Single-channel properties in endoplasmic reticulum membrane of recombinant type 3 inositol trisphosphate receptor

The inositol 1,4,5-trisphosphate receptor (InsP(3)R) is an intracellular Ca(2+)-release channel localized in endoplasmic reticulum (ER) with a central role in complex Ca(2+) signaling in most cell types. A family of InsP(3)Rs encoded by several genes has been identified with different primary sequences, subcellular locations, variable ratios of expression, and heteromultimer formation. This diversity suggests that cells require distinct InsP(3)Rs, but the functional correlates of this diversity are largely unknown. Lacking are single-channel recordings of the recombinant type 3 receptor (InsP(3)R-3), a widely expressed isoform also implicated in plasma membrane Ca(2+) influx and apoptosis. Here, we describe functional expression and single-channel recording of recombinant rat InsP(3)R-3 in its native membrane environment. The approach we describe suggests a novel strategy for expression and recording of recombinant ER-localized ion channels in the ER membrane. Ion permeation and channel gating properties of the rat InsP(3)R-3 are strikingly similar to those of Xenopus type 1 InsP(3)R in the same membrane. Using two different two-electrode voltage clamp protocols to examine calcium store-operated calcium influx, no difference in the magnitude of calcium influx was observed in oocytes injected with rat InsP(3)R-3 cRNA compared with control oocytes. Our results suggest that if cellular expression of multiple InsP(3)R isoforms is a mechanism to modify the temporal and spatial features of [Ca(2+)](i) signals, then it must be achieved by isoform-specific regulation or localization of various types of InsP(3)Rs that have relatively similar Ca(2+) permeation properties.     

Calcium and inositol trisphosphate receptors

Work from the authors' laboratory is supported by the Wellcome Trust, and the Medical, and Agricultural and Food Research Councils. CWT is a Lister Institute Research Fellow.     

An endogenous sulfated inhibitor of neuronal inositol trisphosphate receptors

In cerebellum, inositol trisphosphate- (InsP(3)-) gated Ca channels play a key role in learning, though they exhibit a low sensitivity to InsP(3) compared to peripheral tissues. In the present study, the cerebellar InsP(3) receptor is shown to be associated with a novel inhibitor of InsP(3) binding. (3)H-InsP(3) binding studies indicated that this inositol trisphosphate receptor inhibitor (IRI) could completely inhibit InsP(3) binding to the purified cerebellar InsP(3) receptor and acted as a competitive inhibitor. Gel filtration of IRI showed a predominant peak at 6500 Da, though this peak appeared to be an aggregate (with a monomeric molecular mass of approximately 1500 Da). Mass spectrometry of IRI showed a predominant peak at 1635 m/z, consistent with this low molecular mass estimate. The inhibitory activity of IRI was prevented by pretreatment with aryl sulfatase, suggesting the presence of a critical sulfo ester in IRI. IRI was insensitive to proteases and organic extraction but bound to concanavalin A, suggesting that IRI is a sulfated glycan. IRI was present in cerebellum but below the level of detection in aorta. IRI was also present in the neuronal cell line N1E115 (which exhibits a low sensitivity to InsP(3)). We conclude that IRI is a novel endogenous sulfated inhibitor of the InsP(3) receptor that modulates the sensitivity of the InsP(3) receptor and thus may explain the low InsP(3) sensitivity of neurons.     

Interactions between inositol trisphosphate receptors and fluorescent Ca2+ indicators.

Fura-2 and BAPTA were previously shown to be competitive antagonists of inositol trisphosphate (InsP3) receptors, but for practical reasons the analyses were performed at pH 8.3. We recently developed a scintillation proximity assay (SPA) for pure cerebellar InsP3 receptors which allows low affinity interactions to be characterized and is readily applicable to scarce or expensive ligands. In the present study, we use SPA to demonstrate that at pH 7.2, many of the commonly used fluorescent Ca2+ indicators reversibly displace 3H-InsP3 from its receptor and that they differ substantially in their affinities for the InsP3 receptor (IC50 = 6.5-137 microM). Recombinant type 1 InsP3 receptors expressed in Sf9 cells were used to examine 3H-InsP3 binding in cytosol-like medium: both fura-2 (IC50 = 796 +/- 86 microM) and Ca Green-5N (IC50 = 62 +/- 7 microM) completely inhibited the binding, but only in their Ca(2+)-free forms. Similar results were obtained with type 3 InsP3 receptors. We conclude that many Ca2+ indicators in their Ca(2+)-free forms compete with InsP3 for binding to its receptor, and that for Ca Green-5N the interaction occurs with sufficient affinity to significantly perturb physiological responses.     

