Calcium Regulation of Cyclic Nucleotide Signaling in Lobster Olfactory Receptor Neurons

An elevated free Ca2+ concentration reduces odor-stimulated production of cyclic AMP (cAMP) in the outer dendritic membranes of lobster olfactory receptor neurons in vitro. This effect can occur within 50 ms of odor stimulation. The effect is concentration-dependent at submicromolar concentrations of free Ca2+. An elevated free Ca2+ concentration also reduces basal and forskolin-stimulated cAMP levels in a concentration-dependent manner, suggesting that Ca2+ is not targeting the activation of the odor receptor/G protein complex. The degradation of synthetic cAMP by phosphodiesterases is not enhanced by an increased free Ca2+ concentration, suggesting that Ca2+ acts by down-regulating the olfactory adenylyl cyclase. Western blot analysis of the lobster olfactory sensilla that contain the outer dendrites reveals a protein in the transduction zone with a molecular mass of approximately 138 kDa that is immunoreactive to an antiserum against adenylyl cyclase type III. Given earlier evidence that Ca2+ potentially enters the receptor cell through odor-activated inositol 1,4,5-trisphosphate-gated channels, our results suggest a possible route for cross talk between the cyclic nucleotide and the inositol phospholipid signaling pathways in lobster olfactory receptor neurons.     

Characterization of a phosphoinositide-mediated odor transduction pathway reveals plasma membrane localization of an inositol 1,4, 5-trisphosphate receptor in lobster olfactory receptor neurons

The role of phosphoinositide signaling in olfactory transduction is still being resolved. Compelling functional evidence for the transduction of odor signals via phosphoinositide pathways in olfactory transduction comes from invertebrate olfactory systems, in particular lobster olfactory receptor neurons. We now provide molecular evidence for two components of the phosphoinositide signaling pathway in lobster olfactory receptor neurons, a G protein alpha subunit of the G(q) family and an inositol 1,4, 5-trisphosphate-gated channel or an inositol 1,4,5-trisphosphate (IP(3)) receptor. Both proteins localize to the site of olfactory transduction, the outer dendrite of the olfactory receptor neurons. Furthermore, the IP(3) receptor localizes to membranes in the ciliary transduction compartment of these cells at both the light microscopic and electron microscopic levels. Given the absence of intracellular organelles in the sub-micron diameter olfactory cilia, this finding indicates that the IP(3) receptor is associated with the plasma membrane and provides the first definitive evidence for plasma membrane localization of an IP(3)R in neurons. The association of the IP(3) receptor with the plasma membrane may be a novel mechanism for regulating intracellular cations in restricted cellular compartments of neurons.     

Renal cystic disease proteins play critical roles in the organization of the olfactory epithelium

It was reported that some proteins known to cause renal cystic disease (NPHP6; BBS1, and BBS4) also localize to the olfactory epithelium (OE), and that mutations in these proteins can cause anosmia in addition to renal cystic disease. We demonstrate here that a number of other proteins associated with renal cystic diseases - polycystin 1 and 2 (PC1, PC2), and Meckel-Gruber syndrome 1 and 3 (MKS1, MKS3) - localize to the murine OE. PC1, PC2, MKS1 and MKS3 are all detected in the OE by RT-PCR. We find that MKS3 localizes specifically to dendritic knobs of olfactory sensory neurons (OSNs), while PC1 localizes to both dendritic knobs and cilia of mature OSNs. In mice carrying mutations in MKS1, the expression of the olfactory adenylate cyclase (AC3) is substantially reduced. Moreover, in rats with renal cystic disease caused by a mutation in MKS3, the laminar organization of the OE is perturbed and there is a reduced expression of components of the odor transduction cascade (G(olf), AC3) and α-acetylated tubulin. Furthermore, we show with electron microscopy that cilia in MKS3 mutant animals do not manifest the proper microtubule architecture. Both MKS1 and MKS3 mutant animals show no obvious alterations in odor receptor expression. These data show that multiple renal cystic proteins localize to the OE, where we speculate that they work together to regulate aspects of the development, maintenance or physiological activities of cilia.     

