The inositol 1,4,5‐trisphosphate receptor is required for maintenance of olfactory adaptation in Drosophila antennae

A role for inositol 1,4,5‐trisphosphate (IP₃) as a second messenger during olfactory transduction has been postulated in both vertebrates and invertebrates. However, given the absence of either suitable pharmacological reagents or mutant alleles specific for the IP₃ signaling pathway, an unequivocal demonstration of IP₃ function in olfaction has not been possible. Here we have investigated the role of a well‐established cellular target of IP₃—the IP₃ receptor (IP₃R)—in olfactory transduction in Drosophila. For this purpose we tested existing viable combinations of IP₃R mutant alleles, as well as a newly generated set of viable itpr alleles, for olfactory function. In all of the viable allelic combinations primary olfactory responses were found to be normal. However, a subset of itpr alleles (including a null allele) exhibit faster recovery after a strong pulse of odor, indicating that the IP₃R is required for maintenance of olfactory adaptation. Interestingly, this defect in adaptation is dominant for two of the alleles tested, suggesting that the mechanism of adaptation is sensitive to levels of the IP₃R. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 282–288, 2000     

Genetic dissection of itpr gene function reveals a vital requirement in aminergic cells of Drosophila larvae

Signaling by the second messenger inositol 1,4,5-trisphosphate is thought to affect several developmental and physiological processes. Mutants in the inositol 1,4,5-trisphosphate receptor (itpr) gene of Drosophila exhibit delays in molting while stronger alleles are also larval lethal. In a freshly generated set of EMS alleles for the itpr locus we have sequenced and identified single point mutations in seven mutant chromosomes. The predicted allelic strength of these mutants matches the observed levels of lethality. They range from weak hypomorphs to complete nulls. Interestingly, lethality in three heteroallelic combinations has a component of cold sensitivity. The temporal focus of cold sensitivity lies in the larval stages, predominantly at second instar. Coupled with our earlier observation that an itpr homozygous null allele dies at the second instar stage, it appears that there is a critical period for itpr gene function in second instar larvae. Here we show that the focus of this critical function lies in aminergic cells by rescue with UAS-itpr and DdCGAL4. However, this function does not require synaptic activity, suggesting that InsP(3)-mediated Ca(2+) release regulates the neurohormonal action of serotonin.     

Inositol 1,4,5-trisphosphate transduction cascade in taste reception of the fleshfly, Boettcherisca peregrina

The role of an inositol 1,4,5-trisphosphate (IP3)-mediated transduction cascade in the response of taste receptor cells of the fleshfly Boettcherisca peregrina was investigated by using the following reagents: neomycin (an inhibitor of IP3 production), U73122 (an inhibitor of phospholipase C), adenophostin A (an agonist of the IP3-gated channel), IP3, ruthenium red (a blocker of the IP3-gated channel), and 2-aminoethoxydiphenylborate (2-APB; an antagonist of the IP3-gated channel). For introduction into the receptor cell, the reagents were mixed with a detergent, deoxycholate (DOC). After treatment with neomycin + DOC or U73122 + DOC, the response of the sugar receptor cell to sugars was depressed compared with responses after treatment with DOC alone. During the treatment of adenophostin A + DOC, the response of the sugar receptor cell was elicited. After treatment with IP3 + DOC, the response of the sugar receptor cell to sugars and to amino acids was apparently enhanced. When taste stimuli were administered in the presence of ruthenium red or 2-APB, the response of the sugar receptor cell to glucose were inhibited. The expression of genes for substances involved in the IP3 transduction cascade, such as G protein alpha subunit (dGqalpha), phospholipase C (norpA), and IP3 receptor (itpr), were examined in the taste receptor cell of the fruitfly Drosophila melanogaster by using the pox-neuro70 mutant (poxn70), which lacks taste receptor cells. The expressed levels of dGqalpha and itpr in the tarsus of poxn70 mutant flies were reduced compared with those of wild-type flies. These results suggest that the IP3 transduction cascade is involved in the response of the sugar receptor cell of the fly.     

