Insect olfactory receptors are heteromeric ligand-gated ion channels

In insects, each olfactory sensory neuron expresses between one and three ligand-binding members of the olfactory receptor (OR) gene family, along with the highly conserved and broadly expressed Or83b co-receptor. The functional insect OR consists of a heteromeric complex of unknown stoichiometry but comprising at least one variable odorant-binding subunit and one constant Or83b family subunit. Insect ORs lack homology to G-protein-coupled chemosensory receptors in vertebrates and possess a distinct seven-transmembrane topology with the amino terminus located intracellularly. Here we provide evidence that heteromeric insect ORs comprise a new class of ligand-activated non-selective cation channels. Heterologous cells expressing silkmoth, fruitfly or mosquito heteromeric OR complexes showed extracellular Ca2+ influx and cation-non-selective ion conductance on stimulation with odorant. Odour-evoked OR currents are independent of known G-protein-coupled second messenger pathways. The fast response kinetics and OR-subunit-dependent K+ ion selectivity of the insect OR complex support the hypothesis that the complex between OR and Or83b itself confers channel activity. Direct evidence for odorant-gated channels was obtained by outside-out patch-clamp recording of Xenopus oocyte and HEK293T cell membranes expressing insect OR complexes. The ligand-gated ion channel formed by an insect OR complex seems to be the basis for a unique strategy that insects have acquired to respond to the olfactory environment.     

A cyclic nucleotide-gated conductance in olfactory receptor cilia

Olfactory transduction is thought to be initiated by the binding of odorants to specific receptor proteins in the cilia of olfactory receptor cells. The mechanism by which odorant binding could initiate membrane depolarization is unknown, but the recent discovery of an odorant-stimulated adenylate cyclase in purified olfactory cilia suggests that cyclic AMP may serve as an intracellular messenger for olfactory transduction. If so, then there might be a conductance in the ciliary plasma membrane which is controlled by cAMP. Here we report that excised patches of ciliary plasma membrane, obtained from dissociated receptor cells, contain a conductance which is gated directly by cAMP. This conductance resembles the cyclic GMP-gated conductance that mediates phototransduction in rod and cone outer segments, but differs in that it is activated by both cAMP and cGMP. Our data provide a mechanistic basis by which an odorant-stimulated increase in cyclic nucleotide concentration could lead to an increase in membrane conductance and therefore, to membrane depolarization. These data suggest a remarkable similarity between the mechanisms of olfactory and visual transduction and indicate considerable conservation of sensory transduction mechanisms.     

Primary structure and functional expression of a cyclic nucleotide-activated channel from olfactory neurons.

Odorant signal transduction occurs in the specialized cilia of the olfactory sensory neurons. Considerable biochemical evidence now indicates that this process could be mediated by a G protein-coupled cascade using cyclic AMP as an intracellular second messenger. A stimulatory G protein alpha subunit is expressed at high levels in olfactory neurons and is specifically enriched in the cilia, as is a novel form of adenylyl cyclase. This implies that the olfactory transduction cascade might involve unique molecular components. Electrophysiological studies have identified a cyclic nucleotide-activated ion channel in olfactory cilia. These observations provide evidence for a model in which odorants increase intracellular cAMP concentration, which in turn activates this channel and depolarizes the sensory neuron. An analogous cascade regulating a cGMP-gated channel mediates visual transduction in photoreceptor cells. The formal similarities between olfactory and visual transduction suggest that the two systems might use homologous channels. Here we report the molecular cloning, functional expression and characterization of a channel that is likely to mediate olfactory transduction.     

A second class of chemosensory receptors in the olfactory epithelium

The mammalian olfactory system detects chemicals sensed as odours as well as social cues that stimulate innate responses. Odorants are detected in the nasal olfactory epithelium by the odorant receptor family, whose approximately 1,000 members allow the discrimination of a myriad of odorants. Here we report the discovery of a second family of receptors in the mouse olfactory epithelium. Genes encoding these receptors, called 'trace amine-associated receptors' (TAARs), are present in human, mouse and fish. Like odorant receptors, individual mouse TAARs are expressed in unique subsets of neurons dispersed in the epithelium. Notably, at least three mouse TAARs recognize volatile amines found in urine: one detects a compound linked to stress, whereas the other two detect compounds enriched in male versus female urine-one of which is reportedly a pheromone. The evolutionary conservation of the TAAR family suggests a chemosensory function distinct from odorant receptors. Ligands identified for TAARs thus far suggest a function associated with the detection of social cues.     

