IP3R Channels Research Articles

Requirement of Caspase-3 for Apoptotic Calcium Release

Calcium is known to be an important second messenger in regulation of various physiological processes including execution of programmed cell death. Calcium release from endoplasmic reticulum (ER) triggers activation of multiple pro-apoptotic factors resulting in apoptosis progression and, ultimately, cell death. Apoptotic calcium release from ER stores is mediated by inositol 1,4,5-trisphosphate receptor (IP3R) channels. Different models have confirmed the importance of IP3R channels in modulation of apoptosis, however many details of IP3R-dependent calcium release still remain enigmatic. Previously, we have demonstrated that calcium release from IP3R channels in response to apoptotic stimuli occurs in two temporally distinct phases. A rapid oscillatory cytosolic calcium release during initiation of apoptotic signaling is followed by delayed and sustained increase in calcium levels associated with persistent opening of IP3R channels. Here we show that early elevations in cytosolic calcium do not require caspase-3 activity. Using MCF-7 cells deficient in caspase-3, we detected robust increase in calcium levels in response to staurosporine treatment indicating that calcium release during initiation of apoptosis occurs independently of caspase-3. To further determine contribution of caspase-3 in regulation of calcium release we generated a MCF-7 cell line stably expressing pro-caspase-3. Additionally, we used TAT-based transducing peptide to deliver active recombinant caspase-3 directly into living MCF-7 cells. Utilizing both methodologies, we found that caspase-3 has a marginal effect on the early events leading to cytosolic calcium elevations and irreversible commitment to apoptotic cell death. These results suggest that caspase-3 mediated truncation of IP3R channels is a consequence, not causative, of apoptotic signaling events.

Calcium-mediated Stress Kinase Activation by DMP1 Promotes Osteoblast Differentiation

Calcium signaling and calcium transport play a key role during osteoblast differentiation and bone formation. Here, we demonstrate that DMP1 mediated calcium signaling, and its downstream effectors play an essential role in the differentiation of preosteoblasts to fully functional osteoblasts. DMP1, a key regulatory bone matrix protein, can be endocytosed by preosteoblasts, triggering a rise in cytosolic levels of calcium that initiates a series of downstream events leading to cellular stress. These events include release of store-operated calcium that facilitates the activation of stress-induced p38 MAPK leading to osteoblast differentiation. However, chelation of intracellular calcium and inhibition of the p38 signaling pathway by specific pharmacological inhibitors and dominant negative plasmid suppressed this activation. Interestingly, activated p38 MAPK can translocate to the nucleus to phosphorylate transcription factors that coordinate the expression of downstream target genes such as Runx 2, a key modulator of osteoblast differentiation. These studies suggest a novel paradigm by which DMP1-mediated release of intracellular calcium activates p38 MAPK signaling cascade to regulate gene expression and osteoblast differentiation.

Recording single-channel activity of inositol trisphosphate receptors in intact cells with a microscope, not a patch clamp.

Optical single-channel recording is a novel tool for the study of individual Ca2+-permeable channels within intact cells under minimally perturbed physiological conditions. As applied to the functioning and spatial organization of IP3Rs, this approach complements our existing knowledge, which derives largely from reduced systems - such as reconstitution into lipid bilayers and patch clamping of IP3Rs on the membrane of excised nuclei - where the spatial arrangement and interactions among IP3Rs via CICR are disrupted. The ability to image the activity of single IP3R channels with millisecond resolution together with localization of their positions with a precision of a few tens of nanometers both raises several intriguing questions and holds promise of answers. In particular, what mechanism underlies the anchoring of puffs and blips to static locations; why do these Ca2+ release events appear to involve only a very small fraction of the IP3Rs within a cell; and how can we reconcile the relative immotility of functional IP3Rs with numerous studies reporting free diffusion of IP3R protein in the ER membrane?