Functional properties of the Drosophila melanogaster inositol 1,4,5-trisphosphate receptor mutants

The inositol (1,4,5)-trisphosphate receptor (InsP(3)R) is an intracellular calcium (Ca(2+)) release channel that plays a crucial role in cell signaling. In Drosophila melanogaster a single InsP(3)R gene (itpr) encodes a protein (DmInsP(3)R) that is approximately 60% conserved with mammalian InsP(3)Rs. A number of itpr mutant alleles have been identified in genetic screens and studied for their effect on development and physiology. However, the functional properties of wild-type or mutant DmInsP(3)Rs have never been described. Here we use the planar lipid bilayer reconstitution technique to describe single-channel properties of embryonic and adult head DmInsP(3)R splice variants. The three mutants chosen in this study reside in each of the three structural domains of the DmInsP(3)R-the amino-terminal ligand binding domain (ug3), the middle-coupling domain (wc703), and the channel-forming region (ka901). We discovered that 1), the major functional properties of DmInsP(3)R (conductance, gating, and sensitivity to InsP(3) and Ca(2+)) are remarkably conserved with the mammalian InsP(3)R1; 2), single-channel conductance of the adult head DmInsP(3)R isoform is 89 pS and the embryonic DmInsP(3)R isoform is 70 pS; 3), ug3 mutation affects sensitivity of the DmInsP(3)Rs to activation by InsP(3), but not their InsP(3)-binding properties; 4), wc703 channels have increased sensitivity to modulation by Ca(2+); and 5), homomeric ka901 channels are not functional. We correlated the results obtained in planar lipid bilayer experiments with measurements of InsP(3)-induced Ca(2+) fluxes in microsomes isolated from wild-type and heterozygous itpr mutants. Our study validates the use of D. melanogaster as an appropriate model for InsP(3)R structure-function studies and provides novel insights into the fundamental mechanisms of the InsP(3)R function.     

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.     

Regulation of the type 1 inositol 1,4,5-trisphosphate receptor by phosphorylation at tyrosine 353

The inositol 1,4,5-trisphosphate receptor (IP3R) plays an essential role in Ca2+ signaling during lymphocyte activation. Engagement of the T cell or B cell receptor by antigen initiates a signal transduction cascade that leads to tyrosine phosphorylation of IP3R by Src family nonreceptor protein tyrosine kinases, including Fyn. However, the effect of tyrosine phosphorylation on the IP3R and subsequent Ca2+ release is poorly understood. We have identified tyrosine 353 (Tyr353) in the IP3-binding domain of type 1 IP3R (IP3R1) as a phosphorylation site for Fyn both in vitro and in vivo. We have developed a phosphoepitope-specific antibody and shown that IP3R1-Y353 becomes phosphorylated during T cell and B cell activation. Furthermore, tyrosine phosphorylation of IP3R1 increased IP3 binding at low IP3 concentrations (<10 nm). Using wild-type IP3R1 or an IP3R1-Y353F mutant that cannot be tyrosine phosphorylated at Tyr353 or expressed in IP3R-deficient DT40 B cells, we demonstrated that tyrosine phosphorylation of Tyr353 permits prolonged intracellular Ca2+ release during B cell activation. Taken together, these data suggest that one function of tyrosine phosphorylation of IP3R1-Y353 is to enhance Ca2+ signaling in lymphocytes by increasing the sensitivity of IP3R1 to activation by low levels of IP3.     

Sequencing and exon mapping of the inositol 1,4,5-trisphosphate receptor cDNA from Drosophila embryos suggests the presence of differentially regulated forms of RNA and protein.

A single gene appears to code for the inositol 1,4,5-trisphosphate receptor (itpr) in Drosophila melanogaster, as compared to three known genes in mammals. Expression of the itpr gene in Drosophila occurs in a wide range of tissues and developmental stages, suggesting its requirement during diverse cellular and physiological processes. A head cDNA for the Drosophila IP3R has previously been cloned and sequenced. Here we present and analyse the sequence of cDNAs encoding the complete IP3R, obtained from embryonic stages. The embryonic cDNA is 10525bp long and is a splice variant of the head cDNA. It differs from the latter in three main respects. It has longer 5' and 3' untranslated regions, two potential casein kinase II sites are missing in the embryo form and it contains an alternate exon which results in the replacement of three residues (VHF) in the head form by five residues (GVGHSV) in the embryo form. The significance of these changes is discussed. An exon-intron map of the gene derived from sequencing of intron-containing genomic fragments is also presented. The gene has a total of 11 introns, of which more than half are clustered in a region of the modulatory domain of the IP3R.     