Enantioselectivity of the Musk Odor Sensation

1 Introduction Chiral recognition of substrates is one of the most characteristic phenomena of biological activity. And one of the most fundamental biological activities of chemical substances is their smell. In 1991, Linda Buck and Richard Axel~([1]) discovered a large multigene family that en codes odorant receptors, for which they were awarded the 2004 Nobel Prize in Medicine and Physiology. These odorant receptors are highly homologous, consist of ca. 320 amino acids, and show a heptah…     

Odorant receptors instruct functional circuitry in the mouse olfactory bulb

The mammalian olfactory system detects and discriminates thousands of odorants using many different receptors expressed by sensory neurons in the nasal epithelium. Axonal projections from these neurons to the main olfactory bulbs form reproducible patterns of glomeruli in two widely separated regions of each bulb, creating two mirror-symmetric maps of odorant receptor projections. To investigate whether odorant receptors organize neural circuitry in the olfactory bulb, we have examined a genetically modified mouse line, rI7 --> M71, in which a functionally characterized receptor, rI7, has been substituted into the M71 receptor locus. Here we show that despite their ectopic location the resulting glomeruli are responsive to known ligands of the rI7 receptor, attract postsynaptic innervation by mitral/tufted cell dendrites, and endow these cells with responses that are characteristic of the rI7 receptor. External tufted cells receiving input from rI7 --> M71 glomeruli form precise intrabulbar projections that link medial and lateral rI7 --> M71 glomeruli anatomically, thus providing a substrate for coordinating isofunctional glomeruli. We conclude that odorant receptor identity in epithelial neurons determines not only glomerular convergence and function, but also functional circuitry in the olfactory bulb.     

Comparative chemosensation from receptors to ecology

Odour perception is initiated by specific interactions between odorants and a large repertoire of receptors in olfactory neurons. During the past few years, considerable progress has been made in tracing olfactory perception from the odorant receptor protein to the activity of olfactory neurons to higher processing centres and, ultimately, to behaviour. The most complete picture is emerging for the simplest olfactory system studied--that of the fruitfly Drosophila melanogaster. Comparison of rodent, insect and nematode olfaction reveals surprising differences and unexpected similarities among chemosensory systems.     

Innate versus learned odour processing in the mouse olfactory bulb

The mammalian olfactory system mediates various responses, including aversive behaviours to spoiled foods and fear responses to predator odours. In the olfactory bulb, each glomerulus represents a single species of odorant receptor. Because a single odorant can interact with several different receptor species, the odour information received in the olfactory epithelium is converted to a topographical map of multiple glomeruli activated in distinct areas in the olfactory bulb. To study how the odour map is interpreted in the brain, we generated mutant mice in which olfactory sensory neurons in a specific area of the olfactory epithelium are ablated by targeted expression of the diphtheria toxin gene. Here we show that, in dorsal-zone-depleted mice, the dorsal domain of the olfactory bulb was devoid of glomerular structures, although second-order neurons were present in the vacant areas. The mutant mice lacked innate responses to aversive odorants, even though they were capable of detecting them and could be conditioned for aversion with the remaining glomeruli. These results indicate that, in mice, aversive information is received in the olfactory bulb by separate sets of glomeruli, those dedicated for innate and those for learned responses.     

