IP3 Rs Research Articles

Calcium mobilization via type III inositol 1,4,5-trisphosphate receptors is not altered by PKA-mediated phosphorylation of serines 916, 934, and 1832

Several studies have shown that PKA-mediated phosphorylation of IP 3R1 at serines S 1588 and S 1755 enhances the receptor's ability to mobilize Ca 2+. In contrast, much less is known about whether Ca 2+ mobilization via IP 3R2 and IP 3R3 is regulated by PKA. We report here that IP 3R2 is only very weakly phosphorylated in response to PKA activation and is probably not a physiological substrate for this kinase. IP 3R3, however, is known to be phosphorylated by PKA at three sites (S 916, S 934, and S 1832) and, thus, we examined how phosphorylation of these sites affects Ca 2+ mobilization in DT40-3KO cells stably expressing either exogenous wild-type or mutant IP 3R3s; an antibody raised against phospho-serine 934 of IP 3R3 was used to demonstrate that the exogenous IP 3R3s are strongly phosphorylated in response to PKA activation. Surprisingly, our data show that IP 3R3-mediated Ca 2+ mobilization is unaffected by phosphorylation of S 916, S 934, and S 1832. In contrast, phosphorylation of exogenous IP 3R1 (monitored with an antibody against phospho-serine 1755) enhances Ca 2+ mobilization, indicating that DT40-3KO cells have the capacity to respond to phosphorylation of IP 3Rs. Overall, these data suggest that modification of Ca 2+ flux may not be the primary effect of IP 3R3 phosphorylation by PKA.

Differential inositol 1,4,5-trisphosphate receptor signaling in a neuronal cell line

Differential intracellular distribution of the three pharmacologically and biophysically distinct types of IP 3Rs can lead to different subcellular Ca 2+ transients each coupled to discrete intracellular functions. Here, we report the functional localization of differentially distributed IP 3 receptor types in the commonly-used hippocampal cell line HT22. The distinct subcellular localization and Ca 2+ signaling properties of these receptors determine the potential role of specific IP 3 receptor types in cellular function. By utilizing immunochemistry, we conclude that HT22 cells express all three IP 3 receptors with types 1 and 3 being expressed predominantly in the endoplasmic reticulum and perinuclear regions and type 2 being expressed predominantly in the nuclear envelope. Optical imaging studies using the Ca 2+-sensitive indicator dye fluo-3 show that nuclear IP 3 responses have greater amplitude and faster kinetics than cytosolic IP 3 responses corresponding to the biophysical characteristics of the differentially distributed receptor types. These results support the hypothesis that differentially distributed IP 3R isotypes mediate distinct cellular functions through differential, organelle-specific Ca 2+ signaling.

‘Trigger’ Events Precede Calcium Puffs in Xenopus Oocytes

The liberation of calcium ions sequestered in the endoplasmic reticulum through inositol 1,4,5-trisphosphate receptors/channels (IP 3Rs) results in a spatiotemporal hierarchy of calcium signaling events that range from single-channel openings to local Ca 2+ puffs believed to arise from several to tens of clustered IP 3Rs to global calcium waves. Using high-resolution confocal linescan imaging and a sensitive Ca 2+ indicator dye (fluo-4-dextran), we show that puffs are often preceded by small, transient Ca 2+ elevations that we christen “trigger events”. The magnitude of triggers is consistent with their arising from the opening of a single IP 3 receptor/channel, and we propose that they initiate puffs by recruiting neighboring IP 3Rs within the cluster by a regenerative process of Ca 2+-induced Ca 2+ release. Puff amplitudes (fluorescence ratio change) are on average ∼6 times greater than that of the triggers, suggesting that at least six IP 3Rs may simultaneously be open during a puff. Trigger events have average durations of ∼12 ms, as compared to 19 ms for the mean rise time of puffs, and their spatial extent is ∼3 times smaller than puffs (respective widths at half peak amplitude 0.6 and 1.6 μm). All these parameters were relatively independent of IP 3 concentration, although the proportion of puffs showing resolved triggers was greatest (∼80%) at low [IP 3]. Because Ca 2+ puffs constitute the building blocks from which cellular IP 3-mediated Ca 2+ signals are constructed, the events that initiate them are likely to be of fundamental importance for cell signaling. Moreover, the trigger events provide a useful yardstick by which to derive information regarding the number and spatial arrangement of IP 3Rs within clusters.