Superresolution Localization of Single Functional IP3R Channels Utilizing Ca2+ Flux as a Readout

The subcellular localization of membrane Ca2+ channels is crucial for their functioning, but is difficult to study because channels may be distributed more closely than the resolution of conventional microscopy is able to detect. We describe a technique, stochastic channel Ca2+ nanoscale resolution (SCCaNR), employing Ca2+-sensitive fluorescent dyes to localize stochastic openings and closings of single Ca2+-permeable channels within <50 nm, and apply it to examine the clustered arrangement of inositol trisphosphate receptor (IP3R) channels underlying local Ca2+ puffs. Fluorescence signals (blips) arising from single functional IP3Rs are almost immotile (diffusion coefficient <0.003 μm2 s−1), as are puff sites over prolonged periods, suggesting that the architecture of this signaling system is stable and not subject to rapid, dynamic rearrangement. However, rapid stepwise changes in centroid position of fluorescence are evident within the durations of individual puffs. These apparent movements likely result from asynchronous gating of IP3Rs distributed within clusters that have an overall diameter of ∼400 nm, indicating that the nanoscale architecture of IP3R clusters is important in shaping local Ca2+ signals. We anticipate that SCCaNR will complement superresolution techniques such as PALM and STORM for studies of Ca2+ channels as it obviates the need for photoswitchable labels and provides functional as well as spatial information.

TEMPERATURE EFFECTS ON THE STOCHASTIC GATING OF THE IP3R CALCIUM RELEASE CHANNEL: A NUMERICAL SIMULATION STUDY

The importance of the kinetic study of endoplasmatic calcium ion channels in different intracellular processes is known today. Although there are few experimental reports on the temperature dependency of IP3R channel functions, we did not find any detailed theoretical study on this subject. For this purpose, we used a modified Gillespie algorithm to investigate the effect of temperature on the conditions affecting the open state of a single subunit of the De Young-Keizer (DYK) model. Population of the states was considered as the subject of fluctuation. Key features of the channel, such as bell-shaped dependency of open probability to the Calcium concentrations were modeled at different temperatures, too. The range of temperature variation was selected by regarding the experimental data on IP3R channel.By increasing the temperature, we had the very slow time domains (t: 10-1s ) and the much slower time domains (t: 100s ) in addition to other time domains, which could be seen as new time categories in InsP3R studies, and so the results were reported in these time domains, as well.We found out that increase in temperature declined the open probability in some concentrations of Ca2+and/or IP3. Also, by introducing the intensity graphs, broadening of the range of fluctuations and lowering of the order of frequency of fluctuations for the population of each state were observed due to the temperature increments.The temperature effects on the activation and inactivation states of the channel were studied in the framework of the reaction paths. We did not find similar paths at different time domains; several paths observed which were totally different all together. These time-dependent reaction paths are also depending on the Ca2+and/or the IP3 concentrations. So, one can predict the most probable reaction paths at different concentrations and temperatures and also determine which kind of the path it is; a path for closing the channel or a path to open it.Finally, the temperature effects on the calcium inhibited states were studied. We found out that calcium ion inhibitions were shifted to lower calcium concentration by increasing the temperature. The results suggests that inhibiting role of calcium is not only [ Ca2+] and/or [IP3] dependent, but also temperature dependent.

Super-resolution Imaging Of Ca2+ Flux Through IP3Rs With Millisecond Temporal Resolution And Nanometer Spatial Resolution

Advanced imaging techniques such as PALM and STORM have broken the diffraction limit of conventional optical microscopy through their ability to turn fluorescent molecules on and off at low enough densities such that the positions of single molecules can be determined, one at a time, with a precision of ∼10 nm (Gustafsson, 2008). However, these techniques involve the use of fluorescently tagged proteins or antibodies, which may alter protein properties and provide only positional, not functional information. Thus, we have developed a technique termed Single Channel Ca2+ Nanoscale Resolution (SCCaNR), based on similar principles except that it generates a super-resolution image by using Ca2+ sensitive fluorescent dyes to image the stochastic openings and closings of Ca2+ permeable ion channels. Subsequently, the point spread function resulting from the diffusion of calcium bound to the indicator dye can be fit to a 2-D Gaussian function, allowing the position of functional calcium channels to be localized with much higher precision (∼40 nm) than previously possible.The inositol triphosphate receptor (IP3R) is an ER Ca2+ channel that is both facilitated and inhibited by Ca2+ itself. This property enables a functional coupling between IP3Rs, which underlies the generation of localized Ca2+ events known as puffs (Yao, et al, 1995). This same property makes IP3Rs highly dependent on their spatial proximity to one another. Using our SCCaNR technique, we have found that the concerted opening of 4-10 IP3R channels likely underlies the generation of Ca2+ puffs in SH-SY5Y neuroblastoma cells. These puffs arise from clusters of IP3Rs approximately 300 nm in diameter, a dimension below the resolution limit of conventional optical microscopy.

