Structural and functional characterization of an inositol polyphosphate receptor from cerebellum.

An inositol polyphosphate receptor has been purified from bovine cerebellum which consists of three different polypeptides with Mr of 111,000, 102,000, and 52,000. Negative staining electron microscopy reveals globular-like structures 10-13 nm in diameter. The receptor has a Stokes radius of 400,000 daltons as determined by molecular sieve high performance liquid chromatography. The receptor preparation binds inositol 1,3,4,5-tetrakisphosphate, inositol hexaphosphate (or phytol), and inositol 1,4,5-trisphosphate (IP4, IP6, and IP3, respectively) with submicromolar affinity (0.19, 0.15, and 0.54 microM, respectively) at conditions approximating physiological ionic strength and pH. The purified receptor preparation, when reconstituted into planar bilayers, displays ion channel activity, preferentially permeable to K+. Permeability ratios of the channel are PK+/PNa+ approximately 5 and PK+/PCl approximately 19. In symmetrical 100 mM KCl, the channel is characterized by long open times (minutes) with a conductance of 7.2 picosiemens. The channel is selectively modulated by IP4. That is, at 1 microM IP4, the mean open time decreased substantially to rapid flicker behavior and the channel is completely closed at 10 microM IP4. IP6 and IP3 did not modulate the channel under similar conditions. Thus, the channel appears to be an IP4-modulated K+ channel.     

Postnatal expression of the inositol 1,4,5-trisphosphate receptor in canine cerebellum.

1. Inositol 1,4,5-trisphosphate (IP3), an intracellular second messenger, has been shown to be the link between activation of several plasma membrane receptors and Ca2+ release from intracellular, membrane-bound compartments. In this study, the postnatal expression of the canine cerebellum IP3 receptor was investigated by biochemical, ligand binding and immunocytochemical methods. 2. Specific receptor sites for IP3 and the extent of IP3-induced Ca2+ release were quantitated in microsomal fractions isolated from cerebella of developing (0-28 day-old) and adult dogs. The IP3 receptor was detected in newborn animals and adult levels were attained within 3-4 weeks. 3. The time-course of IP3 receptor ontogeny paralleled both growth of Purkinje neurons, as indicated by immunofluorescence of cerebellum cortex cryosections with anti-IP3 receptor antibodies, and synaptogenesis, as judged by Western blotting of the microsomal fractions with anti-synaptophysin antibodies.     

Functional characterization of mammalian inositol 1,4,5-trisphosphate receptor isoforms

Inositol 1,4,5-trisphosphate receptors (InsP3R) play a key role in intracellular calcium (Ca2+) signaling. Three mammalian InsP3R isoforms--InsP3R type 1 (InsP3R1), InsP3R type 2 (InsP3R2), and InsP3R type 3 (InsP3R3) are expressed in mammals, but the functional differences between the three mammalian InsP3R isoforms are poorly understood. Here we compared single-channel behavior of the recombinant rat InsP3R1, InsP3R2, and InsP3R3 expressed in Sf9 cells, reconstituted into planar lipid bilayers and recorded with 50 mM Ba2+ as a current carrier. We found that: 1), for all three mammalian InsP3R isoforms the size of the unitary current is 1.9 pA and single-channel conductance is 74-80 pS; 2), in optimal recording conditions the maximal single-channel open probability for all three mammalian InsP3R isoforms is in the range 30-40%; 3), in optimal recording conditions the mean open dwell time for all three mammalian InsP3R isoforms is 7-8 ms, the mean closed dwell time is approximately 10 ms; 4), InsP3R2 has the highest apparent affinity for InsP(3) (0.10 microM), followed by InsP3R1 (0.27 microM), and then by InsP3R3 (0.40 microM); 5), InsP3R1 has a high-affinity (0.13 mM) ATP modulatory site, InsP3R2 gating is ATP independent, and InsP3R3 has a low-affinity (2 mM) ATP modulatory site; 6), ATP modulates InsP3R1 gating in a noncooperative manner (n(Hill) = 1.3); 7), ATP modulates InsP3R3 gating in a highly cooperative manner (n(Hill) = 4.1). Obtained results provide novel information about functional properties of mammalian InsP3R isoforms.     

