Inositol 1,4,5,-tris-phosphate receptor: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - Inositol 1,4,5,-tris-phosphate receptor

Synonyms - InsP3R

Cytological map position - 83A5--83A9

Function - calcium channel

Keywords - calcium dependent signaling, receptor, channel

Symbol - Itp-r83A

FlyBase ID:FBgn0010051

Genetic map position - 3-[47.5]

Classification - Inositol 1,4,5,triphosphate receptor

Cellular location - intracellular membranes

NCBI links: Precomputed BLAST | Entrez Gene

Recent literature
Murmu, M. S. and Martin, J. R. (2016). Interaction between cAMP and intracellular Ca-signaling pathways during odor-perception and adaptation in Drosophila. Biochim Biophys Acta 1863: 2156-2174. PubMed ID: 27212269
Binding of an odorant to olfactory receptors triggers cascades of second messenger systems in olfactory receptor neurons (ORNs). The transduction mechanism at ORNs is mediated by cAMP and/or inositol,1,4,5-triphosphate (InsP3)-signaling pathways in an odorant-dependent manner. This study used interfering-RNAi to disrupt the level of cAMP alone or in combination with the InsP3-signaling pathway cellular targets, InsP3 receptor (InsP3R) or ryanodine receptor (RyR) in ORNs, and quantify at ORN axon terminals in the antennal lobe, the odor-induced Ca2+-response. In-vivo functional bioluminescence Ca2+-imaging indicates that a single 5s application of an odor increased Ca2+-transients at ORN axon terminals. However, compared to wild-type controls, the magnitude and duration of ORN Ca2+-response was significantly diminished in cAMP-defective flies. In a behavioral assay, perception of odorants was defective in flies with a disrupted cAMP level suggesting that the ability of flies to correctly detect an odor depends on cAMP. Simultaneous disruption of cAMP level and InsP3R or RyR further diminished the magnitude and duration of ORN response to odorants and affected the flies' ability to detect an odor. In conclusion, this study provides functional evidence that cAMP and InsP3-signaling pathways act in synergy to mediate odor processing within the ORN axon terminals, which is encoded in the magnitude and duration of ORN response.

