PI3K in the olfactory cilia within 2s of odorant stimulation. Odorant-dependent activation of PLC and PI3K in the olfactory epithelium could be blocked by enzyme-specific inhibitors. Odorants activated PLC and PI3K with partially overlapping specificity. These results provide direct evidence that odorants indeed activate PI signaling in mammalian ORNs in a manner that is consistent with the idea that PI signaling plays a role in olfactory transduction.

PI3Kγ-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.

The pore properties of human nociceptor channel TRPA1 evaluated in single channel recordings

TRPA channels detect stimuli of different sensory modalities, including a broad spectrum of chemosensory stimuli, noxious stimuli associated with tissue damage and inflammation, mechanical stimuli, and thermal stimuli. Despite a growing understanding of potential modulators, agonists, and antagonists for these channels, the exact mechanisms of channel regulation and activation remain mostly unknown or controversial and widely debated. Relatively little is also known about the basic biophysical parameters of both native and heterologously expressed TRPA channels. Here we use conventional single channel inside-out and outside-out patch recording from the human TRPA1 channel transiently expressed in human embryonic kidney 293T cells to characterize the selectivity of the channel for inorganic mono-/divalent and organic monovalent cations in the presence of allylisothiocyanate (AITC). We show the relative permeability of the hTRPA1 channel to inorganic cations to be:and to organic cations:Na(+)(1.0)≥ dimethylamine (0.99)>trimethylamine (0.7)>tetramethylammonium (0.4)>N-methyl-d-glucamine (0.1). Activation of the hTRPA1 channels by AITC appears to recruit the channels to a conformational state with an increased permeability to large organic cations. The pore of the channels in this state can be characterized as dilated by approximately 1-2.5 Å. These findings provide important insight into the basic fundamental properties and function of TRPA1 channels in general and human TRPA1 channel in particular.

Phosphoinositide 3-kinase dependent inhibition as a broad basis for opponent coding in Mammalian olfactory receptor neurons.

Phosphoinositide 3-kinase (PI3K) signaling has been implicated in mediating inhibitory odorant input to mammalian olfactory receptor neurons (ORNs). To better understand the breadth of such inhibition in odor coding, we screened a panel of odorants representing different chemical classes, as well as odorants known to occur in a natural odor object (tomato), for their ability to rapidly activate PI3K-dependent inhibitory signaling. Odorants were screened on dissociated native rat ORNs before and after pre-incubation with the PI3K-isoform specific blockers AS252424 and TGX221. Many different odorants increased their excitatory strength for particular ORNs following PI3K blockade in a manner consistent with activating PI3K-dependent inhibitory signaling in those cells. The PI3K-dependent inhibitory odorants overlapped with conventional excitatory odorants, but did not share the same bias, indicating partial partitioning of the odor space. Finding that PI3K-dependent inhibition can be activated by a wide range of otherwise conventional excitatory odorants strongly implies PI3K-dependent inhibition provides a broad basis for opponent coding in mammalian ORNs.

Candidate pheromone receptors of codling moth Cydia pomonella respond to pheromones and kairomones

Olfaction plays a dominant role in the mate-finding and host selection behaviours of the codling moth (Cydia pomonella), an important pest of apple, pear and walnut orchards worldwide. Antennal transcriptome analysis revealed a number of abundantly expressed genes related to the moth olfactory system, including those encoding the olfactory receptors (ORs) CpomOR1, CpomOR3 and CpomOR6a, which belong to the pheromone receptor (PR) lineage, and the co-receptor (CpomOrco). Using heterologous expression, in both Drosophila olfactory sensory neurones and in human embryonic kidney cells, together with electrophysiological recordings and calcium imaging, we characterize the basic physiological and pharmacological properties of these receptors and demonstrate that they form functional ionotropic receptor channels. Both the homomeric CpomOrco and heteromeric CpomOrco + OR complexes can be activated by the common Orco agonists VUAA1 and VUAA3, as well as inhibited by the common Orco antagonists amiloride derivatives. CpomOR3 responds to the plant volatile compound pear ester ethyl-(E,Z)-2,4-decadienoate, while CpomOR6a responds to the strong pheromone antagonist codlemone acetate (E,E)-8,10-dodecadien-1-yl acetate. These findings represent important breakthroughs in the deorphanization of codling moth pheromone receptors, as well as more broadly into insect ecology and evolution and, consequently, for the development of sustainable pest control strategies based on manipulating chemosensory communication.

Phosphoinositide 3-kinase mediated signaling in lobster olfactory receptor neurons

J. Neurochem. (2010) 113, 341–350.

An inhibitor of Na+/Ca2+ exchange blocks activation of insect olfactory receptors

Earlier we showed that the Na(+)/Ca(2+) exchanger inhibitor, KB-R7943, potently blocks the odor-evoked activity of lobster olfactory receptor neurons. Here we extend that finding to recombinant mosquito olfactory receptors stably expressed in HEK cells. Using whole-cell and outside-out patch clamping and calcium imaging, we demonstrate that KB-R7943 blocks both the odorant-gated current and the odorant-evoked calcium signal from two different OR complexes from the malaria vector mosquito, Anopheles gambiae, AgOr48+AgOrco and AgOr65+AgOrco. Both heteromeric and homomeric (Orco alone) OR complexes were susceptible to KB-R7943 blockade when activated by VUAA1, an agonist that targets the Orco channel subunit, suggesting the Orco subunit may be the target of the drugs action. KB-R7943 represents a valuable tool to further investigate the functional properties of arthropod olfactory receptors and raises the interesting specter that activation of these ionotropic receptors is directly or indirectly linked to a Na(+)/Ca(2+) exchanger, thereby providing a template for drug design potentially allowing improved control of insect pests and disease vectors.