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.     

Regulation of inositol 1,4,5-trisphosphate receptor function during mouse oocyte maturation

At the time of fertilization, an increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) underlies egg activation and initiation of development in all species studied to date. The inositol 1,4,5-trisphosphate receptor (IP(3)R1), which is mostly located in the endoplasmic reticulum (ER) mediates the majority of this Ca(2+) release. The sensitivity of IP(3)R1, that is, its Ca(2+) releasing capability, is increased during oocyte maturation so that the optimum [Ca(2+)](i) response concurs with fertilization, which in mammals occurs at metaphase of second meiosis. Multiple IP(3)R1 modifications affect its sensitivity, including phosphorylation, sub-cellular localization, and ER Ca(2+) concentration ([Ca(2+)](ER)). Here, we evaluated using mouse oocytes how each of these factors affected IP(3)R1 sensitivity. The capacity for IP(3)-induced Ca(2+) release markedly increased at the germinal vesicle breakdown stage, although oocytes only acquire the ability to initiate fertilization-like oscillations at later stages of maturation. The increase in IP(3)R1 sensitivity was underpinned by an increase in [Ca(2+)](ER) and receptor phosphorylation(s) but not by changes in IP(3)R1 cellular distribution, as inhibition of the former factors reduced Ca(2+) release, whereas inhibition of the latter had no impact. Therefore, the results suggest that the regulation of [Ca(2+)](ER) and IP(3)R1 phosphorylation during maturation enhance IP(3)R1 sensitivity rendering oocytes competent to initiate oscillations at the expected time of fertilization. The temporal discrepancy between the initiation of changes in IP(3)R1 sensitivity and acquisition of mature oscillatory capacity suggest that other mechanisms that regulate Ca(2+) homeostasis also shape the pattern of oscillations in mammalian eggs.     

Kinetics of calcium channel opening by inositol 1,4,5-trisphosphate.

The subsecond mobilization of intracellular Ca2+ by IP3 was measured with rapid mixing techniques to determine how cells achieve rapid rises in cytosolic [Ca2+] during receptor-triggered calcium spiking. In permeabilized rat basophilic leukemia cells at 11 degrees C, more than 80% of the 0.7 fmol of Ca2+/cell sequestered by the ATP-driven pump could be released by IP3. Half of the stored Ca2+ was released within 200 ms after addition of saturating (1 microM) IP3. The flux rate was half-maximal at 120 nM IP3. Ca2+ release from fully loaded stores was highly cooperative; the Hill coefficient over the 2-40 nM range was greater than 3. The delay time of channel opening was inversely proportional to [IP3], increasing from 150 ms at 100 nM IP3 to 1 s at 15 nM, indicating that the rate-limiting step in channel opening is IP3 binding. Multiple binding steps are required to account for the observed delay and nonexponential character of channel opening. A simple model is proposed in which the binding of four IP3 molecules to identical and independent sites leads to channel opening. The model agrees well with the data for KD = 18 nM, kon = 1.2 X 10(8) M-1 s-1, and koff = 2.2 s-1. The approximately 1-s exchange time of bound IP3 indicates that the channel gating sites are distinct from binding sites having approximately 100-s exchange times that were previously found with radiolabeled IP3. The approximately 1-1s response time of [Ca2+] to a rapid increase in IP3 level can account for observed rise times of calcium spikes.     