Microfilament and microtubule assembly is required for the propagation of inositol trisphosphate receptor‐induced Ca2+ waves in bovine aortic endothelial cells

Ca2+ is a highly versatile second messenger that plays a key role in the regulation of numerous cell processes. One-way cells ensure the specificity and reliability of Ca2+ signals is by organizing them spatially in the form of waves that propagate throughout the cell or within a specific subcellular region. In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP3R) is responsible for the release of Ca2+ from the endoplasmic reticulum. The spatial aspect of the Ca2+ signal depends on the organization of various elements of the Ca2+ signaling toolkit and varies from tissue to tissue. Ca2+ is implicated in many of endothelium functions that thus depend on the versatility of Ca2+ signaling. In the present study, we showed that the disruption of caveolae microdomains in bovine aortic endothelial cells (BAEC) with methyl-beta-cyclodextrin was not sufficient to disorganize the propagation of Ca2+ waves when the cells were stimulated with ATP or bradykinin. However, disorganizing microfilaments with latrunculin B and microtubules with colchicine both prevented the formation of Ca2+ waves. These results suggest that the organization of the Ca2+ waves mediated by IP3R channels does not depend on the integrity of caveolae in BAEC, but that microtubule and microfilament cytoskeleton assembly is crucial.

Can You Hear Me Now?

CELL BIOLOGY It's a bit like talking to your neighbor at a dinner party with a megaphone, but Tovey et al . report that the stimulation of calcium release through inositol 1,4,5-trisphosphate receptors (IP3R) results from enormous amounts (1000 times greater than the amount needed to activate protein kinase A) of the second messenger cAMP produced by adenylyl cyclase (AC) molecules that are closely apposed to the IP3R channel. The authors were led to this unorthodox interpretation by their exploration of the mechanisms by which parathyroid hormone (PTH), which itself does not cause the release of calcium, enhanced the effects of other hormones on the release via IP3Rs of calcium from internal stores. Only PTH analogs that activated AC potentiated calcium release. High concentrations of cAMP analogs were sufficient to reproduce the effects of PTH and were not additive with the effects of the hormone. The authors propose that AC and IP3Rs are in such close proximity that activation of the cyclase produces a massive all-or-none response of the channel that is resistant to modulation by agents that alter cytoplasmic concentrations of cAMP; immunoprecipitation experiments confirmed the prediction that IP3Rs and AC were associated physically. Such signaling complexes would have on-off or switchlike properties and could allow graded responses by recruitment of more activated complexes rather than graded response at an individual complex. To add to the complexity, the IP3R-associated isoform of AC is inhibited by calcium. Thus, localized concentrations of cAMP and calcium might oscillate as a result of feedback inhibition. — LBR J. Cell Biol. 183 , 297 (2008).

Intracellular Calcium Release Channels Mediate Their Own Countercurrent: The Ryanodine Receptor Case Study

Intracellular calcium release channels like ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP3Rs) mediate large Ca2+ release events from Ca2+ storage organelles lasting >5ms. To have such long-lasting Ca2+ efflux, a countercurrent of other ions is necessary to prevent the membrane potential from becoming the Ca2+ Nernst potential in <1ms. A recent model of ion permeation through a single, open RyR channel is used here to show that the vast majority of this countercurrent is conducted by the RyR itself. Consequently, changes in membrane potential are minimized locally and instantly, assuring maintenance of a Ca2+-driving force. This RyR autocountercurrent is possible because of the poor Ca2+ selectivity and high conductance for both monovalent and divalent cations of these channels. The model shows that, under physiological conditions, the autocountercurrent clamps the membrane potential near 0mV within ∼150μs. Consistent with experiments, the model shows how RyR unit Ca2+ current is defined by luminal [Ca2+], permeable ion composition and concentration, and pore selectivity and conductance. This very likely is true of the highly homologous pore of the IP3R channel.