Jayakumar, S., Richhariya, S., Reddy, O. V., Texada, M. J. and Hasan, G. (2016). Drosophila larval to pupal switch under nutrient stress requires IP3R/Ca2+ signalling in glutamatergic interneurons. Elife 5 [Epub ahead of print] PubMed ID: 27494275
Neuronal circuits are known to integrate nutritional information, but the identity of the circuit components is not completely understood. Amino acids are a class of nutrients that are vital for the growth and function of an organism. This study reports a neuronal circuit that allows Drosophila larvae to overcome amino acid deprivation and pupariate. Nutrient stress is sensed by the class IV multidendritic cholinergic neurons. Through live calcium imaging experiments, this study shows that these cholinergic stimuli are conveyed to glutamatergic neurons in the ventral ganglion through mAChR. IP3R-dependent calcium transients in the glutamatergic neurons convey this signal to downstream medial neurosecretory cells (mNSCs). The circuit ultimately converges at the ring gland and regulates expression of ecdysteroid biosynthetic genes. Activity in this circuit is thus likely to be an adaptation that provides a layer of regulation to help surpass nutritional stress during development.
Restrepo, S. and Basler, K. (2016). Drosophila wing imaginal discs respond to mechanical injury via slow InsP3R-mediated intercellular calcium waves. Nat Commun 7: 12450. PubMed ID: 27503836
Calcium signalling is a highly versatile cellular communication system that modulates basic functions such as cell contractility, essential steps of animal development such as fertilization and higher-order processes such as memory. This study probed the function of calcium signalling in Drosophila wing imaginal discs through a combination of ex vivo and in vivo imaging and genetic analysis. Wing discs were found display slow, long-range intercellular calcium waves (ICWs) when mechanically stressed in vivo or cultured ex vivo. These slow imaginal disc intercellular calcium waves (SIDICs) are mediated by the inositol-3-phosphate receptor, the endoplasmic reticulum (ER) calcium pump SERCA and the key gap junction component Inx2. The knockdown of genes required for SIDIC formation and propagation negatively affects wing disc recovery after mechanical injury. These results reveal a role for ICWs in wing disc homoeostasis and highlight the utility of the wing disc as a model for calcium signalling studies.
Chakraborty, S. and Hasan, G. (2017). Spontaneous Ca2+ influx in Drosophila pupal neurons is modulated by IP3-receptor function and influences maturation of the flight circuit. Front Mol Neurosci 10: 111. PubMed ID: 28473752
Inositol 1,4,5-trisphosphate receptors (IP3R) are Ca2+ channels on the neuronal endoplasmic reticulum (ER) membrane. They are gated by IP3, produced upon external stimulation and activation of G protein-coupled receptors on the plasma membrane (PM). IP3-mediated Ca2+ release, and the resulting depletion of the ER store, triggers entry of extracellular Ca2+ by store-operated Ca2+ entry (SOCE). Mutations in IP3R attenuate SOCE. Compromised IP3R function and SOCE during pupal development of Drosophila leads to flight deficits and mimics suppression of neuronal activity during pupal or adult development. To understand the effect of compromised IP3R function on pupal neuronal calcium signaling, the effects were examined of mutations in the IP3R gene (itpr) on Ca2+ signals in cultured neurons derived from Drosophila pupae. Increased spontaneous Ca2+ influx across was observed the PM of isolated pupal neurons with mutant IP3R and also a loss of SOCE. Both spontaneous Ca2+ influx and reduced SOCE were reversed by over-expression of dOrai and dSTIM, which encode the SOCE Ca2+ channel and the ER Ca2+-sensor that regulates it, respectively. Expression of voltage-gated Ca2+ channels (cac, Ca-alpha1D and Ca-alphaT) was significantly reduced in itpr mutant neurons. However, expression of trp mRNAs and transient receptor potential (TRP) protein were increased, suggesting that TRP channels might contribute to the increased spontaneous Ca2+ influx in neurons with mutant IP3R. Thus, IP3R/SOCE modulates spontaneous Ca2+ influx and expression of PM Ca2+ channels in Drosophila pupal neurons. Spontaneous Ca2+ influx compensates for the loss of SOCE in Drosophila itpr mutant neurons.
Bollepalli, M. K., Kuipers, M. E., Liu, C. H., Asteriti, S. and Hardie, R. C. (2017). Phototransduction in Drosophila is compromised by Gal4 expression but not by InsP3 receptor knockdown or mutation. eNeuro 4(3). PubMed ID: 28660247
Drosophila phototransduction is mediated by phospholipase C, leading to activation of transient receptor potential (TRP) and TRP-like (TRPL) channels by mechanisms that are unresolved. A role for InsP3 receptors (IP3Rs) had been excluded because IP3R mutants (itpr) appeared to have normal light responses; however, this was recently challenged by Kohn et al. ("Functional cooperation between the IP3 receptor and phospholipase C secures the high sensitivity to light of Drosophila photoreceptors in vivo," Journal of Neuroscience 35:2530), who reported defects in phototransduction after IP3R-RNAi knockdown. They concluded that InsP3-induced Ca2+ release plays a critical role in facilitating channel activation, and that previous failure to detect IP3R phenotypes resulted from trace Ca2+ in electrodes substituting for InsP3-induced Ca2+ release. In an attempt to confirm this, electroretinograms, whole-cell recordings, and GCaMP6f Ca2+ imaging were performed from both IP3R-RNAi flies and itpr-null mutants. Like Kohn et al., this study used GMRGal4 to drive expression of UAS-IP3R-RNAi, but controls expressing GMRGal4 alone were also used. Several GMRGal4 phenotypes are described suggestive of compromised development, including reductions in sensitivity, dark noise, potassium currents, and cell size and capacitance, as well as extreme variations in sensitivity between cells. However, no effect of IP3R RNAi or mutation was found on photoreceptor responses or Ca2+ signals, indicating that the IP3R plays little or no role in Drosophila phototransduction.
Narciso, C. E., Contento, N. M., Storey, T. J., Hoelzle, D. J. and Zartman, J. J. (2017). Release of applied mechanical loading stimulates intercellular calcium waves in Drosophila wing discs. Biophys J 113(2): 491-501. PubMed ID: 28746859
Mechanical forces are critical inputs for organogenesis and wound healing. Calcium ions (Ca2+) are critical second messengers in cells for integrating environmental and mechanical cues. This paper reports a chip-based regulated environment for microorgans that enables systematic investigations of the crosstalk between an organ's mechanical stress environment and biochemical signaling under genetic and chemical perturbations. This method enabled defining the essential conditions for generating organ-scale intercellular Ca2+ waves in Drosophila wing discs that are also observed in vivo during organ development. Mechanically induced intercellular Ca2+ waves were found to require fly extract growth serum as a chemical stimulus. Using the chip-based regulated environment for microorgans, it was demonstrated that not the initial application but instead the release of mechanical loading is sufficient, but not necessary, to initiate intercellular Ca2+ waves. The Ca2+ response depends on the prestress intercellular Ca2+ activity and not on the magnitude or duration of the mechanical stimulation applied. Mechanically induced intercellular Ca2+ waves rely on IP3R-mediated Ca2+-induced Ca2+ release and propagation through gap junctions. Thus, intercellular Ca2+ waves in developing epithelia may be a consequence of stress dissipation during organ growth.

