Mechanism of the excitatory Cl- response in mouse olfactory receptor neurons.
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ABSTRACT:
SUBMITTER: Reisert J
PROVIDER: S-EPMC2877386 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:In mammalian olfactory transduction, odorants activate a cAMP-mediated signaling pathway that leads to the opening of cyclic nucleotide-gated (CNG), nonselective cation channels and depolarization. The Ca2+ influx through open CNG channels triggers an inward current through Ca2+-activated Cl channels (ANO2), which is expected to produce signal amplification. However, a study on an Ano2-/- mouse line reported no elevation in the behavioral threshold of odorant detection compared with wild type (WT). Subsequent studies by others on the same Ano2-/- line, nonetheless, found subtle defects in olfactory behavior and some abnormal axonal projections from the olfactory receptor neurons (ORNs) to the olfactory bulb. As such, the question regarding signal amplification by the Cl current in WT mouse remains unsettled. Recently, with suction-pipette recording, we have successfully separated in frog ORNs the CNG and Cl currents during olfactory transduction and found the Cl current to predominate in the response down to the threshold of action-potential signaling to the brain. For better comparison with the mouse data by others, we have now carried out similar current-separation experiments on mouse ORNs. We found that the Cl current clearly also predominated in the mouse olfactory response at signaling threshold, accounting for ?80% of the response. In the absence of the Cl current, we expect the threshold stimulus to increase by approximately sevenfold.
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Project description:The sense of smell begins with odorant molecules binding to membrane receptors on the cilia of olfactory receptor neurons (ORNs), thereby activating a G protein, G(olf), and the downstream effector enzyme, an adenylyl cyclase (ACIII). Recently, we have found in amphibian ORNs that an odorant-binding event has a low probability of activating sensory transduction at all; even when successful, the resulting unitary response apparently involves a single active Gα(olf)-ACIII molecular complex. This low amplification is in contrast to rod phototransduction in vision, the best-quantified G-protein signaling pathway, where each photoisomerized rhodopsin molecule is well known to produce substantial amplification by activating many G-protein, and hence effector-enzyme, molecules. We have now carried out similar experiments on mouse ORNs, which offer, additionally, the advantage of genetics. Indeed, we found the same low probability of transduction, based on the unitary olfactory response having a fairly constant amplitude and similar kinetics across different odorants and randomly encountered ORNs. Also, consistent with our picture, the unitary response of Gα(olf)(+/-) ORNs was similar to WT in amplitude, although their Gα(olf)-protein expression was only half of normal. Finally, from the action potential firing, we estimated that ≤19 odorant-binding events successfully triggering transduction in a WT mouse ORN will lead to signaling to the brain.
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Project description:The monogenic and monoallelic expression of only one out of >1000 mouse olfactory receptor (ORs) genes requires the formation of large heterochromatic chromatin domains that sequester the OR gene clusters. Within these domains, intergenic transcriptional enhancers evade heterochromatic silencing and converge into interchromosomal hubs that assemble over the transcriptionally active OR. The significance of this nuclear organization in OR choice remains elusive. Here, we show that transcription factors Lhx2 and Ebf specify OR enhancers by binding in a functionally cooperative fashion to stereotypically spaced motifs that defy heterochromatin. Specific displacement of Lhx2 and Ebf from OR enhancers resulted in pervasive, long-range, and trans downregulation of OR transcription, whereas pre-assembly of a multi-enhancer hub increased the frequency of OR choice in cis. Our data provide genetic support for the requirement and sufficiency of interchromosomal interactions in singular OR choice and generate general regulatory principles for stochastic, mutually exclusive gene expression programs.
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Project description: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.
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Project description:Signaling by heterotrimeric GTP-binding proteins (G proteins) drives numerous cellular processes. The number of G protein molecules activated by a single membrane receptor is a determinant of signal amplification, although in most cases this parameter remains unknown. In retinal rod photoreceptors, a long-lived photoisomerized rhodopsin molecule activates many G protein molecules (transducins), yielding substantial amplification and a large elementary (single-photon) response, before rhodopsin activity is terminated. Here we report that the elementary response in olfactory transduction is extremely small. A ligand-bound odorant receptor has a low probability of activating even one G protein molecule because the odorant dwell-time is very brief. Thus, signal amplification in olfactory transduction appears fundamentally different from that of phototransduction.
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Project description:Activation of most primary sensory neurons results in transduction currents that are carried by cations. One notable exception is the vertebrate olfactory receptor neuron (ORN), where the transduction current is carried largely by the anion [Formula: see text] However, it remains unclear why ORNs use an anionic current for signal amplification. We have sought to provide clarification on this topic by studying the so far neglected dynamics of [Formula: see text], [Formula: see text], [Formula: see text], and [Formula: see text] in the small space of olfactory cilia during an odorant response. Using computational modeling and simulations we compared the outcomes of signal amplification based on either [Formula: see text] or [Formula: see text] currents. We found that amplification produced by [Formula: see text] influx instead of a [Formula: see text] efflux is problematic for several reasons: First, the [Formula: see text] current amplitude varies greatly, depending on mucosal ion concentration changes. Second, a [Formula: see text] current leads to a large increase in the ciliary [Formula: see text] concentration during an odorant response. This increase inhibits and even reverses [Formula: see text] clearance by [Formula: see text] exchange, which is essential for response termination. Finally, a [Formula: see text] current increases the ciliary osmotic pressure, which could cause swelling to damage the cilia. By contrast, a transduction pathway based on [Formula: see text] efflux circumvents these problems and renders the odorant response robust and reliable.
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