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.

IP 3 type 1 receptors in the heart: Their predominance in atrial walls with ganglion cells

Previously we have shown that inositol 1,4,5-trisphosphate (IP 3) receptors (IP 3Rs) are abundantly expressed in the atria of rat hearts. Since arrangement of atria is very heterogeneous, in this work we focused on the precise localization of IP 3 receptors in the left atrium, where the gene expression of the type 1 IP 3R was the highest. The mRNA levels of the IP 3 type 1 receptors in the left atrium, left ventricle and myocytes were determined using real-time polymerase chain reaction and Taqman probe. For precise localization, immunohistochemistry with the antibody against type 1 IP 3Rs was performed. The mRNA of type 1 IP 3 receptor was more than three times higher in the left atrium than in the left ventricle, as determined by real-time PCR. Expression of the type 1 IP 3 receptor mRNA was higher in the atria, especially in parts containing cardiac ganglion cells. The atrial auricles, which are particularly free of ganglion cells, and the ventricles (wall of the right and left ventricle and ventricular septum) contained four to five times less IP 3 receptors than atrial samples with ganglia. IP 3R type 1 immunoreactivity detected by a confocal microscope attributed the most condensed signal on ganglionic cells, although light immunoreactivity was also seen in cardiomyocytes. These results show that type 1IP 3 receptors predominate in intrinsic neuronal ganglia of cardiac atria.

Calcium regulation of inositol 1,4,5-trisphosphate receptors

Ca 2+ exerts both a stimulatory and inhibitory effect on type-I IP 3R channel activity. However, the structural determinants of Ca 2+ sensing in IP 3Rs are not fully understood. Previous studies by others have identified eight domains of the type-I IP 3R that bind 45Ca 2+ when expressed as GST-fusion proteins. We have mutated six highly conserved acidic residues within the second of these domains (aa378–450) in the full-length IP 3R and measured the Ca 2+regulation of IP 3-mediated Ca 2+ release in COS-7 cells. 45Ca 2+flux assays measured with a maximal [IP 3] (1 μM) indicate that one of the mutants retained a Ca 2+ sensitivity that was not significantly different from control (E411Q), three of the mutants show an enhanced Ca 2+ inhibition (D426N, E428Q and E439Q) and two of the mutants were relatively insensitive to Ca 2+ inhibition (D442N and D444N). IP 3 dose–response relationships indicated that the sensitivity to Ca 2+ inhibition and affinity for IP 3 were correlated for three of the constructs. Other mutants with enhanced IP 3 sensitivity (e.g. R441Q and a type-II/I IP 3R chimera) were also less sensitive to Ca 2+ inhibition. We conclude that the acidic residues within the aa378–450 segment are unlikely to represent a single functional Ca 2+ binding domain and do not contribute to Ca 2+ activation of the receptor. The different effects of the mutations may be related to their location within two clusters of acidic residues identified in the crystal structure of the ligand-binding domain [I. Bosanac, J.R. Alattia, T.K. Mal, et al., Structure of the inositol 1,4,5-trisphosphate receptor binding core in complex with its ligand, Nature 420 (2002) 696–700]. The data support the view that all IP 3R isoforms may display a range of Ca 2+ sensitivities that are determined by multiple sites within the protein and markedly influenced by the affinity of the receptor for IP 3.