The release of intracellular Ca2+ is an intermediate step in many cellular signaling processes. The soluble messenger known as inositol 1,4,5 triphosphate) acts to release intracellular Ca2+ from the intracellular store maintained within the endoplasmic reticulum. The InsP3 receptor (Itp-r83A), here called InsP3R, is a Ca2+ channel that releases intracellular Ca2+ in response to InsP3. This signal transduction pathway is used in processes as diverse as the responses to hormones, growth factors and neurotransmitters, as well as in various vertebrate sensory systems, such as olfaction, gustation and vision (Hasan, 1992 and references).

Before describing the biology of the Drosophila InsP3 receptor, a short diversion will provide a closer look at Ca2+ signaling and intracellular Ca2+ channels. There are two common motifs for Ca2+ release from intracellular storage depots into the cytosol for signal transduction, depending on whether the cells are excitable or non-excitable.

  1. Nonexcitable cells: Here the slow inositol (1,4,5)-trisphosphate (InsP3)-mediated pathway predominates. Two receptor classes (the G protein-coupled receptor class of seven transmembrane-spanning receptors and the receptor tyrosine kinases), acting indirectly, release InsP3, which in turn acts as an intracellular second messenger by binding to the specialized tetrameric InsP3 receptor (InsP3R) that spans the endoplasmic reticular membrane and triggers release of Ca2+. G protein-coupled receptors activate phospholipase Cbeta while receptor tyrosine kinases stimulate phospholipase Cgamma to convert the lipid known as phosphatidylinositol (4,5)-biphosphate into the small soluble messenger InsP3 and the membrane bound lipid diacylglycerol. Diacylglycerol serves to activate another signaling cascade by activating the enzyme Protein kinase C (Clapham, 1995).

  2. Excitable cells: The Ca2+ release from intracellular storage depots into the cytosol also occurs in excitable cells containing voltage-dependent Ca2+ channels that enable these cells to dramatically increase cytosolic Ca2+ levels. In excitable cells, Ca2+ entering through voltage-dependent Ca2+ channels may directly activate ryanodine receptors, the excitable cell counterparts to the InsP3 receptor, to release Ca2+ from intracellular stores (Clapham, 1995).

The InsP3R of Drosophila (Itp-r83A) is expressed ubiquitously and is involved in multiple aspects of development: it is required for embryonic and larval development, is involved in muscle development and is implicated in chemosensory functions. The InsP3R was identified based on its homology to mammalian InsP3R. The protein is highly conserved and shows the complex structure of a transmembrane protein, with identifiable ligand binding domains, ATP binding domains, a conserved presumptive Ca2+ channel and conserved cysteine residues also found in the ryanodine receptor, but the fly protein shows no Protein kinase A phosphorylation sites, indicating that it is not regulated by the PKA pathway as is the case for the mammalian homologs (Yoshikawa, 1992).

Numerous modulatory signals have been postulated for mammalian InsP3R receptor: the best characterized is known as type 1 InsP3R (Insp3r1). Calmodulin, cyclic GMP-dependent protein kinase and Calcium/calmodulin dependent protein kinase II have all been proposed as modifiers of the activity of the channel protein in addition to ATP, Protein kinase A and ligand. Thus, other second messenger transduction cascades, i.e., cyclic AMP (via PKA), diacyl glycerol (via PKC), and cyclic GMP (via cyclic GMP-dependent protein kinase) all converge on a single modulatory and transducing domain found in the middle portion of the receptor. In addition, alternative splicing results in variations in regulatory regions of the protein. The protein is differentially distributed throughout the adult brain and show preferences for distinct subcellular compartments. Besides being present in cerebellar Purkinje cells, InsP3R1 is present in the olfactory tubercle, cerebral cortex, CA1 region of the hippocampus, caudate-putamen, molecular layer of the cerebellum and choroid plexis (Furuichi, 1995).