Targeting Bcl-2-IP3 Receptor Interaction to Reverse Bcl-2's Inhibition of Apoptotic Calcium Signals

The antiapoptotic protein Bcl-2 inhibits Ca2+ release from the endoplasmic reticulum (ER). One proposed mechanism involves an interaction of Bcl-2 with the inositol 1,4,5-trisphosphate receptor (IP3R) Ca2+ channel localized with Bcl-2 on the ER. Here we document Bcl-2-IP3R interaction within cells by FRET and identify a Bcl-2 interacting region in the regulatory and coupling domain of the IP3R. A peptide based on this IP3R sequence displaced Bcl-2 from the IP3R and reversed Bcl-2-mediated inhibition of IP3R channel activity in vitro, IP3-induced ER Ca2+ release in permeabilized cells, and cell-permeable IP3 ester-induced Ca2+ elevation in intact cells. This peptide also reversed Bcl-2's inhibition of T cell receptor-induced Ca2+ elevation and apoptosis. Thus, the interaction of Bcl-2 with IP3Rs contributes to the regulation of proapoptotic Ca2+ signals by Bcl-2, suggesting the Bcl-2-IP3R interaction as a potential therapeutic target in diseases associated with Bcl-2's inhibition of cell death.

Store-operated Ca2+ Influx Causes Ca2+ Release from the Intracellular Ca2+ Channels That Is Required for T Cell Activation

The precise control of many T cell functions relies on cytosolic Ca(2+) dynamics that is shaped by the Ca(2+) release from the intracellular store and extracellular Ca(2+) influx. The Ca(2+) influx activated following T cell receptor (TCR)-mediated store depletion is considered to be a major mechanism for sustained elevation in cytosolic Ca(2+) concentration ([Ca(2+)](i)) necessary for T cell activation, whereas the role of intracellular Ca(2+) release channels is believed to be minor. We found, however, that in Jurkat T cells [Ca(2+)](i) elevation observed upon activation of the store-operated Ca(2+) entry (SOCE) by passive store depletion with cyclopiazonic acid, a reversible blocker of sarco-endoplasmic reticulum Ca(2+)-ATPase, inversely correlated with store refilling. This indicated that intracellular Ca(2+) release channels were activated in parallel with SOCE and contributed to global [Ca(2+)](i) elevation. Pretreating cells with (-)-xestospongin C (10 microM) or ryanodine (400 microM), the antagonists of inositol 1,4,5-trisphosphate receptor (IP3R) or ryanodine receptor (RyR), respectively, facilitated store refilling and significantly reduced [Ca(2+)](i) elevation evoked by the passive store depletion or TCR ligation. Although the Ca(2+) release from the IP3R can be activated by TCR stimulation, the Ca(2+) release from the RyR was not inducible via TCR engagement and was exclusively activated by the SOCE. We also established that inhibition of IP3R or RyR down-regulated T cell proliferation and T-cell growth factor interleukin 2 production. These studies revealed a new aspect of [Ca(2+)](i) signaling in T cells, that is SOCE-dependent Ca(2+) release via IP3R and/or RyR, and identified the IP3R and RyR as potential targets for manipulation of Ca(2+)-dependent functions of T lymphocytes.

THE INTERACTION OF VASODILATOR‐STIMULATED PHOSPHOPROTEIN (VASP) WITH IP3R AND TRPC CHANNELS IN MADIN‐DARBY CANINE KIDNEY (MDCK) CELLS

Several dedifferentiating disorders of tissues are the result of aberrantly elevated [Ca2+]i leading to increased transcription. Although the cGMP‐activated protein kinase (PKG) pathway attenuates increases in [Ca2+]i and dedifferentiation, the mechanism is not understood. We recently found in mesangial cells that when VASP, an actin associated focal adhesion protein, is phosphorylated at Ser239 by PKG, it associates with TRPC4 channels. This was interesting because Homer, a scaffold protein, interacts with IP3R and TRP channels at the Ena/VASP Homology I (EVHI) domain. Because both VASP and Homer contain the EVH1 domains, we hypothesized that VASP forms a similar interaction with TRPC and IP3R in MDCK cells, which are model epithelia for studying diseases such as polycystic kidney disease (PKD). We identified with RT‐PCR and immunocytochemistry the presence of TRPC1 and TRPC4 in MDCK cells. We also demonstrated interaction between VASP and TRPC1 with immunocytochemistry and an interaction between VASP with IP3R using immunocytochemistry and Co‐IP. Moreover, upon PKG phosphorylation of VASP at Ser239, VASP no longer interacted with TRPC1, but instead interacted with TRPC4 on the cell membrane. These results suggest that PKG controls Ca2+ entry in MDCK cells by phosphorylating VASP at Ser239. This may be part of the mechanism by which PKG attenuates the elevated [Ca2+]i in some dedifferentiating diseases.