The Ras-MAPK pathway is important for olfaction in Caenorhabditis elegans

The Ras-MAPK (mitogen-activated protein kinase) signal transduction pathway is well known to control cellular proliferation and differentiation in response to extracellular signals, but its other functions are less understood. In Caenorhabditis elegans this pathway regulates several developmental events, such as vulval induction and progression of meiosis, but its function in the nervous system is unknown. Here we report that the Ras-MAPK pathway is involved in olfaction in this organism. Mutational inactivation and hyperactivation of this pathway impairs efficiency of chemotaxis to a set of odorants. Experiments in which let-60 ras was expressed using a heat-shock promoter and a cell-specific promoter show that a normal activity of LET-60 Ras is required in mature olfactory neurons. Application of the odorant isoamylalcohol to wild-type animals leads to the activation of MAP kinase in olfactory neurons within 10 seconds. This induction is dependent on the function of the nucleotide-gated channel TAX-2/TAX-4 and the voltage-activated calcium channel subunit UNC-2. These results suggest a dynamic regulatory role for the Ras-MAPK pathway in perception and transmission of sensory signals in olfactory neurons.     

RGS2 regulates signal transduction in olfactory neurons by attenuating activation of adenylyl cyclase III

The heterotrimeric G-protein Gs couples cell-surface receptors to the activation of adenylyl cyclases and cyclic AMP production (reviewed in refs 1, 2). RGS proteins, which act as GTPase-activating proteins (GAPs) for the G-protein alpha-subunits alpha(i) and alpha(q), lack such activity for alpha(s) (refs 3-6). But several RGS proteins inhibit cAMP production by Gs-linked receptors. Here we report that RGS2 reduces cAMP production by odorant-stimulated olfactory epithelium membranes, in which the alpha(s) family member alpha(olf) links odorant receptors to adenylyl cyclase activation. Unexpectedly, RGS2 reduces odorant-elicited cAMP production, not by acting on alpha(olf) but by inhibiting the activity of adenylyl cyclase type III, the predominant adenylyl cyclase isoform in olfactory neurons. Furthermore, whole-cell voltage clamp recordings of odorant-stimulated olfactory neurons indicate that endogenous RGS2 negatively regulates odorant-evoked intracellular signalling. These results reveal a mechanism for controlling the activities of adenylyl cyclases, which probably contributes to the ability of olfactory neurons to discriminate odours.     

A single class of olfactory neurons mediates behavioural responses to a Drosophila sex pheromone

Insects, like many other animals, use sex pheromones to coordinate their reproductive behaviours. Volatile pheromones are detected by odorant receptors expressed in olfactory receptor neurons (ORNs). Whereas fruit odours typically activate multiple ORN classes, pheromones are thought to act through single dedicated classes of ORN. This model predicts that activation of such an ORN class should be sufficient to trigger the appropriate behavioural response. Here we show that the Drosophila melanogaster male-specific pheromone 11-cis-vaccenyl acetate (cVA) acts through the receptor Or67d to regulate both male and female mating behaviour. Mutant males that lack Or67d inappropriately court other males, whereas mutant females are less receptive to courting males. These data suggest that cVA has opposite effects in the two sexes: inhibiting mating behaviour in males but promoting mating behaviour in females. Replacing Or67d with moth pheromone receptors renders these ORNs sensitive to the corresponding moth pheromones. In such flies, moth pheromones elicit behavioural responses that mimic the normal response to cVA. Thus, activation of a single ORN class is both necessary and sufficient to mediate behavioural responses to the Drosophila sex pheromone cVA.     

Movement of 'gating charge' is coupled to ligand binding in a G-protein-coupled receptor

Activation by agonist binding of G-protein-coupled receptors (GPCRs) controls most signal transduction processes. Although these receptors span the cell membrane, they are not considered to be voltage sensitive. Recently it was shown that both the activity of GPCRs and their affinity towards agonists are regulated by membrane potential. However, it remains unclear whether GPCRs intrinsically respond to changes in membrane potential. Here we show that two prototypical GPCRs, the m2 and m1 muscarinic receptors (m2R and m1R), display charge-movement-associated currents analogous to 'gating currents' of voltage-gated channels. The gating charge-voltage relationship of m2R correlates well with the voltage dependence of the affinity of the receptor for acetylcholine. The loop that couples m2R and m1R to their G protein has a crucial function in coupling voltage sensing to agonist-binding affinity. Our data strongly indicate that GPCRs serve as sensors for both transmembrane potential and external chemical signals.     