Purification and characterization of the inositol 1,4,5-trisphosphate receptor protein from rat vas deferens

Among rat peripheral tissues examined, Ins(1,4,5)P(3) receptor binding is highest in the vas deferens, with levels about 25% of those of the cerebellum. We have purified the InsP(3) receptor binding protein from rat vas deferens membranes 600-fold. The purified protein displays a single 260 kDa band on SDS/PAGE, and the native protein has an apparent molecular mass of 1000 kDa, the same as in cerebellum. The inositol phosphate specificity, pH-dependence and influence of various reagents are the same for purified vas deferens and cerebellar receptors. Whereas particulate InsP(3) binding in cerebellum is potently inhibited by Ca(2+), particulate and purified vas deferens receptor binding of InsP(3) is not influenced by Ca(2+). Vas deferens appears to lack calmedin activity, but the InsP(3) receptor is sensitive to Ca(2+) inhibition conferred by brain calmedin. The vas deferens may prove to be a valuable tissue for characterizing functional aspects of InsP(3) receptors.     

A functional interaction between chromogranin B and the inositol 1,4,5-trisphosphate receptor/Ca2+ channel

Chromogranins A and B (CGA and CGB) are high capacity, low affinity calcium (Ca2+) storage proteins found in many cell types most often associated with secretory granules of secretory cells but also with the endoplasmic reticulum (ER) lumen of these cells. Both CGA and CGB associate with inositol 1,4,5-trisphosphate receptor (InsP3R) in a pH-dependent manner. At an intraluminal pH of 5.5, as found in secretory vesicles, both CGA and CGB bind to the InsP3R. When the intraluminal pH is 7.5, as found in the ER, CGA totally dissociates from InsP3R, whereas CGB only partially dissociates. To investigate the functional consequences of the interaction between the InsP3R and CGB monomers or CGA/CGB heteromers, purified mouse InsP3R type I were fused to planar lipid bilayers and activated by 2 microM InsP3. In the presence of luminal CGB monomers or CGA/CGB heteromers the InsP3R/Ca2+ channel open probability and mean open time increased significantly. The channel activity remained elevated when the pH was changed to 7.5, a reflection of CGB binding to the InsP3R even at pH 7.5. These results suggest that CGB may play an important modulatory role in the control of Ca2+ release from the ER. Furthermore, the difference in the ability of CGA and CGB to regulate the InsP3R/Ca2+ channel and the variability of CGA/CGB ratios could influence the pattern of InsP3-mediated Ca2+ release.     

Molecular cloning and characterization of the inositol 1,4,5-trisphosphate receptor in Drosophila melanogaster.

We isolated a cDNA encoding an inositol 1,4,5-trisphosphate receptor (InsP3R) of Drosophila melanogaster. The predicted Drosophila InsP3R (2,833 amino acids) has extensive sequence similarity to the mouse InsP3R. The polypeptide encoded by the cDNA was functionally expressed and showed characteristic InsP3-binding activity. The Drosophila InsP3R gene is located at the region 83A5-9 on the third chromosome and expresses throughout development but predominantly in the adult. Localization of the InsP3R mRNA in adult tissues suggests strong expression in the retina and antenna, indicating the involvement of the InsP3R in visual and olfactory transduction. In addition, the InsP3R mRNA is abundant in the legs and thorax, which are enriched with a muscular system. Such localization is apparently consistent with the quantitatively predominant sites for [3H]InsP3 binding in Drosophila and the fleshfly (Boettcherisca peregrina). The present study points to the likely functional importance of the InsP3/Ca2+ signaling system in Drosophila.     

Molecular target sizes of inositol 1,4,5-trisphosphate receptors in liver and cerebellum.

A peptide corresponding to a surface loop in the C-terminal domain of chicken ovotransferrin (residues 570-584) was made by solid-phase synthesis and used to immunize rabbits. A 15-amino acid-residue disulphide-linked loop occurs in both domains of all five transferrins for which the sequence is available and lies on the opposite side of the iron-binding site from the interdomain cleft. Polyclonal antibodies to the peptide were specific for non-reduced holo-ovotransferrin and the C-terminal domain, as shown by e.l.i.s.a. and immunoblotting. The antibody did not inhibit binding of ovotransferrin to receptors on chick-embryo reticulocytes but was able to bind ovotransferrin bound to the cellular receptors at 0 degree C. The loop composed of residues 570-584 appears to be remote from the transferrin receptor-binding site.     