Molecular Characterization of the Inositol 1,4,5-Trisphosphate Receptor Pore-forming Segment

Specific residues in the putative pore helix, selectivity filter, and S6 transmembrane helix of the inositol 1,4,5-trisphosphate receptor were mutated in order to examine their effects on channel function. Mutation of 5 of 8 highly conserved residues in the pore helix/selectivity filter region inactivated the channel (C2533A, G2541A, G2545A, G2546A, and G2547A). Of the remaining three mutants, C2527A and R2543A were partially active and G2549A behaved like wild type receptor. Mutation of a putative glycine hinge residue in the S6 helix (G2586A) or a putative gating residue at the cytosolic end of S6 helix (F2592A) had minimal effects on function, although channel function was inactivated by G2586P and F2592D mutations. The mutagenesis data are interpreted in the context of a structural homology model of the inositol 1,4,5-trisphosphate receptor.

A Kinetic Model of Single and Clustered IP 3 Receptors in the Absence of Ca 2+ Feedback

Ca 2+ liberation through inositol 1,4,5-trisphosphate receptor (IP 3R) channels generates complex patterns of spatiotemporal cellular Ca 2+ signals owing to the biphasic modulation of channel gating by Ca 2+ itself. These processes have been extensively studied in Xenopus oocytes, where imaging studies have revealed local Ca 2+ signals (“puffs”) arising from clusters of IP 3R, and patch-clamp studies on isolated oocyte nuclei have yielded extensive data on IP 3R gating kinetics. To bridge these two levels of experimental data, we developed an IP 3R model and applied stochastic simulation and transition matrix theory to predict the behavior of individual and clustered IP 3R channels. The channel model consists of four identical, independent subunits, each of which has an IP 3-binding site together with one activating and one inactivating Ca 2+-binding site. The channel opens when at least three subunits undergo a conformational change to an “active” state after binding IP 3 and Ca 2+. The model successfully reproduces patch-clamp data; including the dependence of open probability, mean open duration, and mean closed duration on [IP 3] and [Ca 2+]. Notably, the biexponential distribution of open-time duration and the dependence of mean open time on [Ca 2+] are explained by populations of openings involving either three or four active subunits. As a first step toward applying the single IP 3R model to describe cellular responses, we then simulated measurements of puff latency after step increases of [IP 3]. Assuming that stochastic opening of a single IP 3R at basal cytosolic [Ca 2+] and any given [IP 3] has a high probability of rapidly triggering neighboring channels by calcium-induced calcium release to evoke a puff, optimal correspondence with experimental data of puff latencies after photorelease of IP 3 was obtained when the cluster contained a total of 40–70 IP 3Rs.

The Calponin Homology Domain of Vav1 Associates with Calmodulin and Is Prerequisite to T Cell Antigen Receptor-induced Calcium Release in Jurkat T Lymphocytes

Vav1 is a guanine nucleotide exchange factor that is expressed specifically in hematopoietic cells and plays important roles in T cell development and activation. Vav1 consists of multiple structural domains so as to facilitate both its guanine nucleotide exchange activity and scaffold function following T cell antigen receptor (TCR) engagement. Previous studies demonstrated that the calponin homology (CH) domain of Vav1 is required for TCR-stimulated calcium mobilization and thus downstream activation of nuclear factor of activated T cells. However, it remained obscure how Vav1 functions in regulating calcium flux. In an effort to explore molecules interacting with Vav1, we found that calmodulin bound to Vav1 in a calcium-dependent and TCR activation-independent manner. The binding site was mapped to the CH domain of Vav1. Reconstitution of vav1-null Jurkat T cells (J.Vav1) with CH-deleted Vav1 exhibited a severe deficiency in calcium release to the same extent as that of Jurkat cells treated with the calmodulin inhibitor or J.Vav1 cells. The defect persisted even when phospholipase-Cgamma1 was fully activated, indicating a prerequisite role of Vav1 CH domain in calcium signaling. The results suggest that Vav1 and calmodulin function cooperatively to potentiate TCR-induced calcium release. This study unveiled a mechanism by which the Vav1 CH domain is involved in calcium signaling and provides insight into our understanding of the role of Vav1 in T cell activation.