Isomers of inositol trisphosphate in exocrine pancreas.

In rat pancreatic acinar cells, the Ca2+-mobilizing receptor-agonist, caerulein, at both maximal and submaximal concentrations, stimulated a rapid, transient, increase in [3H]inositol 1,4,5-trisphosphate [(1,4,5)IP3], followed by a slower, sustained, increase in [3H]inositol 1,3,4-trisphosphate [(1,3,4)IP3]. Neither activation of protein kinase C by phorbol dibutyrate nor prevention of the caerulein-stimulated elevation of cytosolic [Ca2+] significantly affected the pattern of formation of the two isomers of IP3. Although carbachol evoked an increase in cytosolic [Ca2+], it did not significantly stimulate [3H](1,4,5)IP3 accumulation, but did promote [3H](1,3,4)IP3 accumulation. Moreover, both carbachol and caerulein maintained hormone-sensitive intracellular Ca2+ pools in a Ca2+-depleted state after [3H](1,4,5)IP3 had returned to basal concentrations. One interpretation of these findings is that total cellular concentrations of [3H](1,4,5)IP3 may not accurately reflect the concentration of this putative mediator in biologically relevant compartments.     

Ryanodine and inositol trisphosphate receptors coexist in avian cerebellar Purkinje neurons

Two intracellular calcium-release channel proteins, the inositol trisphosphate (InsP3), and ryanodine receptors, have been identified in mammalian and avian cerebellar Purkinje neurons. In the present study, biochemical and immunological techniques were used to demonstrate that these proteins coexist in the same avian Purkinje neurons, where they have different intracellular distributions. Western analyses demonstrate that antibodies produced against the InsP3 and the ryanodine receptors do not cross-react. Based on their relative rates of sedimentation in continuous sucrose gradients and SDS-PAGE, the avian cerebellar InsP3 receptor has apparent native and subunit molecular weights of approximately 1,000 and 260 kD, while those of the ryanodine receptors are approximately 2,000 and 500 kD. Specific [3H]InsP3- and [3H]ryanodine-binding activities were localized in the sucrose gradient fractions enriched in the 260-kD and the approximately 500-kD polypeptides, respectively. Under equilibrium conditions, cerebellar microsomes bound [3H]InsP3 with a Kd of 16.8 nM and Bmax of 3.8 pmol/mg protein; whereas, [3H]ryanodine was bound with a Kd of 1.5 nM and a capacity of 0.08 pmol/mg protein. Immunolocalization techniques, applied at both the light and electron microscopic levels, revealed that the InsP3 and ryanodine receptors have overlapping, yet distinctive intracellular distributions in avian Purkinje neurons. Most notably the InsP3 receptor is localized in endomembranes of the dendritic tree, in both the shafts and spines. In contrast, the ryanodine receptor is observed in dendritic shafts, but not in the spines. Both receptors appear to be more abundant at main branch points of the dendritic arbor. In Purkinje neuron cell bodies, both the InsP3 and ryanodine receptors are present in smooth and rough ER, subsurface membrane cisternae and to a lesser extent in the nuclear envelope. In some cases the receptors coexist in the same membranes. Neither protein is observed at the plasma membrane, Golgi complex or mitochondrial membranes. Both the InsP3 and ryanodine receptors are associated with intracellular membrane systems in axonal processes, although they are less abundant there than in dendrites. These data demonstrate that InsP3 and ryanodine receptors exist as unique proteins in the same Purkinje neuron. These calcium-release channels appear to coexist in ER membranes in most regions of the Purkinje neurons, but importantly they are differentially distributed in dendritic processes, with the dendritic spines containing only InsP3 receptors.     