Cellular and molecular constituents of olfactory sensation in vertebrates

Since the discovery of odorant-activated adenylate cyclase in the olfactory receptor cilia, research into the olfactory perception of vertebrates has rapidly expanded. Recent studies have shown how the odor discrimination starts at the receptor level: each of 700-1000 types of the olfactory neurons in the neural olfactory epithelium contains a single type of odor receptor protein. Although the receptors have relatively low specific affinities for odorants, excitation of different types of receptors forms an excitation pattern specific to each odorant in the glomerular layer of the olfactory bulb. It was demonstrated that adenosine 3',5'-cyclic monophosphate (cAMP) is very likely the sole second messenger for olfactory transduction. It was also demonstrated that the affinity of the cyclic nucleotide-gated channel for cAMP regulated by Ca(2+)/calmodulin is solely responsible for the adaptation of the cell. However, many other regulatory components were found in the transduction cascade. Regulated by Ca(2+) and/or the protein-phosphorylation, many of them may serve for the adaptation of the cell, probably on a longer time scale. It may be important to consider the resensitization as a part of this adaptation, as well as to collect kinetic data of each reaction to gain further insight into the olfactory mechanism.     

The proteome of rat olfactory sensory cilia

Olfactory sensory neurons expose to the inhaled air chemosensory cilia which bind odorants and operate as transduction organelles. Odorant receptors in the ciliary membrane activate a transduction cascade which uses cAMP and Ca²⁺ for sensory signaling in the ciliary lumen. Although the canonical transduction pathway is well established, molecular components for more complex aspects of sensory transduction, like adaptation, regulation, and termination of the receptor response have not been systematically identified. Moreover, open questions in olfactory physiology include how the cilia exchange solutes with the surrounding mucus, assemble their highly polarized set of proteins, and cope with noxious substances in the ambient air. A specific ciliary proteome would promote research efforts in all of these fields. We have improved a method to detach cilia from rat olfactory sensory neurons and have isolated a preparation specifically enriched in ciliary membrane proteins. Using LC‐ESI‐MS/MS analysis, we identified 377 proteins which constitute the olfactory cilia proteome. These proteins represent a comprehensive data set for olfactory research since more than 80% can be attributed to the characteristic functions of olfactory sensory neurons and their cilia: signal processing, protein targeting, neurogenesis, solute transport, and cytoprotection. Organellar proteomics thus yielded decisive information about the diverse physiological functions of a sensory organelle.     

昆虫气味受体研究进展

嗅觉在昆虫的多种行为中发挥关键作用。气味分子与嗅觉神经元树突上气味受体的结合,参与了昆虫嗅觉识别的初始过程。昆虫的嗅觉神经元表达两类气味受体： 一是传统气味受体,该类受体同源性较低,在少部分嗅觉神经元中表达; 二是Or83b家族受体,该类受体不感受气味,在不同昆虫间较为保守且在大多数嗅觉神经元中表达。目前,对于单个传统气味受体的气味分子配体特异性所知甚少; 对于Or83b家族受体,一般认为其可能具有将传统气味受体运送至嗅觉神经元树突膜上的功能。此外,有一些实验证据不支持昆虫气味受体为G蛋白偶联受体的观点。     

Failure to implicate cAMP in transduction in lobster olfactory receptor cells

The possible role of adenosine 3',5'-cyclic monophosphate (cAMP)in olfactory transduction in the spiny lobster was investigatedusing radioimmunoassay of cAMP and intracellular recording.Application of forskolin or 1-isobutyl-3-methylxanthine increasedcAMP levels in intact sensilla containing the chemoreceptiveouter dendritic segments of the lobster olfactory receptor cell,thereby demonstrating adenylate cyclase and phosphodiesteraseactivity in the sensilla. A complex odor mixture and identifiedexcitatory odor molecules failed to stimulate the productionof cAMP, however In intracellular recordings, superfusion ofthe outer dendritic segments with forskolin, 1-isobutyl-3-methylxanthineand cyclic nucleotide analogs had no direct effect on odor-responsivecells. These compounds did cause infrequent enhancements (sixof 42 cells) of odor-evoked receptor potentials, but processesother than transduction are the most likely causes of this effect.We conclude that cAMP-dependent transduction mechanisms areunlikely to mediate most odor responses in lobsters, in contrastto transduction mechanisms in amphibians and rats.     