Mutants in the Drosophila InsP3R gene arrest development at the second larval instar; many larval tissues are observed that have not proliferated normally in these mutants. Expression during the embryonic phase in mesoderm and sensory organ precursor cells suggests that Insp3R could be involved in embryonic development but that no embryonic phenotype is observed because of the presumed presence of a maternal transcript. If maternal transcripts play a role in embryonic development, then making germ line clones lacking all InsP3R should lead to early lethality. When such clones are generated no viable eggs or embryos are recovered. It is therefore likely that InsP3R larval lethality is a maternal-effect phenomenon due to the preponderance of maternally contributed message. It is concluded that InsP3R is essential for cell proliferation, growth and differentiation. More specific information about the roles of Insp3R in Drosophila development await analysis of mutant clones where the maternal contribution is diminished (Acharya, 1997).

Availability of Insp3R mutants has allowed an assessment of the role of InsP3R in Drosophila photoreceptor signaling. One of the many unanswered questions stimulating research in the field of invertebrate photoreceptor biology has to do with the gating mechanism of light-activated channels. In Drosophila photoreceptor neurons, light activation of rhodopsin activates a Gqalpha, which in turn activates a Phospholipase C (PLC) encoded by the norpA gene. PLC catalyzes the breakdown of PIP2 into InsP3 and diacyl glycerol; these are thought to lead to the eventual opening (and modulation) of the TRP and TRPL membrane channels and the generation of a receptor potential. It has been proposed that the TRP channel functions as a store-operated channel (opened by Ca2+ released from intracellular stores) that responds to a capacitative calcium entry signal. According to this model, light-activation of rhodopsin results in the production of InsP3, which would lead to the emptying of InsP3-sensitive internal calcium stores and the subsequent gating of plasma membrane channels. It has been thought that TRP channels and their vertebrate homologs represent examples of store-operated channels (Acharya, 1997 and references).

However, TRP (transient receptor potential) and TRPL light-activated ion channels, known to be involved in the light sensitivity of the Drosophila retina, do not localize in close proximity to the internal calcium stores, thus eliminating the possibilty of gating by protein-protein interaction via conformation coupling (Niemeyer, 1996). It has also been shown that all light-induced increases in Ca2+ are entirely dependent on the influx of extracellular Ca2+ (Ranganathan, 1994). These results suggest that internal InsP3-sensitive stores are not involved in activation of the light responses (for a contrary result see InsP3R and visual phototransduction). To catagorically test the involvement of InsP3R in light activation, clones of Insp3R negative photoreceptors were generated in an otherwise wild-type background. Mutant photoreceptors and surrounding tissues undergo retinal degeneration, consistent with defective growth of mutant larval tissue. Intracellular recordings were carried out to study light responses in control and mutant photoreceptor cells. InsP3R mutant photoreceptors are indistinguishable from wild-type controls in sensitivity, kinetics of activation and deactivation, and adaptation. Thus, no evidence was found for a role of the InsP3R in Drosophila phototransduction, and models invoking InsP3-induced calcium release in the activation of this pathway are likely to be invalid (Acharya, 1997).

Functional cooperation between the IP3 receptor and Phospholipase C secures the high sensitivity to light of Drosophila photoreceptors in vivo