DANGER, a Novel Regulatory Protein of Inositol 1,4,5-Trisphosphate-Receptor Activity

We report the cloning and characterization of DANGER, a novel protein which physiologically binds to inositol 1,4,5-trisphosphate receptors (IP(3)R). DANGER is a membrane-associated protein predicted to contain a partial MAB-21 domain. It is expressed in a wide variety of neuronal cell lineages where it localizes to membranes in the cell periphery together with IP(3)R. DANGER interacts with IP(3)R in vitro and co-immunoprecipitates with IP(3)R from cellular preparations. DANGER robustly enhances Ca(2+)-mediated inhibition of IP(3) RCa(2+) release without affecting IP(3) binding in microsomal assays and inhibits gating in single-channel recordings of IP(3)R. DANGER appears to allosterically modulate the sensitivity of IP(3) RtoCa(2+) inhibition, which likely alters IP(3)R-mediated Ca(2+) dynamics in cells where DANGER and IP(3)R are co-expressed.

The Number and Spatial Distribution of IP 3 Receptors Underlying Calcium Puffs in Xenopus Oocytes

Calcium puffs are local Ca 2+ release events that arise from a cluster of inositol 1,4,5-trisphosphate receptor channels (IP 3Rs) and serve as a basic “building block” from which global Ca 2+ waves are generated. Important questions remain as to the number of IP 3Rs that open during a puff, their spatial distribution within a cluster, and how much Ca 2+ current flows through each channel. The recent discovery of “trigger” events—small Ca 2+ signals that immediately precede puffs and are interpreted to arise through opening of single IP 3R channels—now provides a useful yardstick by which to calibrate the Ca 2+ flux underlying puffs. Here, we describe a deterministic numerical model to simulate puffs and trigger events. Based on confocal linescan imaging in Xenopus oocytes, we simulated Ca 2+ release in two sequential stages; representing the trigger by the opening of a single IP 3R in the center of a cluster for 12 ms, followed by the concerted opening of some number of IP 3Rs for 19 ms, representing the rising phase of the puff. The diffusion of Ca 2+ and Ca 2+-bound indicator dye were modeled in a three-dimensional cytosolic volume in the presence of immobile and mobile Ca 2+ buffers, and were used to predict the observed fluorescence signal after blurring by the microscope point-spread function. Optimal correspondence with experimental measurements of puff spatial width and puff/trigger amplitude ratio was obtained assuming that puffs arise from the synchronous opening of 25–35 IP 3Rs, each carrying a Ca 2+ current of ∼0.4 pA, with the channels distributed through a cluster 300–800 nm in diameter.

Reaction Diffusion Modeling of Calcium Dynamics with Realistic ER Geometry

We describe a finite-element model of mast cell calcium dynamics that incorporates the endoplasmic reticulum’s complex geometry. The model is built upon a three-dimensional reconstruction of the endoplasmic reticulum (ER) from an electron tomographic tilt series. Tetrahedral meshes provide volumetric representations of the ER lumen, ER membrane, cytoplasm, and plasma membrane. The reaction-diffusion model simultaneously tracks changes in cytoplasmic and ER intraluminal calcium concentrations and includes luminal and cytoplasmic protein buffers. Transport fluxes via PMCA, SERCA, ER leakage, and Type II IP 3 receptors are also represented. Unique features of the model include stochastic behavior of IP 3 receptor calcium channels and comparisons of channel open times when diffusely distributed or aggregated in clusters on the ER surface. Simulations show that IP 3R channels in close proximity modulate activity of their neighbors through local Ca 2+ feedback effects. Cytoplasmic calcium levels rise higher, and ER luminal calcium concentrations drop lower, after IP 3-mediated release from receptors in the diffuse configuration. Simulation results also suggest that the buffering capacity of the ER, and not restricted diffusion, is the predominant factor influencing average luminal calcium concentrations.