Genetic tracing reveals a stereotyped sensory map in the olfactory cortex.

The olfactory system translates myriad chemical structures into diverse odour perceptions. To gain insight into how this is accomplished, we prepared mice that coexpressed a transneuronal tracer with only one of about 1,000 different odorant receptors. The tracer travelled from nasal neurons expressing that receptor to the olfactory bulb and then to the olfactory cortex, allowing visualization of cortical neurons that receive input from a particular odorant receptor. These studies revealed a stereotyped sensory map in the olfactory cortex in which signals from a particular receptor are targeted to specific clusters of neurons. Inputs from different receptors overlap spatially and could be combined in single neurons, potentially allowing for an integration of the components of an odorant's combinatorial receptor code. Signals from the same receptor are targeted to multiple olfactory cortical areas, permitting the parallel, and perhaps differential, processing of inputs from a single receptor before delivery to the neocortex and limbic system.     

An essential role for a CD36-related receptor in pheromone detection in Drosophila

The CD36 family of transmembrane receptors is present across metazoans and has been implicated biochemically in lipid binding and transport. Several CD36 proteins function in the immune system as scavenger receptors for bacterial pathogens and seem to act as cofactors for Toll-like receptors by facilitating recognition of bacterially derived lipids. Here we show that a Drosophila melanogaster CD36 homologue, Sensory neuron membrane protein (SNMP), is expressed in a population of olfactory sensory neurons (OSNs) implicated in pheromone detection. SNMP is essential for the electrophysiological responses of OSNs expressing the receptor OR67d to (Z)-11-octadecenyl acetate (cis-vaccenyl acetate, cVA), a volatile male-specific fatty-acid-derived pheromone that regulates sexual and social aggregation behaviours. SNMP is also required for the activation of the moth pheromone receptor HR13 by its lipid-derived pheromone ligand (Z)-11-hexadecenal, but is dispensable for the responses of the conventional odorant receptor OR22a to its short hydrocarbon fruit ester ligands. Finally, we show that SNMP is required for responses of OR67d to cVA when ectopically expressed in OSNs not normally activated by pheromones. Because mammalian CD36 binds fatty acids, we suggest that SNMP acts in concert with odorant receptors to capture pheromone molecules on the surface of olfactory dendrites. Our work identifies an unanticipated cofactor for odorant receptors that is likely to have a widespread role in insect pheromone detection. Moreover, these results define a unifying model for CD36 function, coupling recognition of lipid-based extracellular ligands to signalling receptors in both pheromonal communication and pathogen recognition through the innate immune system.     

α-LTX and α-LTXN4C induce [Ca2+]i elevation through different mechanisms in pancreatic β cells

α-latrotoxin (α-LTX) is the only neurotoxin from black-widow spider which has secretagogue effects in the vertebrates. It causes massive neurotransmitter and hormone release via two instinct mechanisms after binding with its high-affinity membrane recep…     

Transduction in taste receptor cells requires cAMP-dependent protein kinase

In taste chemoreception, cyclic adenosine monophosphate (cAMP) appears to be one of the intracellular messengers coupling reception of stimulus to the generation of the response. The recent finding that sweet agents cause a GTP-dependent generation of cAMP poses the question of how this cytosolic messenger acts at the membrane of taste receptor cells. We have shown that cAMP causes a substantial depolarization in these cells. Here we show with whole-cell recordings and inside-out membrane patches that the depolarization caused by cAMP is accounted for by the action of cAMP-dependent protein kinase, which inactivates potassium channels predominantly of 44 pS conductance. Thus, intracellular signalling of the gustatory cells differs from that of olfactory and photoreceptor cells, where cyclic nucleotides control unspecific channels by binding to them rather than by inducing their phosphorylation.     