Drosophila phototransduction is a model system for the ubiquitous phosphoinositide signaling. In complete darkness, spontaneous unitary current events (dark bumps) are produced by spontaneous single Gqα activation, while single-photon responses (quantum bumps) arise from synchronous activation of several Gqα molecules. Recent studies have shown that most of the spontaneous single Gqα activations do not produce dark bumps, because of a critical phospholipase Cβ (PLCβ) activity level required for bump generation. Surpassing the threshold of channel activation depends on both PLC activity and cellular [Ca(2+)], which participates in light excitation via a still unclear mechanism. This study shows that in IP3 receptor (IP3R)-deficient photoreceptors, both light-activated Ca(2+) release from internal stores and light sensitivity were strongly attenuated. This was further verified by Ca(2+) store depletion, linking Ca(2+) release to light excitation. In IP3R-deficient photoreceptors, dark bumps were virtually absent and the quantum-bump rate was reduced, indicating that Ca(2+) release from internal stores is necessary to reach the critical level of PLCβ catalytic activity and the cellular [Ca(2+)] required for excitation. Combination of IP3R knockdown with reduced PLCbeta catalytic activity resulted in highly suppressed light responses that were partially rescued by cellular Ca(2+) elevation, showing a functional cooperation between IP3R and PLCβ via released Ca(2+). These findings suggest that in contrast to the current dogma that Ca(2+) release via IP3R does not participate in light excitation, this study shows that released Ca(2+) plays a critical role in light excitation. The positive feedback between PLCβ and IP3R found here may represent a common feature of the inositol-lipid signaling (Kohn, 2015).

In this study, in vivo light-response suppression was accompanied by reduced Ca2+ release from IP3-sensitive stores. In addition, the rate of spontaneously produced dark bumps, which is highly sensitive to Gqα-dependent PLCβ catalytic activity and cellular Ca2+ level, was virtually abolished in IP3R-deficient photoreceptors. This dark-bump elimination indicates that the suppressed Ca2+ release from IP3-sensitive stores underlies the suppressed catalytic activity of PLCβ, leading to suppressed light response in IP3R-deficient photoreceptors. Further evidence that the suppressed light response arises from inhibition of Ca2+ release from IP3-sensitive stores came from blocking the Ca2+ pump by Tg, which mimicked the phenotype of the IP3R-deficient photoreceptors in WT flies. The above findings indicate that IP3R-mediated Ca2+ release has a critical role in light excitation of Drosophila photoreceptors. The combination of the PLCβ mutant norpAH43 with IP3R-deficient photoreceptors, which synergistically suppressed the light response, strongly suggests that there is functional cooperation between the IP3R and PLCβ in generation of the light response (Kohn, 2015).

It has been previously shown that an increase in cytosolic Ca2+ participates in light excitation as evidenced by enhancement of the light response following photo release of caged Ca2+ at the rising phase of the light response. The target of Ca2+ action has not been entirely resolved. PLCβ is an important target for Ca2+ action and the regulation of its catalytic activity by Ca2+ has been thoroughly investigated. These studies showed that the positive charge of Ca2+ is used to counterbalance local negative charges formed in the active site during the course of the catalytic reaction. Accordingly, Ca2+ performs electrostatic stabilization of both the substrate and the transition state, thus providing a twofold contribution to lower the activation energy of the enzyme reaction (Kohn, 2015).

The following model explains how functional cooperation between the IP3R and PLCβ via the released Ca2+ operates and secures quantum-bump production: absorption of a single photon, which induces activation of several PLCβ molecules, is initially insufficient at resting Ca2+ levels to reach the critical level of PLCβ activity required for TRP/TRPL channel activation. Nevertheless, the IP3 molecules produced by the given PLCβ activity are able to activate the nearby IP3Rs, mobilize Ca2+ from the stores, and elevate PLCβ activity above the threshold required for TRP/TRPL channel activation. In addition, the released Ca2+ may also reduce the threshold of TRP/TRPL channel activation and allow bump generation. According to this model, the following enzymatic reactions may explain the current findings. Each Gqα-activated PLCβ has low catalytic activity due to the relatively low (<160 nM) resting Ca2+ concentration in the cytosol. In addition, each activated PLCβ remains active for only a short (approximately several tens of milliseconds) time due to the GTPase-activating protein activity of PLCβ that causes a rapid hydrolysis of Gqα-GTP followed by inactivation of PLCβ. The initial low catalytic activity of PLCβ is apparently below the threshold required for activation of the TRP and TRPL channels, but this low activity still results in hydrolysis of PIP2 producing IP3. Since there are no IP3 buffers in the microvilli and the IP3 degradation time is relatively slow (~1 s), the produced IP3 molecules diffuse fast along the microvillus at an estimated time of ~1 ms along 1 microm long microvillus and bind to IP3R located at the nearby submicrovillar cisternae (SMC; the photoreceptors' extensions of smooth ER). IP3R channels residing at the SMC, which are large channels with high sensitivity for IP3 and thus can be activated at low PLCβ activity, open and release Ca2+ juxtaposed to the base of the microvillus. The released Ca2+ steeply raises the local Ca2+ concentration, probably to the microM range, because of the very small aqueous volume of the microvillus and the relatively large local Ca2+ elevation via the release mechanism (Kohn, 2015).