Odorant-Dependent Generation of Nitric Oxide in Mammalian Olfactory Sensory Neurons

The gaseous signalling molecule nitric oxide (NO) is involved in various physiological processes including regulation of blood pressure, immunocytotoxicity and neurotransmission. In the mammalian olfactory bulb (OB), NO plays a role in the formation of olfactory memory evoked by pheromones as well as conventional odorants. While NO generated by the neuronal isoform of NO synthase (nNOS) regulates neurogenesis in the olfactory epithelium, NO has not been implicated in olfactory signal transduction. We now show the expression and function of the endothelial isoform of NO synthase (eNOS) in mature olfactory sensory neurons (OSNs) of adult mice. Using NO-sensitive micro electrodes, we show that stimulation liberates NO from isolated wild-type OSNs, but not from OSNs of eNOS deficient mice. Integrated electrophysiological recordings (electro-olfactograms or EOGs) from the olfactory epithelium of these mice show that NO plays a significant role in modulating adaptation. Evidence for the presence of eNOS in mature mammalian OSNs and its involvement in odorant adaptation implicates NO as an important new element involved in olfactory signal transduction. As a diffusible messenger, NO could also have additional functions related to cross adaptation, regeneration, and maintenance of MOE homeostasis.     

Type-specific inositol 1,4,5-trisphosphate receptor localization in the vomeronasal organ and its interaction with a transient receptor potential channel,TRPC2

The vomeronasal organ (VNO) is the receptor portion of the accessory olfactory system and transduces chemical cues that identify social hierarchy, reproductive status, conspecifics and prey. Signal transduction in VNO neurons is apparently accomplished via an inositol 1,4,5-trisphosphate (IP3)-activated calcium conductance that includes a different set of G proteins than those identified in vertebrate olfactory sensory neurons. We used immunohistochemical (IHC) and SDS-PAGE/western analysis to localize three IP3 receptors (IP3R) in the rat VNO epithelium. Type-I IP3R expression was weak or absent. Antisera for type-II and -III IP3R recognized appropriate molecular weight proteins by SDS-PAGE, and labeled protein could be abolished by pre-adsorption of the respective antibody with antigenic peptide. In tissue sections, type-II IP3R immunoreactivity was present in the supporting cell zone but not in the sensory cell zone. Type-III IP3R immunoreactivity was present throughout the sensory zone and overlapped that of transient receptor potential channel 2 (TRPC2) in the microvillar layer of sensory epithelium. Co-immunoprecipitation of type-III IP3R and TRPC2 from VNO lysates confirmed the overlapping immunoreactivity patterns. The protein-protein interaction complex between type-III IP3R and TRPC2 could initiate calcium signaling leading to electrical signal production in VNO neurons.     

Gated conductances in native and reconstituted membranes from frog olfactory cilia.

Although cAMP is well established as a second messenger for olfactory transduction in vertebrates, the role of inositol 1,4,5-trisphosphate (IP3) in this process remains controversial. We addressed this issue by comparing currents evoked by cAMP and IP3 in native and reconstituted membranes from olfactory cilia. We detected only a cyclic nucleotide-gated conductance in the native membrane but both cyclic nucleotide-gated and IP3-gated conductances in the reconstituted membrane. The magnitudes of the cyclic nucleotide- and IP3-gated conductances were not correlated with each other in reconstituted membranes, suggesting that cyclic nucleotide- and IP3-gated channels originate in different cellular compartments.     

Interactions between the inositol 1,4,5-trisphosphate and cyclic AMP signaling pathways regulate larval molting in Drosophila

Larval molting in Drosophila, as in other insects, is initiated by the coordinated release of the steroid hormone ecdysone, in response to neural signals, at precise stages during development. In this study we have analyzed, using genetic and molecular methods, the roles played by two major signaling pathways in the regulation of larval molting in Drosophila. Previous studies have shown that mutants for the inositol 1,4,5-trisphosphate receptor gene (itpr) are larval lethals. In addition they exhibit delays in molting that can be rescued by exogenous feeding of 20-hydroxyecdysone. Here we show that mutants for adenylate cyclase (rut) synergize, during larval molting, with itpr mutant alleles, indicating that both cAMP and InsP(3) signaling pathways function in this process. The two pathways act in parallel to affect molting, as judged by phenotypes obtained through expression of dominant negative and dominant active forms of protein kinase A (PKA) in tissues that normally express the InsP(3) receptor. Furthermore, our studies predict the existence of feedback inhibition through protein kinase A on the InsP(3) receptor by increased levels of 20-hydroxyecdysone.     