Mechanism of proteasomal degradation of inositol trisphosphate receptors in CHO-K1 cells

myo-Inositol 1,4,5-trisphosphate receptor (IP3R) degradation occurs in response to carbachol (Cch) stimulation of CHO-K1 cells. The response was mediated by endogenous muscarinic receptors and was blocked by atropine or proteasomal inhibitors. We have used these cells to identify the sites of ubiquitination on IP3Rs and study the role of Ca2+ and substrate recognition properties of the degradation system using exogenously expressed IP3R constructs. Employing caspase-3 for IP3R cleavage, we show that Cch promotes polyubiquitination in the N-terminal domain and monoubiquitination in the C-terminal domain. The addition of extracellular Ca2+ to Ca2+-depleted Chinese hamster ovary (CHO) cells initiates IP3R degradation provided Cch is present. This effect is inhibited by thapsigargin. The data suggest that both a sustained elevation of IP3 and a minimal content of Ca2+ in the endoplasmic reticulum lumen is required to initiate IP3R degradation. Transient transfection of IP3R constructs into CHO cells indicated the selective degradation of only the SI+ splice variant of the type I IP3R. This was also the splice form present endogenously in these cells. A pore-defective, nonfunctional SI+ IP3R mutant (D2550A) was also degraded in Cch-stimulated cells. The Cch-mediated response in CHO cells provides a convenient model system to further analyze the Ca2+ dependence and structural requirements of the IP3R proteasomal degradation pathway.     

Participation of inositol trisphosphate and ryanodine receptors in Bufo arenarum oocyte activation

Calcium is considered the most important second messenger at fertilization. Transient release from intracellular stores is modulated through both agonist-gated channels, IP?Rs and RyRs, which can be found individually or together depending on the oocyte species. Using the four commonly used compounds (thimerosal, caffeine, heparin and ruthenium red), we investigated the existence and interdependence of both IP?Rs and RyRs in mature Bufo arenarum oocytes. We found that caffeine, a well known specific RyRs agonist, was able to trigger oocyte activation in a dose-dependent manner. Microinjection of 10 mM caffeine showed 100% of oocytes exhibiting characteristic morphological criteria of egg activation. Ruthenium red, the specific RyR blocker, was able to inhibit oocyte activation induced either by sperm or caffeine. Our present findings provide the first reported evidence of the existence of RyR in frogs. We further explored the relationship between IP?Rs and RyRs in B. arenarum oocytes by exposing them to the agonists of one class after injecting a blocker of the other class of receptor. We found that thimerosal overcame the inhibitory effect of RyR on oocyte activation, indicating that IP?Rs function as independent receptors. In contrast, previous injection of heparin delayed caffeine-induced calcium release, revealing a relative dependence of RyRs on functional IP?Rs, probably through a CICR mechanism. Both receptors play a role in Ca2+ release mechanisms although their relative contribution to the activation process is unclear.     

Single-channel function of recombinant type 2 inositol 1,4, 5-trisphosphate receptor

A full-length rat type 2 inositol 1,4,5-trisphosphate (InsP(3)) receptor cDNA construct was generated and expressed in COS-1 cells. Targeting of the full-length recombinant type 2 receptor protein to the endoplasmic reticulum was confirmed by immunocytochemistry using isoform specific affinity-purified antibodies and InsP(3)R-green fluorescent protein chimeras. The receptor protein was solubilized and incorporated into proteoliposomes for functional characterization. Single-channel recordings from proteoliposomes fused into planar lipid bilayers revealed that the recombinant protein formed InsP(3)- and Ca(2+)-sensitive ion channels. The unitary conductance ( approximately 250 pS; 220/20 mM Cs(+) as charge carrier), gating, InsP(3), and Ca(2+) sensitivities were similar to those previously described for the native type 2 InsP(3)R channel. However, the maximum open probability of the recombinant channel was slightly lower than that of its native counterpart. These data show that our full-length rat type 2 InsP(3)R cDNA construct encodes a protein that forms an ion channel with functional attributes like those of the native type 2 InsP(3)R channel. The possibility of measuring the function of single recombinant type 2 InsP(3)R is a significant step toward the use of molecular tools to define the determinants of isoform-specific InsP(3)R function and regulation.     

Vasopressin receptors and inositol trisphosphate production in blood vessels of spontaneously hypertensive rats

To understand the regulation of vasopressin (AVP) receptors in spontaneous hypertension, we investigated the pressor response of AVP in the perfused mesenteric vasculature, AVP binding sites in the membrane preparation of the same vascular bed, and the production of inositol trisphosphate (InsP3) stimulated by AVP in the aorta of spontaneously hypertensive rats (SHR), Wistar-Kyoto rats (WKY), and Wistar rats (WR) at different ages (4-16 weeks). Plasma AVP concentrations were similar in SHR, WKY, and WR at all ages. The density of AVP vascular binding sites was significantly higher in WKY than in SHR and WR at 12 weeks. Receptor affinity was similar in all strains. The pressor response of the mesenteric vasculature to AVP was similar in the three strains of rats at 4 weeks (prehypertensive stage) and increased progressively in SHR compared with WKY and WR at 8 and 12 weeks of age by 43 and 35%, respectively, and by more than 80% at 16 weeks of age (established hypertensive stage). There was no difference in vascular sensitivity to AVP. A significantly increased pressor response to a supramaximal dose of norepinephrine was also found at 16 weeks in SHR, but not in younger rats. InsP3 production in the aorta in response to AVP was increased in SHR at 8, 12, and 16 weeks, compared with WKY and WR. These results suggest that the vascular response to AVP is increased in SHR, in spite of decreased or normal density of binding sites compared with WKY or WR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Markov chain Monte Carlo fitting of single-channel data from inositol trisphosphate receptors