Accordingly, a single IP3R channel can release ∼104 Ca2+ ions in 1 ms channel opening and Ca2+-induced Ca2+ release mechanism is a property of the IP3R channels and of the ryanodine receptors, which reside in the ER. Ca2+ released via IP3R of the WT SMC diffuse back toward the activated PLCβ and the TRP/TRPL channels in the microvillus. Although Ca2+ diffuses ∼20-fold slower than IP3 due to strong buffering, the diffusion constant strongly depends on Ca2+ concentration. Accordingly, at ~250 μM the Ca2+ diffusion coefficient is as large as that of IP3. Once a single TRP channel is activated, the large Ca2+ influx through this channel is sufficient to facilitate the rest of the active PLCβ molecules or reduce the threshold for TRP/TRPL channel activation in this microvillus and produce a bump that reflects activation of the entire microvillus . When there is abnormally low Ca2+ release via the IP3R because of low IP3R expression levels (IP3R-RNAi), there is not enough Ca2+ to increase PLC activity or to reduce TRP activation threshold, and activated PLC in this microvillus does not produce a bump, leading to abnormally low frequency of dark bumps (Kohn, 2015).

The invasive whole-cell recording technique, which was used in previous studies and avoided Ca2+ buffering of the pipette solution, most likely resulted in abnormally elevated cytosolic Ca2+ concentration, which also allowed the Ca2+ pump to keep the stores full. This artificially elevated cytosolic [Ca2+] together with the constitutive Ca2+ leak from the full stores, bypassed the need to mobilize Ca2+ via functional IP3R to facilitate PLCβ activity and reach its critical catalytic activity level needed to activate the TRP/TRPL channels. In the present study in the intact eye, a significant reduction in light-response amplitude was observed when the IP3R level was reduced. Furthermore, when cellular [Ca2+] was reduced by prolonged extracellular EGTA application, the light response of the IP3R-deficient flies was further suppressed. Moreover, when using invasive patch-clamp whole-cell recordings without Ca2+ buffering of the pipette solution, no significant difference between WT and IP3R-deficient flies was observed, as found in the previous study. However, when pipette Ca2+ was reduced with EGTA, the phenotype of reduced light excitation was observed in both reduced quantum-bump frequency as well as in macroscopic light-response suppression. Unlike quantum bumps, dark bumps were virtually eliminated even without buffering the pipette Ca2+ in IP3R-deficient flies, indicating that in the dark the IP3R-deficiency led to abnormally low cytosolic [Ca2+], possibly due to reduced Ca2+ leak from stores leading to cellular [Ca2+] below the critical level required for PLC activation observed in WT flies. Alternatively, the positive feedback between the released Ca2+ and PLC may function at single PLC molecules. Hence the nominal pipette Ca2+ is not sufficient to allow PLC activity to pass the threshold of channel activation, but the released Ca2+ via IP3R activation together with pipette Ca2+ allows PLC activity to pass this threshold and generate dark-bump (Kohn, 2015).

There is a striking functional similarity between, the cerebellar Purkinje cell (PC) proteins of the IP signaling and Drosophila photoreceptors, but the link of cerebellar mGluR1 receptor to TRPC3 activation is not clear. Interestingly, in PC neurons, stromal interaction molecule 1 (STIM1) was proved an essential regulator of Ca2+ level in neuronal endoplasmic reticulum Ca2+ stores. Accordingly, STIM1-specific deletion caused impairments in slow synaptic current and cerebellar motor behavior. Strikingly, refilling empty Ca2+ stores through increased Ca2+ level in the cytosol partially rescued the phenotype of the stim1 knock-out mice, reminiscent of the rescue of the phenotype of the IP3R-deficient fly by artificially elevated cytosolic Ca2+. Thus, the facilitatory role of released Ca2+ on PLC in light excitation of Drosophila photoreceptors represents an essential mechanism that operates in other PI systems (Kohn, 2015).