Spatial microenvironment defines Ca 2+ entry and Ca 2+ release in salivary gland cells

The difference of Ca 2+ mobilization induced by muscarinic receptor activation between parotid acinar and duct cells was examined. Oxotremorine, a muscarinic–cholinergic agonist, induced intracellular Ca 2+ release and extracellular Ca 2+ entry through store-operated Ca 2+ entry (SOC) and non-SOC channels in acinar cells, but it activated only Ca 2+ entry from non-SOC channels in duct cells. RT-PCR experiments showed that both types of cells expressed the same muscarinic receptor, M3. Given that ATP activated the intracellular Ca 2+ stores, the machinery for intracellular Ca 2+ release was intact in the duct cells. By immunocytochemical experiments, IP 3R2 colocalized with M3 receptors in the plasma membrane area of acinar cells; in duct cells, IP 3R2 resided in the region on the opposite side of the M3 receptors. On the other hand, purinergic P2Y2 receptors were found in the apical area of duct cells where they colocalized with IP 3R2. These results suggest that the expression of the IP 3Rs near G-protein-coupled receptors is necessary for the activation of intracellular Ca 2+ stores. Therefore, the microenvironment probably affects intracellular Ca 2+ release and Ca 2+ entry.

Involvement of calcium and calmodulin in the regulation of ovarian steroidogenesis in Atlantic croaker ( Micropogonias undulatus) and modulation by Aroclor 1254

The involvement of calcium-dependent signal transduction pathways in the regulation of ovarian steroidogenesis was investigated in Atlantic croaker. Treatment with the calcium ionophores A23187 and ionomycin caused a 2- to 5-fold increase in basal steroid accumulation by croaker ovarian tissue in vitro. A23187 potentiated human chorionic gonadotropin (hCG)-induced testosterone (T) accumulation, whereas it inhibited accumulation of estradiol-17β (E 2) and the conversion of T to E 2, suggesting that intracellular calcium modulates aromatase enzyme activity. Gonadotropin stimulation of ovarian steroidogenesis was decreased in the presence of EGTA and inhibitors of voltage-sensitive calcium channels (VSCCs) and inositol-1,4,5-triphosphate-receptors (IP 3Rs), indicating that releases of calcium from both intracellular and extracellular stores are components of the signal transduction pathways initiated by gonadotropin. Calmodulin is also involved in the regulation of ovarian steroidogenesis in croaker, since the calmodulin inhibitors W-7 and trifluoperazine (TFP) attenuated hCG-stimulated T and E 2 accumulation. These results are broadly similar to those reported previously in goldfish and suggest that the major calcium-dependent signaling pathways involved in gonadotropin stimulation of ovarian steroidogenesis in tetrapods are also present in teleosts. In addition, the involvement of calcium in the regulation of aromatase activity was demonstrated for the first time in a vertebrate ovary. Finally, acute exposure to 0.001–1 ppm Aroclor 1254 induced up to a 5-fold increase in hCG-stimulated E 2 accumulation, and this effect was attenuated by co-treatment with inhibitors of VSCCs and calmodulin, suggesting the existence of a novel mechanism of endocrine disruption by an environmental contaminant involving alteration of calcium-dependent signaling pathways regulating steroidogenesis.

The dynamics of stochastic attrition viewed as an absorption time on a terminating Markov chain

Localized Ca 2+ elevations known as Ca 2+ puffs and sparks are cellular signals that arise from the cooperative activity of clusters of inositol 1,4,5-trisphosphate receptors and ryanodine receptors clustered at Ca 2+ release sites on the surface of the endoplasmic reticulum or sarcoplasmic reticulum. When Markov chain models of these intracellular Ca 2+-regulated Ca 2+ channels are coupled via a mathematical representation of Ca 2+ microdomain, simulated Ca 2+ release sites may exhibit the phenomenon of “stochastic Ca 2+ excitability” where the inositol 1,4,5-trisphosphate receptors (IP 3Rs) or ryanodine receptors (RyRs) open and close in a concerted fashion. Interestingly, under some conditions simulated puffs and sparks can be observed even when the single-channel model used does not include slow Ca 2+ inactivation or, indeed, any long-lived closed/refractory state [V. Nguyen, R. Mathias, G. Smith, Stochastic automata network descriptor for Markov chain models of instantaneously-coupled intracellular Ca 2+ channels, Bull. Math. Biol. 67 (2005) 393–432]. In this case, termination of the localized Ca 2+ elevation occurs when all of the intracellular channels at a release site simultaneously close through a process referred to as stochastic attrition [M. Stern, Theory of excitation-contraction coupling in cardiac muscle, Biophys. J. 63 (1992) 497–517]. In this paper, we investigate the statistical properties of stochastic attrition viewed as an absorption time on a terminating Markov chain that represents a Ca 2+ release site composed of N two-state channels that are activated by Ca 2+. Assuming that the local [Ca 2+] experienced by a channel depends only on the number of open channels at the Ca 2+ release site (i.e., instantaneous mean-field coupling [ibid.], we derive the probability distribution function for the time until stochastic attrition occurs and present an analytical formula for the expectation of this random variable. We explore how the contribution of stochastic attrition to the termination of Ca 2+ puffs and sparks depends on the number of channels at a release site, the source amplitude of the channels (i.e., the strength of the coupling), the background [Ca 2+], channel kinetics, and the cooperactivity of Ca 2+ binding. Because we explicitly model the Ca 2+ regulation of the intracellular channels, our results differ markedly from (and in fact generalize) preliminary analyses that assume the intracellular Ca 2+ channels are uncoupled and consequently independent.