In many cell types, the inositol trisphosphate receptor (IPR) is one of the important components that control intracellular calcium dynamics, and an understanding of this receptor (which is also a calcium channel) is necessary for an understanding of calcium oscillations and waves. Recent advances in experimental techniques now allow for the measurement of single-channel activity of the IPR in conditions similar to its native environment, and these data can be used to determine the rate constants in Markov models of the IPR. We illustrate a parameter estimation method based on Markov chain Monte Carlo, which can be used to fit directly to single-channel data, and determining, as an intrinsic part of the fit, the times at which the IPR is opening and closing. We show, using simulated data, the most complex Markov model that can be unambiguously determined from steady-state data and show that non-steady-state data is required to determine more complex models.     

Molecular and functional characterization of inositol trisphosphate receptors during early zebrafish development

Fluctuations in cytosolic Ca(2+) are crucial for a variety of cellular processes including many aspects of development. Mobilization of intracellular Ca(2+) stores via the production of inositol trisphosphate (IP(3)) and the consequent activation of IP(3)-sensitive Ca(2+) channels is a ubiquitous means by which diverse stimuli mediate their cellular effects. Although IP(3) receptors have been well studied at fertilization, information regarding their possible involvement during subsequent development is scant. In the present study we examined the role of IP(3) receptors in early development of the zebrafish. We report the first molecular analysis of zebrafish IP(3) receptors which indicates that, like mammals, the zebrafish genome contains three distinct IP(3) receptor genes. mRNA for all isoforms was detectable at differing levels by the 64 cell stage, and IP(3)-induced Ca(2+) transients could be readily generated (by flash photolysis) in a controlled fashion throughout the cleavage period in vivo. Furthermore, we show that early blastula formation was disrupted by pharmacological blockade of IP(3) receptors or phospholipase C, by molecular inhibition of the former by injection of IRBIT (IP(3) receptor-binding protein released with IP(3)) and by depletion of thapsigargin-sensitive Ca(2+) stores after completion of the second cell cycle. Inhibition of Ca(2+) entry or ryanodine receptors, however, had little effect. Our work defines the importance of IP(3) receptors during early development of a genetically and optically tractable model vertebrate organism.     

Fast biphasic regulation of type 3 inositol trisphosphate receptors by cytosolic calcium

In cytosol-like medium (CLM) with a free [Ca(2+)] of 200 nm, a supramaximal concentration of inositol 1,4,5-trisphosphate (IP(3)) (30 microm) evoked (45)Ca(2+) release from type 3 IP(3) receptors only after a latency of 48 +/- 6 ms; this latency could not be reduced by increasing the IP(3) concentration. In CLM containing a low free [Ca(2+)] ( approximately 4 nm), 300 microm IP(3) evoked (45)Ca(2+) release after a latency of 66 +/- 11 ms; this was reduced to 14 +/- 3 ms when the [Ca(2+)] was 1 mm. Preincubation with CLM containing 100 microm Ca(2+) caused a rapid (half-time = 33 +/- 9 ms), complete, and fully reversible inhibition that could not be overcome by a high concentration of IP(3) (300 microm). Hepatic (type 2) IP(3) receptors were not inhibited by Ca(2+) once they had bound IP(3), but 100 microm Ca(2+) rapidly inhibited type 3 IP(3) receptors whether it was delivered before addition of IP(3) or at any stage during a response to IP(3). Ca(2+) increases the affinity of IP(3) for hepatic receptors by slowing IP(3) dissociation, but Ca(2+) had no effect on IP(3) binding to type 3 receptors. The rate of inhibition of type 3 IP(3) receptors by Ca(2+) was faster than the rate of IP(3) dissociation, and occurred at similar rates whether receptors had bound a high (adenophostin) or low affinity (3-deoxy-3-fluoro-IP(3)) agonist. Dissociation of agonist is not therefore required for Ca(2+) to inhibit type 3 IP(3) receptors. We conclude that type 2 and 3 IP(3) receptors are each biphasically regulated by Ca(2+), but by different mechanisms. For both, IP(3) binding causes a stimulatory Ca(2+)-binding site to be exposed allowing Ca(2+) to bind and open the channel. IP(3) binding protects type 2 receptors from Ca(2+) inhibition, but type 3 receptors are inhibited by Ca(2+) whether or not they have IP(3) bound. Increases in cytosolic [Ca(2+)] will immediately inhibit type 3 receptors, but inhibit type 2 receptors only after IP(3) has dissociated.     