Two Functions for IP 3 Receptor Partner

Inositol 1,4,5-trisphosphate (IP 3 ), a second messenger formed in response to stimulation of cell surface receptors, binds to and activates IP 3 receptors (IP 3 Rs, internal calcium release channels) to liberate calcium from intracellular stores. Phosphorylated IRBIT (IP 3 R-binding protein released with inositol 1,4,5-trisphosphate) binds to the IP 3 -binding region of the IP 3 R, from which it is displaced by IP 3 . Ando et al. , who initially identified IRBIT, used an in vitro binding assay to show that IRBIT displaced IP 3 from a fusion protein containing the IP 3 R lacking its channel domain. Scatchard analysis indicated that IRBIT acted as a competitive inhibitor that decreased the affinity of the IP 3 R for IP 3 , and mutational analysis indicated that most of the amino acid residues necessary for IP 3 binding to the IP 3 R were also involved in IRBIT binding. Both IRBIT that had been exposed to alkaline phosphatase and a mutant form (S68A, in which serine 68 was substituted with alanine) that showed decreased phosphorylation and less efficient binding to the IP 3 R than wild-type IRBIT failed to suppress IP 3 binding to the IP 3 R. IRBIT also inhibited IP 3 binding and IP 3 -dependent calcium release in cerebellar microsomes. Calcium imaging of HeLa cells in which IRBIT was depleted with siRNA revealed enhanced release of calcium compared with wild-type cells in response to stimulation with an IP 3 -generating agonist. Overexpression of S68A (which bound to endogenous IRBIT, forming a heteromultimer with weaker affinity for the IP 3 R than that of the wild-type homomultimer) also enhanced IP 3 -dependent release. Thus, the authors propose that IRBIT acts as a "pseudoligand" regulated by phosphorylation that inhibits IP 3 binding and thereby calcium release. In a second paper, another group from the Mikoshiba lab proposes a second function of IRBIT based on the idea that when IP 3 binds to IP 3 Rs, displaced IRBIT might serve to carry a signal to other partners. Shirakabe et al. therefore searched for proteins that interacted with IRBIT in extracts of mouse cerebellum. A prominent interacting protein from membrane fractions was identified as NBC1, the Na + /HCO 3 – cotransporter that shuttles HCO 3 – and Na + ions across the plasma membrane. Reciprocal immunoprecipitation experiments verified that the endogenous proteins interact. Like its binding to the IP 3 R, binding of IRBIT to NBC1 was dependent on phosphorylation of IRBIT. The functional consequence of the interaction was tested in a Xenopus oocyte system. Electrophysiological measurements showed that expression of IRBIT along with NBC1 was necessary to allow full activity of the transporter. IRBIT interacted only with the NBC1 splicing variant called pNBC1, which is present in the pancreas, where it is thought to promote HCO 3 – transport in pancreatic duct cells. Thus, the authors propose that, at least in the pancreas, physiological concentrations of IP 3 , which cause dissociation of IRBIT from IP 3 Rs, could not only promote release of calcium through the IP 3 R channel but also mobilize an IRBIT-mediated signal that modulates acid-base balance. H. Ando, A. Mizutani, H. Kiefer, D. Tsuzurugi, T. Michikawa, K. Mikoshiba, IRBIT suppresses IP 3 receptor activity by competing with IP 3 for the common binding site on the IP 3 receptor. Mol. Cell 22 , 795-806 (2006). [Online Journal] K. Shirakabe, G. Priori, H. Yamada, H. Ando, S. Horita, T. Fujita, I. Fujimoto, A. Mizutani, G. Seki, K. Mikoshiba, IRBIT, an inositol 1,4,5-trisphosphate receptor-binding protein, specifically binds to and activates pancreas-type Na + /HCO 3 – cotransporter 1 (pNBC1). Proc. Natl. Acad. Sci. U.S.A. 103 , 9542-9547 (2006). [Abstract] [Full Text]

Modeling of ATP-mediated signal transduction and wave propagation in astrocytic cellular networks

Astrocytes, a special type of glial cells, were considered to have supporting role in information processing in the brain. However, several recent studies have shown that they can be chemically stimulated by neurotransmitters and use a form of signaling, in which ATP acts as an extracellular messenger. Pathological conditions, such as spreading depression, have been linked to abnormal range of wave propagation in astrocytic cellular networks. Nevertheless, the underlying intra- and inter-cellular signaling mechanisms remain unclear. Motivated by the above, we constructed a model to understand the relationship between single-cell signal transduction mechanisms and wave propagation and blocking in astrocytic networks. The model incorporates ATP-mediated IP 3 production, the subsequent Ca 2+ release from the ER through IP 3R channels and ATP release into the extracellular space. For the latter, two hypotheses were tested: Ca 2+- or IP 3-dependent ATP release. In the first case, single astrocytes can exhibit excitable behavior and frequency-encoded oscillations. Homogeneous, one-dimensional astrocytic networks can propagate waves with infinite range, while in two dimensions, spiral waves can be generated. However, in the IP 3-dependent ATP release case, the specific coupling of the driver ATP–IP 3 system with the driven Ca 2+ subsystem leads to one- and two-dimensional wave patterns with finite range of propagation.