Postsynaptic IP 3 receptor-mediated Ca 2+ release modulates synaptic transmission in hippocampal neurons

Ca 2+-dependent mechanisms are important in regulating synaptic transmission. The results herein indicate that whole-cell perfusion of inositol 1,4,5-trisphosphate receptor (IP 3R) agonists greatly enhanced excitatory postsynaptic current (EPSC) amplitudes in postsynaptic hippocampal CA1 neurons. IP 3R agonist-mediated increases in synaptic transmission changed during development and paralleled age-dependent increases in hippocampal type-1 IP 3Rs. IP 3R agonist-mediated increases in EPSC amplitudes were inhibited by postsynaptic perfusion of inhibitors of Ca 2+/calmodulin, PKC and Ca 2+/calmodulin-dependent protein kinase II. Postsynaptic perfusion of inhibitors of smooth endoplasmic reticulum (SER) Ca 2+-ATPases, which deplete intracellular Ca 2+ stores, also enhanced EPSC amplitudes. Postsynaptic perfusion of the IP 3R agonist adenophostin (AdA) during subthreshold stimulation appeared to convert silent to active synapses; synaptic transmission at these active synapses was completely blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Postsynaptic IP 3R-mediated Ca 2+ release also produced a significant increase in spontaneous EPSC frequency. These results indicate that Ca 2+ release from intracellular stores play a key role in regulating the function of postsynaptic AMPARs.

Involvement of ryanodine receptors in IP 3-mediated calcium signalling in neurons : A modelling approach

Phospholipase C (PLC)-coupled metabotropic receptors trigger the release of intracellular Ca 2+ through activation of IP 3 receptors (IP 3Rs). Increasing evidence suggests that they can also and perhaps more efficiently mobilize Ca 2+ through ryanodine receptors (RyRs). We constructed a model allowing a variable PLC stimulation level (via the parameter γ) as well as a variable involvement of RyRs (via the parameter A). The sole presence of RyRs ( A ≠ 0) affected the basal Ca 2+ concentration [Ca 2+] i. To keep Ca 2+ below 160 nM, we fixed the upper limit of A to 0.2, a value that is compatible with the numerical ratio between RyRs and IP 3Rs in cerebellar Purkinje neurons. Metabotropic responses were simulated by abruptly raising the value of γ to various levels. In the absence of RyRs, the model starts to oscillate with γ = 0.4. For lower levels of PLC stimulation ( γ ≤ 0.3), the presence of RyR is capable of triggering an oscillatory signal. When A ≤ 0.18, the frequency of the Ca 2+ oscillations augments from 0.1 to 1.3 Hz as a function of γ. Conversely, as the frequency increases, the amplitude of the oscillations is reduced from 1 μM to 50 nM. With higher values of A, the oscillating pattern is definitively inhibited. It is concluded that RyRs have the potentiality to strikingly affect the temporal pattern of the Ca 2+ signalling triggered by IP 3-related metabotropic responses.