Expression of gustducin overlaps with that of type III IP3 receptor in taste buds of the rat soft palate

Type III IP3 receptor (IP3R3) is one of the common critical calcium-signaling molecules for sweet, umami, and bitter signal transduction in taste cells, and the total IP3R3-expressing cell population represents all cells mediating these taste modalities in the taste buds. Although gustducin, a taste cell-specific G-protein, is also involved in sweet, umami, and bitter signal transduction, the expression of gustducin is restricted to different subsets of IP3R3-expressing cells by location in the tongue. Based on the expression patterns of gustducin and taste receptors in the tongue, the function of gustducin has been implicated primarily in bitter taste in the circumvallate (CV) papillae and in sweet taste in the fungiform (FF) papillae. However, in the soft palate (SP), the expression pattern of gustducin remains unclear and little is known about its function. In the present paper, the expression patterns of gustducin and IP3R3 in taste buds of the SP and tongue papillae in the rat were examined by double-color whole-mount immunohistochemistry. Gustducin was expressed in almost all (96.7%) IP3R3-expressing cells in taste buds of the SP, whereas gustducin-positive cells were 42.4% and 60.1% of IP3R3-expressing cells in FF and CV, respectively. Our data suggest that gustducin is involved in signal transduction of all the tastes of sweet, umami, and bitter in the SP, in contrast to its limited function in the tongue.     

pipsqueak encodes a novel nuclear protein required downstream of seven-up for the development of photoreceptors R3 and R4.

Photoreceptor induction in the Drosophila eye is mediated by activation of the Ras signal transduction cascade. Although this process is well understood, little is known about how the diversity of photoreceptor subtypes is generated. The pipsqueak (psq) gene is expressed at high levels in the R3/R4 precursors during eye development and this expression depends on seven-up (svp) gene function. Moreover, strong psq alleles are dominant suppressors of a svp-induced cone cell transformation phenotype. Although the gene was previously identified and described as a member of the maternal posterior group of genes, the strong semilethal alleles isolated here demonstrate a specific requirement for psq function downstream of svp for the development of photoreceptors R3/R4. The gene has three independent 5' ends and codes for several nuclear protein isoforms, some containing the POZ domain which has been implicated in protein-protein interactions. Interestingly, all viable alleles with a maternal posterior group phenotype cluster around one specific 5' exon, while all semilethal alleles have lesions which map to a different alternative 5' exon.     

The IP3 receptor/Ca2+ channel and its cellular function

The IP3R [IP3 (inositol 1,4,5-trisphosphate) receptor] is responsible for Ca2+ release from the ER (endoplasmic reticulum). We have been working extensively on the P400 protein, which is deficient in Purkinje-neuron-degenerating mutant mice. We have discovered that P400 is an IP3R and we have determined the primary sequence. Purified IP3R, when incorporated into a lipid bilayer, works as a Ca2+ release channel and overexpression of IP3R shows enhanced IP3 binding and channel activity. Addition of an antibody blocks Ca2+ oscillations indicating that IP3R1 works as a Ca2+ oscillator. Studies on the role of IP3R during development show that IP3R is involved in fertilization and is essential for determination of dorso-ventral axis formation. We found that IP3R is involved in neuronal plasticity. A double homozygous mutant of IP3R2 (IP3R type 2) and IP3R3 (IP3R type 3) shows a deficit of saliva secretion and gastric juice secretion suggesting that IP3Rs are essential for exocrine secretion. IP3R has various unique properties: cryo-EM (electron microscopy) studies show that IP3R contains multiple cavities; IP3R allosterically and dynamically changes its form reversibly (square form-windmill form); IP3R is functional even though it is fragmented by proteases into several pieces; the ER forms a meshwork but also forms vesicular ER and moves along microtubules using a kinesin motor; X ray analysis of the crystal structure of the IP3 binding core consists of an N-terminal beta-trefoil domain and a C-terminal alpha-helical domain. We have discovered ERp44 as a redox sensor in the ER which binds to the luminal part of IP3R1 and regulates its activity. We have also found the role of IP3 is not only to release Ca2+ but also to release IRBIT which binds to the IP3 binding core of IP3R.     