Flash photolysis of caged compounds in the cilia of olfactory sensory neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP or caged Ca. We use a flash lamp to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration.     

The stimulatory Gα(s) protein is involved in olfactory signal transduction in Drosophila

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 Gα_s 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 Gα_s 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 Gα_s plays a role in the OR mediated signaling cascade in Drosophila.     

Go contributes to olfactory reception in Drosophila melanogaster

Background  

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 and function as ligand-gated cation channels. Consequently, the involvement of cyclic nucleotides and G proteins in insect odor reception is controversial. Since the heterotrimeric G_oα subunit is expressed in Drosophila olfactory receptor neurons, we reasoned that G_o acts together with insect odorant receptor cation channels to mediate odor-induced physiological responses.     

Modelling and sensitivity analysis of the reactions involving receptor,G-protein and effector in vertebrate olfactory receptor neurons

A biochemical model of the receptor, G-protein and effector (RGE) interactions during transduction in the cilia of vertebrate olfactory receptor neurons (ORNs) was developed and calibrated to experimental recordings of cAMP levels and the receptor current (RC). The model describes the steps from odorant binding to activation of the effector enzyme which catalyzes the conversion of ATP to cAMP, and shows how odorant stimulation is amplified and delayed by the RGE transduction cascade. A time-dependent sensitivity analysis was performed on the model. The model output—the cAMP production rate—is particularly sensitive to a few, dominant parameters. During odorant stimulation it depends mainly on the initial density of G-proteins and the catalytic constant for cAMP production.     

腺苷酸环化酶3缺失对小鼠主要嗅觉表皮组织内相关因子及信号通路的影响

主要嗅觉表皮(main olfactory epithelium, MOE)是哺乳动物感知气味分子的主要嗅觉器官。在MOE组织内,大多数嗅觉神经元通过cAMP信号传导通路感知气味信息。作为嗅觉cAMP信号通路的主要成员之一,腺苷酸环化酶3（adenylyl cyclase 3, ac3）基因敲除小鼠嗅觉探测功能丧失。除cAMP信号传导通路外,MOE内AC3相关因子AC2和AC4,以及肌醇1,4,5-三磷酸（inositol 1,4,5-trisphosphate,IP3）信号通路和Sonic Hedgehog（Shh）信号通路均有表达。然而,敲除ac3是否会对ac2和ac4以及IP3和Shh信号通路成员产生影响,尚不清楚。本文以AC3缺失（AC3-/-）及其野生型小鼠（AC3+/+）MOE为材料,采用实时荧光定量PCR（qRT-PCR）和免疫荧光组织化学方法,发现AC3缺失后,MOE内的ac2和ac4,以及IP3信号通路中的IP3受体ip3r1及钙调蛋白calm1和calm2表达水平均明显降低。Shh信号通路中的受体patched(ptch)与smoothened(smo)、以及核转录因子gli1与gli2的表达也受到了影响。总之,AC3基因缺失不但导致小鼠MOE组织中cAMP信号通路受损,同时AC3相关因子,IP3信号通路和Shh信号通路的传导也受到抑制。本文对于阐明AC3基因敲除小鼠嗅觉丧失的原因及其嗅觉探测机制具有重要启示作用。     

Two second messengers mediate amino acid responses in olfactory sensory neurons of the salamander,Necturus maculosus