Determinants of postsynaptic Ca 2+ signaling in Purkinje neurons

Neuronal integration in Purkinje neurons involves many forms of Ca 2+ signaling. Two afferent synaptic inputs, the parallel and the climbing fibers, provide a major drive for these signals. These two excitatory synaptic inputs are both glutamatergic. Postsynaptically they activate alpha-amino-3-hydroxy-5-methyl-4-propionic acid (AMPA) receptors (AMPARs) and metabotropic glutamate receptors (mGluRs). Unlike most other types of central neurons, Purkinje neurons do not express NMDA ( N-methyl- d-aspartate) receptors (NMDARs). AMPARs in Purkinje neurons are characterized by a low permeability for Ca 2+ ions. AMPAR-mediated synaptic depolarization may activate voltage-gated Ca 2+ channels, mostly of the P/Q-type. The resulting intracellular Ca 2+ signals are shaped by the Ca 2+ buffers calbindin and parvalbumin. Ca 2+ clearance from the cytosol is brought about by Ca 2+-ATPases in the plasma membrane and the endoplasmic reticulum, as well as the Na +–Ca 2+-exchanger. Binding of glutamate to mGluRs induces postsynaptic Ca 2+-transients through two G protein-dependent pathways: involving (1) the release of Ca 2+ ions from intracellular Ca 2+ stores and (2) the opening of the cation channel TRPC1. Homer proteins appear to play an important role in postsynaptic Ca 2+ signaling by providing a direct link between the plasma membrane-resident elements (mGluRs and TRPC1) and their intracellular partners, including the IP 3Rs.

Subtype-Specific and ER Lumenal Environment-Dependent Regulation of Inositol 1,4,5-Trisphosphate Receptor Type 1 by ERp44

Inositol 1,4,5-trisphosphate receptors (IP 3Rs) are intracellular channel proteins that mediate Ca 2+ release from the endoplasmic reticulum (ER) and are involved in many biological processes and diseases. IP 3Rs are differentially regulated by a variety of cytosolic proteins, but their regulation by ER lumenal protein(s) remains largely unexplored. In this study, we found that ERp44, an ER lumenal protein of the thioredoxin family, directly interacts with the third lumenal loop of IP 3R type 1 (IP 3R1) and that the interaction is dependent on pH, Ca 2+ concentration, and redox state: the presence of free cysteine residues in the loop is required. Ca 2+-imaging experiments and single-channel recording of IP 3R1 activity with a planar lipid bilayer system demonstrated that IP 3R1 is directly inhibited by ERp44. Thus, ERp44 senses the environment in the ER lumen and modulates IP 3R1 activity accordingly, which should in turn contribute to regulating both intralumenal conditions and the complex patterns of cytosolic Ca 2+ concentrations.

Regulation of muscarinic cationic current in myocytes from guinea-pig ileum by intracellular Ca 2+ release: a central role of inositol 1,4,5-trisphosphate receptors

The dynamics of carbachol (CCh)-induced [Ca 2+] i changes was related to the kinetics of muscarinic cationic current (mI cat) and the effect of Ca 2+ release through ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP 3Rs) on mI cat was evaluated by fast x– y or line-scan confocal imaging of [Ca 2+] i combined with simultaneous recording of mI cat under whole-cell voltage clamp. When myocytes freshly isolated from the longitudinal layer of the guinea-pig ileum were loaded with the Ca 2+-sensitive indicator fluo-3, x– y confocal imaging revealed CCh (10 μM)-induced Ca 2+ waves, which propagated from the cell ends towards the myocyte centre at 45.9±8.8 μm s −1 ( n=13). Initiation of the Ca 2+ wave preceded the appearance of any measurable mI cat by 229±55 ms ( n=7). Furthermore, CCh-induced [Ca 2+] i transients peaked 1.22±0.11 s ( n=17) before mI cat reached peak amplitude. At −50 mV, spontaneous release of Ca 2+ through RyRs, resulting in Ca 2+ sparks, had no effect on CCh-induced mI cat but activated BK channels leading to spontaneous transient outward currents (STOCs). In addition, Ca 2+ release through RyRs induced by brief application of 5 mM caffeine was initiated at the cell centre but did not augment mI cat ( n=14). This was not due to an inhibitory effect of caffeine on muscarinic cationic channels (since application of 5 mM caffeine did not inhibit mI cat when [Ca 2+] i was strongly buffered with Ca 2+/BAPTA buffer) nor was it due to an effect of caffeine on other mechanisms possibly involved in the regulation of Ca 2+ sensitivity of muscarinic cationic channels (since in the presence of 5 mM caffeine, photorelease of Ca 2+ upon cell dialysis with 5 mM NP–EGTA/3.8 mM Ca 2+ potentiated mI cat in the same way as in control). In contrast, IP 3R-mediated Ca 2+ release upon flash photolysis of “caged” IP 3 (30 μM in the pipette solution) augmented mI cat ( n=15), even though [Ca 2+] i did not reach the level required for potentiation of mI cat during photorelease of Ca 2+ ( n=10). Intracellular calcium stores were visualised by loading of the myocytes with the low-affinity Ca 2+ indicator fluo-3FF AM and consisted of a superficial sarcoplasmic reticulum (SR) network and some perinuclear formation, which appeared to be continuous with the superficial SR. Immunostaining of the myocytes with antibodies to IP 3R type 1 and to RyRs revealed that IP 3Rs are predominant in the superficial SR while RyRs are confined to the central region of the cell. These results suggest that IP 3R-mediated Ca 2+ release plays a central role in the modulation of mI cat in the guinea-pig ileum and that IP 3 may sensitise the regulatory mechanisms of the muscarinic cationic channels gating to Ca 2+.