An IP3R3- and NPY-Expressing Microvillous Cell Mediates Tissue Homeostasis and Regeneration in the Mouse Olfactory Epithelium

Calcium-dependent release of neurotrophic factors plays an important role in the maintenance of neurons, yet the release mechanisms are understudied. The inositol triphosphate (IP3) receptor is a calcium release channel that has a physiological role in cell growth, development, sensory perception, neuronal signaling and secretion. In the olfactory system, the IP3 receptor subtype 3 (IP3R3) is expressed exclusively in a microvillous cell subtype that is the predominant cell expressing neurotrophic factor neuropeptide Y (NPY). We hypothesized that IP3R3-expressing microvillous cells secrete sufficient NPY needed for both the continual maintenance of the neuronal population and for neuroregeneration following injury. We addressed this question by assessing the release of NPY and the regenerative capabilities of wild type, IP3R3^+/−, and IP3R3^−/− mice. Injury, simulated using extracellular ATP, induced IP3 receptor-mediated NPY release in wild-type mice. ATP-evoked NPY release was impaired in IP3R3^−/− mice, suggesting that IP3R3 contributes to NPY release following injury. Under normal physiological conditions, both IP3R3^−/− mice and explants from these mice had fewer progenitor cells that proliferate and differentiate into immature neurons. Although the number of mature neurons and the in vivo rate of proliferation were not altered, the proliferative response to the olfactotoxicant satratoxin G and olfactory bulb ablation injury was compromised in the olfactory epithelium of IP3R3^−/− mice. The reductions in both NPY release and number of progenitor cells in IP3R3^−/− mice point to a role of the IP3R3 in tissue homeostasis and neuroregeneration. Collectively, these data suggest that IP3R3 expressing microvillous cells are actively responsive to injury and promote recovery.     

Plasma membrane inositol 1,4,5-trisphosphate-activated channels mediate signal transduction in lobster olfactory receptor neurons.

Inositol 1,4,5-trisphosphate (IP3) selectively evokes an inward (excitatory) current in cultured lobster olfactory receptor neurons (ORNs) and directly activates two types of channels in cell-free patches of plasma membrane from the ORNs. The IP3-activated channels have kinetic properties of odor-activated channels in the ORNs and pharmacological properties of intracellular IP3-activated channels in other systems. An antibody directed against an intracellular, cerebellar IP3 receptor recognizes a protein with a molecular weight similar to the mammalian receptor in the ORNs. The antibody selectively increases odor-evoked inward currents and IP3-activated unitary currents in the ORNs. The data provide further evidence for IP3 as an olfactory second messenger and implicate at least one and possibly two novel plasma membrane IP3 receptors in olfactory transduction.     

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.     

Cross-adaptation between olfactory responses induced by two subgroups of odorant molecules

It has long been believed that vertebrate olfactory signal transduction is mediated by independent multiple pathways (using cAMP and InsP3 as second messengers). However, the dual presence of parallel pathways in the olfactory receptor cell is still controversial, mainly because of the lack of information regarding the single-cell response induced by odorants that have been shown to produce InsP3 exclusively (but not cAMP) in the olfactory cilia. In this study, we recorded activities of transduction channels of single olfactory receptor cells to InsP3-producing odorants. When the membrane potential was held at -54 mV, application of InsP3-producing odorants to the ciliary region caused an inward current. The reversal potential was 0 +/- 7 mV (mean +/- SD, n = 10). Actually, InsP3-producing odorants generated responses in a smaller fraction of cells (lilial, 3.4%; lyral, 1.7%) than the cAMP-producing odorant (cineole, 26%). But, fundamental properties of responses were surprisingly homologous; namely, spatial distribution of the sensitivity, waveforms, I-V relation, and reversal potential, dose dependence, time integration of stimulus period, adaptation, and recovery. By applying both types of odorants alternatively to the same cell, furthermore, we observed cells to exhibit symmetrical cross-adaptation. It seems likely that even with odorants with different modalities adaptation occurs completely depending on the amount of current flow. The data will also provide evidence showing that olfactory response generation and adaptation are regulated by a uniform mechanism for a wide variety of odorants.     

Regulation of the phosphorylation of the inositol 1,4,5-trisphosphate receptor by protein kinase C

The various inositol 1,4,5-trisphosphate receptor (IP(3)R) isoforms are potential substrates for several protein kinases. We compared the in vitro phosphorylation of purified IP(3)R1 and IP(3)R3 by the catalytic subunit of protein kinase C (PKC). Phosphorylation of IP(3)R1 by PKC was about eight times stronger than that of IP(3)R3 under identical conditions. Protein kinase A strongly stimulated the PKC-induced phosphorylation of IP(3)R1. In contrast, Ca(2+) inhibited its phosphorylation (IC(50)相似文献