IP3R mediated Ca2+ release regulates protein metabolism in Drosophila neuroendocrine cells: implications for development under nutrient stress

Successful completion of animal development is fundamentally reliant on nutritional cues. Adaptations for surviving nutritional loss are coordinated in part by neural circuits. As neuropeptides secreted by neuroendocrine (NE) cells critically modulate neural circuits (see Mapping peptidergic cells in Drosophila: where DIMM fits in), this study investigated NE cell function during development under nutrient stress. Starved Drosophila larvae exhibited reduced pupariation, if either insulin signaling or IP3/Ca2+ signaling, were down-regulated in NE cells. Moreover, an IP3R (Inositol 1,4,5-trisphosphate receptor) loss-of-function mutant displayed reduced protein synthesis, which was rescued by over-expression of either InR (insulin receptor) or IP3R in NE cells of the mutant, suggesting that the two signaling pathways may be functionally compensatory. Furthermore, cultured IP3R mutant NE cells, but not neurons, exhibited reduced protein translation. Thus cell-specific regulation of protein synthesis by IP3R in NE cells influences protein metabolism. It is proposed that this regulation helps developing animals survive poor nutritional conditions (Megha, 2017).

Nutritional poverty during development has long-lasting effects on the growth and behavior of an animal. Although under-nutrition causes overall body size to decrease, the brain grows to near-normal size, a process termed 'brain sparing'. This suggests unique mechanisms in neuronal tissues to weather nutritional stress. Drosophila is an attractive model system to uncover these mechanisms because larvae subjected to nutrient restriction exhibit 'brain sparing' and nutritional effects on larval-to-pupal development are easily monitored. Additionally, growth signaling pathways activated by dietary cues such as insulin receptor (InR) and TOR signaling, are conserved in Drosophila (Megha, 2017).

When starved, larval neural stem cells (NSCs) continue to proliferate by using an InR ortholog, Alk (Anaplastic lymphoma kinase). This study focuses on neuroendocrine (NE) cells, which, unlike NSCs, are differentiated and non-dividing. Importantly, neuropeptides released by NE cells modulate neural circuits that regulate processes associated with animal physiology and behavior, eventually influencing how animals adapt to external or internal stimuli. Crucially, NE cells produce peptide hormones that regulate feeding behavior and metabolism, processes required for larvae to complete development successfully (Megha, 2017).

IP3R (Itp-r83A in Drosophila) is an endoplasmic reticulum (ER) channel that releases stored Ca2+ and acts downstream of G protein-coupled receptor activation. The ER-resident protein STIM (Stromal interaction molecule) conveys loss of stored Ca2+ to Orai (Olf186-F in Drosophila), a plasma membrane Ca2+ channel, thereby enabling store-operated Ca2+ entry (SOCE) from the extracellular milieu. SOCE occurs in both mammals. Thus, all three molecules - IP3R, STIM and Orai - function during stimulus-dependent elevation of cytosolic Ca2+ that potentiates diverse signaling outcomes, depending on the cellular context (Megha, 2017).

Loss of IP3R and STIM leads to obesity in adult Drosophila. Importantly, adults of a hypomorphic IP3R mutant heteroallelic combination, itprka1091/ug3 (hereafter: itprku) exhibit obesity, starvation resistance and hyperphagia, which are all rescued by overexpression of IP3R in NE cells. This adult metabolic phenotype prompted an investigation of the role of IP3R and InR in NE cells during larval development (Megha, 2017).

In summary, IP3R-mediated Ca2+ signaling helps maintain normal protein translation levels in NE cells, and this activity promotes systemic protein metabolism during larval development. On a nutrient-rich diet, loss of IP3R signaling is not detrimental, because dietary cues maintain insulin/TOR signaling, and thereby keep protein levels normal for completing development. Under starvation, dietary cues are lost. IP3/Ca2+ signaling possibly provides a nutrient-independent mechanism in order to maintain protein synthesis in cells essential to surviving nutrient stress, such as NE cells in which increased levels of cell surface receptors or neuropeptides might be required for modulating relevant neural circuits. As yet, there are no receptors or neuropeptides reported to be upregulated upon starvation in dimm+ NE cells, but there is precedence to suggest that they might exist. For example, in starved Drosophila, the receptor for short Neuropeptide F is upregulated in the antenna, and in starved mammals levels of agouti-related peptide, which affects appetite and feeding, are increased. A recent screen identified IP3/Ca2+-coupled neuropeptide receptors, on glutamatergic neurons, that are required for larval adaptation to nutrient stress. Neuropeptides from NE cells that couple to such receptors might function during starvation in this model (Megha, 2017).