Interactions between inositol 1,4,5-trisphosphate receptors and ryanodine receptors in smooth muscle: one store or two?

This short review proposes a system of simplified functional models describing possible interactions between Ca 2+-release channels associated with IP 3Rs and RyRs in smooth muscle, and considers each of these models in the light of the available experimental evidence. Complete separation of IP 3R- and RyR-gated stores seems to be unusual. Where both receptors release Ca 2+ from a common pool, simple interactions can occur since changes in the activation of one receptor type affects the availability of Ca 2+ for release through the other. Alterations in [Ca 2+] within the sarcoplasmic reticulum can also affect the open probability of the release channels, and not just the Ca 2+-flux through the channels when open, e.g., Ca 2+-release through tonically active IP 3Rs appears to limit SR Ca 2+-content in some myocytes, and this modulates RyR activity, as indicated by changes in Ca 2+-spark frequency. There is also evidence that intracellular release channels may co-operate, leading to positive feedback during activation. In particular, agonist-dependent activation of IP 3Rs can promote activation of RyRs, amplifying and shaping the resulting Ca 2+-signal. While there is little direct evidence as to the mechanism responsible for this interaction, some form of Ca 2+-induced Ca 2+-release in response to local increases in [Ca 2+] c seems likely.

Role of FKBP12.6 in hypoxia- and norepinephrine-induced Ca 2+ release and contraction in pulmonary artery myocytes

The cellular and molecular processes underlying the regulation of ryanodine receptor (RyR) Ca 2+ release in smooth muscle cells (SMCs) are incompletely understood. Here we show that FKBP12.6 proteins are expressed in pulmonary artery (PA) smooth muscle and associated with type-2 RyRs (RyR2), but not RyR1, RyR3, or IP 3 receptors (IP 3Rs) in PA sarcoplasmic reticulum. Application of FK506, which binds to FKBPs and dissociates these proteins from RyRs, induced an increase in [Ca 2+] i and Ca 2+-activated Cl − and K + currents in freshly isolated PASMCs, whereas cyclosporin, an agent known to inhibit calcineurin but not to interact with FKBPs, failed to induce an increase in [Ca 2+] i. FK506-induced [Ca 2+] i increase was completely blocked by the RyR antagonist ruthenium red and ryanodine, but not the IP 3R antagonist heparin. Hypoxic Ca 2+ response and hypoxic vasoconstriction were significantly enhanced in FKBP12.6 knockout mouse PASMCs. FK506 or rapamycin pretreatment also enhanced hypoxic increase [Ca 2+] i, but did not alter caffeine-induced Ca 2+ release (SR Ca 2+ content) in PASMCs. Norepinephrine-induced Ca 2+ release and force generation were also markedly enhanced in PASMCs from FKBP12.6 null mice. These findings suggest that FKBP12.6 plays an important role in hypoxia- and neurotransmitter-induced Ca 2+ and contractile responses by regulating the activity of RyRs in PASMCs.