Odorant-sensitive adenylate cyclase may mediate olfactory reception

The mechanism of the sense of smell has long been a subject for theory and speculation. More recently, the notion of odorant recognition by stereospecific protein receptors has gained wide acceptance, but the receptor molecules remained elusive. The recognition molecules are believed to be quite diverse, which would partly explain the unusual difficulties encountered in their isolation by conventional ligand-binding techniques. An alternative approach would be to probe the receptors through transductory components that may be common to all receptor types. Here we report the identification of one such transductory molecular component. This is an odorant-sensitive adenylate cyclase, present in very large concentrations in isolated dendritic membranes of olfactory sensory neurones. Odorant activation of the enzyme is ligand and tissue specific, and occurs only in the presence of GTP, suggesting the involvement of receptor(s) coupled to a guanine nucleotide binding protein (G-protein). The olfactory G-protein is independently identified by labelling with bacterial toxins, and found to be similar to stimulatory G-proteins in other systems. Our results suggest a role for cyclic nucleotides in olfactory transduction, and point to a molecular analogy between olfaction and visual, hormone and neurotransmitter reception. Most importantly, the present findings reveal new ways to identify and isolate olfactory receptor proteins.     

基于人工嗅觉的呈香物质识别方法

研究了人工嗅觉在物质识别中的应用，概述了使用模式识别辨别物质气味的基本原理，详细介绍了人工嗅觉系统的硬件设计，算法和软件实现方法，最后使用本系统对不同香气物质进行了识别，取得了较好的结果。     

A natural polymorphism alters odour and DEET sensitivity in an insect odorant receptor

Blood-feeding insects such as mosquitoes are efficient vectors of human infectious diseases because they are strongly attracted by body heat, carbon dioxide and odours produced by their vertebrate hosts. Insect repellents containing DEET (N,N-diethyl-meta-toluamide) are highly effective, but the mechanism by which this chemical wards off biting insects remains controversial despite decades of investigation. DEET seems to act both at close range as a contact chemorepellent, by affecting insect gustatory receptors, and at long range, by affecting the olfactory system. Two opposing mechanisms for the observed behavioural effects of DEET in the gas phase have been proposed: that DEET interferes with the olfactory system to block host odour recognition and that DEET actively repels insects by activating olfactory neurons that elicit avoidance behaviour. Here we show that DEET functions as a modulator of the odour-gated ion channel formed by the insect odorant receptor complex. The functional insect odorant receptor complex consists of a common co-receptor, ORCO (ref. 15) (formerly called OR83B; ref. 16), and one or more variable odorant receptor subunits that confer odour selectivity. DEET acts on this complex to potentiate or inhibit odour-evoked activity or to inhibit odour-evoked suppression of spontaneous activity. This modulation depends on the specific odorant receptor and the concentration and identity of the odour ligand. We identify a single amino-acid polymorphism in the second transmembrane domain of receptor OR59B in a Drosophila melanogaster strain from Brazil that renders OR59B insensitive to inhibition by the odour ligand and modulation by DEET. Our data indicate that natural variation can modify the sensitivity of an odour-specific insect odorant receptor to odour ligands and DEET. Furthermore, they support the hypothesis that DEET acts as a molecular 'confusant' that scrambles the insect odour code, and provide a compelling explanation for the broad-spectrum efficacy of DEET against multiple insect species.     

Rapid kinetics of second messenger formation in olfactory transduction

Olfactory transduction is thought to be mediated by a membrane-bound receptor protein initiating a multistep reaction cascade which ultimately leads to a depolarizing generator current. There is considerable evidence for the involvement of adenylate cyclase in vertebrate olfactory transduction, and some data indicate that phospholipase C may have a central role in insect olfaction. However, one must show that odorants not only stimulate enzyme activity but also induce changes in concentrations of relevant second messengers. One important criterion for a candidate second messenger of chemo-electrical transduction is that its formation must precede the onset of the odorant-induced membrane permeability changes which proceed on a subsecond time-scale. Here we report an odorant-induced, transient accumulation of cyclic AMP in isolated olfactory cilia from rats, and the generation of inositol trisphosphate in antennal preparations from insects, both of which show subsecond time courses that are sufficiently rapid to mediate the odorant-regulated permeability of olfactory receptor cells.