Differential intracellular distributions of inositol trisphosphate and ryanodine receptors within and among hematopoietic cells.

To better understand the mechanism(s) of leukocyte Ca(2+) signaling, we have studied the intracellular locations of two Ca(2+)-mobilizing receptors, the inositol 1,4,5-trisphosphate receptor and ryanodine receptor, by immunofluorescence microscopy. Our results show that localization differs not only between receptor classes within a cell, but among leukocyte types as well. We also illustrate the importance of preserving labile cellular filaments in maintaining cell integrity by fixation with the Safiejko-Mroczka and Bell protocol, because conventional fixation methods distort receptor patterns. We suggest that the observed differences influence intracellular Ca(2+) signaling.     

Calcium transients in 1B5 myotubes lacking ryanodine receptors are related to inositol trisphosphate receptors

Potassium depolarization of skeletal myotubes evokes slow calcium waves that are unrelated to contraction and involve the cell nucleus (Jaimovich, E., Reyes, R., Liberona, J. L., and Powell, J. A. (2000) Am. J. Physiol. 278, C998-C1010). Studies were done in both the 1B5 (Ry53-/-) murine "dyspedic" myoblast cell line, which does not express any ryanodine receptor isoforms (Moore, R. A., Nguyen, H., Galceran, J., Pessah, I. N., and Allen, P. D. (1998) J. Cell Biol. 140, 843-851), and C(2)C(12) cells, a myoblast cell line that expresses all three isoforms. Although 1B5 cells lack ryanodine binding, they bind tritiated inositol (1,4,5)-trisphosphate. Both type 1 and type 3 inositol trisphosphate receptors were immuno-located in the nuclei of both cell types and were visualized by Western blot analysis. After stimulation with 47 mm K(+), inositol trisphosphate mass raised transiently in both cell types. Both fast calcium increase and slow propagated calcium signals were seen in C(2)C(12) myotubes. However, 1B5 myotubes (as well as ryanodine-treated C(2)C(12) myotubes) displayed only a long-lasting, non-propagating calcium increase, particularly evident in the nuclei. Calcium signals in 1B5 myotubes were almost completely blocked by inhibitors of the inositol trisphosphate pathway: U73122, 2-aminoethoxydiphenyl borate, or xestospongin C. Results support the hypothesis that inositol trisphosphate mediates slow calcium signals in muscle cell ryanodine receptors, having a role in their time course and propagation.     

Models of the inositol trisphosphate receptor

The inositol (1,4,5)-trisphosphate receptor (IPR) plays a crucial role in calcium dynamics in a wide range of cell types, and is often a central feature in quantitative models of calcium oscillations and waves. We review deterministic and stochastic mathematical models of the IPR, from the earliest ones of the 1970s and 1980s, to the most recent. The effects of IPR stochasticity on Ca2+ dynamics are briefly discussed.     

Two types of inositol trisphosphate binding in cardiac microsomes.

Two distinct types of [3H]IP3 binding were found in canine cardiac microsomes with high (Kd = 21 nM, Bmax = 0.66 pmol/mg) and low affinity (Kd = 230 nM, Bmax = 2.9 pmol/mg). Also found were low affinity [3H]IP4 binding (Kd = 190 nM, Bmax = 4.5 pmol/mg) and high affinity [3H]IP6 binding (Kd = 10 nM, Bmax = 4.9 pmol/mg). The rank order of potency to displace these radioligands indicates that binding of IP3 and IP6 is ligand-specific. Sucrose gradient centrifugation of the detergent-solubilized cardiac microsomes indicates that the molecular size of the cardiac high affinity IP3 receptor is similar to that of the aortic smooth muscle IP3 receptor and smaller than that of the ryanodine receptor which migrates more rapidly. The IP4 and IP6 binding migrates more slowly than the IP3 receptor.