Subcellular distribution of the inositol 1,4,5-trisphosphate receptors: functional relevance and molecular determinants

The inositol 1,4,5-trisphosphate receptor (IP 3R) is an intracellular Ca 2+ channel that is for the largest part expressed in the endoplasmic reticulum. Its precise subcellular localization is an important factor for the correct initiation and propagation of Ca 2+ signals. The relative position of the IP 3Rs, and thus of the IP 3-sensitive Ca 2+ stores, to mitochondria, nucleus or plasma membrane determines in many cases the physiological consequences of IP 3-induced Ca 2+ release. Most cell types express more than one IP 3R isoform and their subcellular distribution is cell-type dependent. Moreover, it was recently demonstrated that depending on the physiological status of the cell redistribution of IP 3Rs and/or of IP 3-sensitive Ca 2+ stores could occur. This indicates that the cell must be able to regulate not only IP 3R expression but also its distribution. The various proteins potentially determining IP 3R localization and redistribution will therefore be discussed.

Roles of ryanodine and inositol triphosphate receptors in regulation of plateau potentials in turtle spinal motoneurons

Generation of plateau potentials in spinal motoneurons depends on activation of voltage sensitive L-type Ca 2+ channels. These channels are facilitated by metabotropic receptors known to promote release of Ca 2+ from intracellular stores. The aim of this study is to determine if Ca 2+-release receptors in the endoplasmic reticulum (ER) that are sensitive to ryanodine (RyRs) and to inositol triphosphate receptors (IP 3Rs) contribute to the generation of plateau potentials. The effects of antagonists to RyRs, IP 3Rs and phospholipase C (PLC) were tested on discharge patterns associated with plateau potentials in motoneurons in slices from the spinal cord of the turtle. Plateau-related discharge patterns, un-facilitated or facilitated by agonists for group I glutamate metabotropic receptors, muscarine-sensitive cholinergic receptors or L-type Ca 2+ channels were inhibited by blockade of RyRs. In contrast, antagonists of IP 3Rs or PLC preferentially inhibited plateau-related discharge patterns when facilitated by activation of metabotropic receptors but in only half of the cells when promoted in the absence of metabotropic facilitators. Our findings show that RyRs and IP 3Rs regulate the generation of plateau potentials in motoneurons and suggest that RyRs may be directly involved with activation of the plateau potential.

Presence of the inositol 1,4,5-triphosphate receptor isoforms in the nucleoplasm

Although the inositol 1,4,5-triphosphate (IP 3)-induced nuclear Ca 2+ release has been shown to play key roles in nuclear functions, the presence of IP 3 receptor (IP 3R)/Ca 2+ channels in the nucleoplasm has not been found. Recently, the IP 3R/Ca 2+ channels were reported to exist in the nucleoplasmic reticulum structure, an extension of the nuclear envelope. Here we investigated the potential existence of the IP 3Rs in the nucleoplasm and found the presence of all three IP 3R isoforms in neuroendocrine and non-neuroendocrine cells. The IP 3Rs were widely scattered in the nucleoplasm, localizing in both the heterochromatin and euchromatin regions.

Homer Binds TRPC Family Channels and Is Required for Gating of TRPC1 by IP 3 Receptors

Receptor signaling at the plasma membrane often releases calcium from intracellular stores. For example, inositol triphosphate (IP3) produced by receptor-coupled phospholipase C activates an intracellular store calcium channel, the IP 3R. Conversely, stores can induce extracellular calcium to enter the cell through plasma membrane channels, too. How this “reverse” coupling works was unclear, but store IP 3Rs were proposed to bind and regulate plasma membrane TRP cation channels. Here, we demonstrate that the adaptor protein, termed Homer, facilitates a physical association between TRPC1 and the IP 3R that is required for the TRP channel to respond to signals. The TRPC1-Homer-IP 3R complex is dynamic and its disassembly parallels TRPC1 channel activation. Homer's action depends on its ability to crosslink and is blocked by the dominant-negative immediate early gene form, H1a. Since H1a is transcriptionally regulated by cellular activity, this mechanism can affect both short and long-term regulation of TRPC1 function.