Odor transduction mediated by the adenylyl cyclase/cAMP pathway has been well studied, but it is still uncertain whether this pathway mediates the transduction of all odors in vertebrates. We isolated olfactory sensory neurons from the salamander Necturus maculosus and used calcium imaging with the indicator dye fura-2 to examine olfactory responses elicited by amino acids. The properties of approximately two-thirds of the odor responses suggested they were mediated by the adenylyl cyclase/cAMP pathway, but one-third of the responses were not mimicked by cAMP analogs nor blocked by inhibition of adenylyl cyclase, suggesting that these odor responses were mediated differently. Responses that were unaffected by inhibition of adenylyl cyclase were blocked by neomycin, an inhibitor of phospholipase C, implying that they were transduced by activation of phospholipase C. Some cells which responded to more than one amino acid appeared to employ both pathways, but each was used to transduce different odors. In addition, many responses that were mediated by the adenylyl cyclase/cAMP pathway were enhanced following inhibition of phospholipase C, suggesting that the phospholipase C pathway has a role not only in odor transduction, but also in the modulation of olfactory responses.     

Ca2+-Activated Cl? Channels of the ClCa Family Express in the Cilia of a Subset of Rat Olfactory Sensory Neurons

The Ca²⁺-activated Cl⁻ channel is considered a key constituent of odor transduction. Odorant binding to a specific receptor in the cilia of olfactory sensory neurons (OSNs) triggers a cAMP cascade that mediates the opening of a cationic cyclic nucleotide-gated channel (CNG), allowing Ca²⁺ influx. Ca²⁺ ions activate Cl⁻ channels, generating a significant Cl⁻ efflux, with a large contribution to the receptor potential. The Anoctamin 2 channel (ANO2) is a major constituent of the Cl⁻ conductance, but its knock-out has no impairment of behavior and only slightly reduces field potential odorant responses of the olfactory epithelium. Likely, an additional Ca²⁺-activated Cl⁻ channel of unknown molecular identity is also involved. In addition to ANO2, we detected two members of the ClCa family of Ca²⁺-activated Cl⁻ channels in the rat olfactory epithelium, ClCa4l and ClCa2. These channels, also expressed in the central nervous system, may correspond to odorant transduction channels. Whole Sprague Dawley olfactory epithelium nested RT-PCR and single OSNs established that the mRNAs of both channels are expressed in OSNs. Real time RT-PCR and full length sequencing of amplified ClCa expressed in rat olfactory epithelium indicated that ClCa4l is the most abundant. Immunoblotting with an antibody recognizing both channels revealed immunoreactivity in the ciliary membrane. Immunochemistry of olfactory epithelium and OSNs confirmed their ciliary presence in a subset of olfactory sensory neurons. The evidence suggests that ClCa4l and ClCa2 might play a role in odorant transduction in rat olfactory cilia.     

Proteins associated with soluble adenylyl cyclase in sea urchin sperm flagella

Adenylyl cyclases (ACs) synthesize cAMP and are present in cells as transmembrane AC and soluble AC (sAC). In sperm, the cAMP produced regulates ion channels and it also activates protein kinase-A that in turn phosphorylates specific axonemal proteins to activate flagellar motility. In mammalian sperm, sAC localizes to the midpiece of flagella, whereas in sea urchin sperm sAC is along the entire flagellum. Here we show that in sea urchin sperm, sAC is complexed with proteins of the plasma membrane and axoneme. Immunoprecipitation shows that a minimum of 10 proteins is tightly associated with sAC. Mass spectrometry of peptides derived from these proteins shows them to be: axonemal dynein heavy chains 7 and 9, sperm specific Na+/H+ exchanger, cyclic nucleotide-gated ion channel, sperm specific creatine kinase, membrane bound guanylyl cyclase, cyclic GMP specific phosphodiesterase 5A, the receptor for the egg peptide speract, and alpha- and beta-tubulins. The sAC-associated proteins could be important in linking membrane signal transduction to energy utilisation in the regulation of flagellar motility.     