By focusing on animal development, this study integrates cellular observations and organismal phenotype. Therefore, it sets the framework for the discovery of mechanistic details of how stimulus-coupled increases in cytosolic Ca2+ can regulate protein synthesis in a cell-specific manner, and how that consequently regulates protein metabolism in the whole animal (Megha, 2017).


Two bands of hybridization, differing very slightly in size, are seen with the Drosophila InsP3R probe. The smaller-sized band (approx. 9.7 kb) is seen only in early embryos of 0-6 hours. A larger band of approx. 10 kb is expressed in mid-late embryos. The basis for the difference between these two bands is unknown (Hasan, 1992). The InsP3R gene is on the third chromsome at position 83A4-5. Two transcriptional units can be detected in this region: InsP3R and the NMDA receptor (Acharya, 1997).

cDNA clone length - 10 kb

Bases in 5' UTR - 845


Amino Acids - 2833

Structural Domains

The Drosophila InsP3R has striking similarity in its overall structure to the mouse InsP3R, although the homologous sequences are interspersed by some stretches of insert, deleted, and diverged sequences. Among the sequence differences, it is notable that a 40-amino acid deletion of the mouse sequence (residues 1692-1731) is found after Lys-1792 in the Drosophila sequence. The amino acids in Drosophila share 57% identity with those of the mouse sequence. The sequence similarity between the mouse InsP3R and the ryanodine receptor is also well conserved overall in Drosophila, with 8 conserved Cys residues (Cys-6555, -1345, -2052, -2123, -2614, -2620, -2697, -2700) in the Drosophila receptor (Yoshikawa, 1992).

A comparison of the InsP3 receptor and the Drosophila ryanodine receptors shows that they share significant homology near their C-terminal amino acids. The 300 C-terminal amino acids of the cloned Drosophila ryanodine receptor and InsP3R are 62.6% similar and 33.6% identical. Rodent and fly ryanodine receptor genes appear equally related to the rodent and fly InsP3 receptor genes, indicating than an ancient duplication event probable gave rise to these two classes of intracellular Ca2+-releasing channel genes (Hasan, 1992).

Hydrophobicity analysis of the InsP3R receptor indicates at least 6 main peaks of hydrophobic amino acids that could represent multiple membrane-spanning sequences (residues 2367-3676). It is likely that the mouse receptor traverses the membrane 6 times as well. The large N-terminal region (83.5% of the protein) is located on the cytoplasmic side, and a short C-terminal region is likewise found on the cytoplasmic face. There is significant homology in the N-terminal regions (residues 1-695 in Drosophila and 1-664 in the mouse), including the N-terminal InsP3-binding region of rodent receptors. The Drosophila and mouse sequences have 12% postively charged amino acids (30 Arg and 51 Lys in 677 amino acids of the Drosophila receptor) that may be involved in binding to a negatively charged InsP3 molecule. These N-terminal homologous regions contain fragmentary but significant similarity to the ryanodine receptor. In contrast to the mouse InsP3R receptor, which is phosphorylated and regulated by protein kinase A, Drosophila InsP3R diverges in the PKA target site and has no potential protein kinase A phosphorylation site (Yoshikawa, 1992).

ATP is known to promote InsP3-induced Ca2+ release in rodents. The ATP binding residues consist of a Gly-rich stretch in Drosophila (residues 2092-97) and in mouse (2016-21). It is assumed that the rodent InsP3Rs form a Ca2+ channel structure in the C-terminal transmembrane regions as a homotetramer. Within this transmembrane region (M1-M6) there is significant homology, in particular from residues 2499 and 2411 onwards, in Drosophila and mouse respectively. The last two membrane-spanning sequences (M5 and M6) are, respectively, 87% and 95% homologous. In addition, they resemble the corresponding sequences M3 and M4 of the ryanodine receptors. Within the luminal loop between M5 and M6, there is a peculiar distribution of negative charges, 20 and 28% in Drosophila and mouse receptors, respectively. These negative charges may be involved in efficient Ca2+ permeation by concentrating Ca2+ near the channel pore (Yoshikawa, 1992).

Inositol 1,4,5,-tris-phosphate receptor: | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 16 July 97  

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