The mAKAP signaling complex: integration of cAMP, calcium, and MAP kinase signaling pathways

Following its production by adenylyl cyclases, the second messenger cAMP is in involved in pleiotrophic signal transduction. The effectors of cAMP include the cAMP-dependent protein kinase (PKA), the guanine nucleotide exchange factor Epac (exchange protein activated by cAMP), and cAMP-dependent ion channels. In turn, cAMP signaling is attenuated by phosphodiesterase-catalyzed degradation. The association of cAMP effectors and the enzymes that regulate cAMP concentration into signaling complexes helps to explain the differential signaling initiated by members of the G(s)-protein coupled receptor family. The signal transduction complex formed by the scaffold protein mAKAP (muscle A kinase-anchoring protein) at the nuclear envelope of both striated myocytes and neurons contains three cAMP-binding proteins, PKA, Epac1, and the phosphodiesterase PDE4D3. In addition, the mAKAP complex also contains components of the ERK5 MAP kinase signaling pathway, the calcium release channel ryanodine receptor and the phosphatases PP2A as well as calcineurin. Analysis of the mAKAP complex illustrates how a macromolecular complex can serve as a node in the intracellular signaling network of cardiac myocytes to integrate multiple cAMP signals with those of calcium and MAP kinases to regulate the hypertrophic actions of several hormones.     

Olfactory receptor responses to sex pheromone components in the redbanded leafroller moth

Electrical responses of single olfactory receptor neurons of the male redbanded leafroller moth were elicited by each of the principle components of the sex pheromone and six other behaviorally active compounds. Response frequencies to equal intensities of each of these compounds and changes in response frequency with increasing amounts of any one compound, varied from receptor to receptor. These differences in response characteristics appear to be due to factors intrinsic to the olfactory recptor neuron and not to factors external to it. The encoding of odor quality by these receptor neurons cannot be in the simple presence or absence of activity in any one of them. Rather, odor quality may be encoded by the pattern of activity which invariably arises across an ensemble of receptor neurons, each having its own distribution of sensitivities.     

Activation of the sensory current in salamander olfactory receptor neurons depends on a G protein-mediated cAMP second messenger system.

Olfactory receptor neurons respond to odor stimulation with an inward cationic current. Under whole-cell patch clamp, individual, isolated olfactory receptors were exposed to pharmacological agents known to interact with distinct enzymes in a putative second messenger cascade, and their response to odors was measured. IBMX prolonged the odor-evoked current and also reduced its amplitude. cAMP and cGMP induced a current electrically identical to the odor current, but the current showed desensitization only with cAMP. GTP-gamma-s prolonged and GDP-beta-s interfered with the odor-evoked current. The long latency seen in the odor response appears to be mainly due to the loading of the G protein and secondarily to the requirement for cAMP accumulation. The main source of the response decay appears to be cyclic nucleotide hydrolysis.     

Odor suppression of voltage-gated currents contributes to the odor-induced response in olfactory neurons

Olfactorychemotransduction involves a signaling cascade. In addition totriggering transduction, odors suppress ion conductances. Bystimulating with brief odorant pulses, we observed a current associatedwith odor-induced suppression of voltage-gated conductances and studiedits time dependence. We characterized this suppression current inisolated Caudiverbera caudiverberaolfactory neurons. All four voltage-gated currents are suppressed byodor pulses in almost every neuron, and suppression is caused by odorsinducing excitation and by those inducing inhibition, indicating anonselective phenomenon, in contrast to transduction. Suppression has a10-fold shorter latency than transduction. Suppression was morepronounced when odors were applied to the soma than to the cilia,opposite to transduction. Suppression was also present in rat olfactory neurons. Furthermore, we could induce it inDrosophila photoreceptor cells,demonstrating its independence from the chemotransduction cascade. Weshow that odor concentrations causing suppression are similar to thosetriggering chemotransduction and that both suppression and transductioncontribute to the odor response in isolated olfactory neurons.Furthermore, suppression affects spiking, implying a possiblephysiological role in olfaction.