Novel regulation of calcium inhibition of the inositol 1,4,5-trisphosphate receptor calcium-release channel

The inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R), a Ca2+-release channel localized to the endoplasmic reticulum, plays a critical role in generating complex cytoplasmic Ca2+ signals in many cell types. Three InsP3R isoforms are expressed in different subcellular locations, at variable relative levels with heteromultimer formation in different cell types. A proposed reason for this diversity of InsP3R expression is that the isoforms are differentially inhibited by high cytoplasmic free Ca2+ concentrations ([Ca2+]i), possibly due to their different interactions with calmodulin. Here, we have investigated the possible roles of calmodulin and bath [Ca2+] in mediating high [Ca2+]i inhibition of InsP3R gating by studying single endogenous type 1 InsP3R channels through patch clamp electrophysiology of the outer membrane of isolated Xenopus oocyte nuclei. Neither high concentrations of a calmodulin antagonist nor overexpression of a dominant-negative Ca2+-insensitive mutant calmodulin affected inhibition of gating by high [Ca2+]i. However, a novel, calmodulin-independent regulation of [Ca2+]i inhibition of gating was revealed: whereas channels recorded from nuclei kept in the regular bathing solution with [Ca2+] approximately 400 nM were inhibited by 290 muM [Ca2+]i, exposure of the isolated nuclei to a bath solution with ultra-low [Ca2+] (<5 nM, for approximately 300 s) before the patch-clamp experiments reversibly relieved Ca2+ inhibition, with channel activities observed in [Ca2+]i up to 1.5 mM. Although InsP3 activates gating by relieving high [Ca2+]i inhibition, it was nevertheless still required to activate channels that lacked high [Ca2+]i inhibition. Our observations suggest that high [Ca2+]i inhibition of InsP3R channel gating is not regulated by calmodulin, whereas it can be disrupted by environmental conditions experienced by the channel, raising the possibility that presence or absence of high [Ca2+]i inhibition may not be an immutable property of different InsP3R isoforms. Furthermore, these observations support an allosteric model in which Ca2+ inhibition of the InsP3R is mediated by two Ca2+ binding sites, only one of which is sensitive to InsP3.     

Solubilization of rat liver inositol 1,4,5-trisphosphate receptor

High affinity Ins(1,4,5)P₃-binding sites of permeabilized hepatocytes are probably the ligand recognition sites of the receptors that mediate the effects of Ins91,4,5)P₃ on intracellular Ca²⁺ mobilization. We have now solubilized these sites from rat liver membranes in the zwitterionic detergent, CHAPS, and shown that the solubilized bind Ins(1,4,5)P₃ with an affinity (K_d = 7.26 ± 0.52 nM, Hill coefficient H = 1.05 ± 0.06) similar to that of the sites in native membranes (K_d = 6.02 ± 0.02). ATP and a range of inositol phosphates (Ins(2,4,5)P₃ Ins(4,5)P₂, and inositol 1,4,5-trisphosphorothioate) also bound with similar affinities to the native and solubilized sites. Solubilization of the liver InsP₃ receptor will allow its further characterization, purification, and comparison of its properties with those of InsP₃ receptors already purified from cerebellum and smooth muscle.     

Isoform-specific function of single inositol 1,4,5-trisphosphate receptor channels.

The inositol 1,4,5-trisphosphate receptor (InsP3R) family of Ca2+ release channels is central to intracellular Ca2+ signaling in mammalian cells. The InsP3R channels release Ca2+ from intracellular compartments to generate localized Ca2+ transients that govern a myriad of cellular signaling phenomena (Berridge, 1993. Nature. 361:315-325; Joseph, 1996. Cell Signal. 8:1-7; Kume et al., 1997. Science. 278:1940-1943; Berridge, 1997. Nature. 368:759-760). express multiple InsP3R isoforms, but only the function of the single type 1 InsP3R channel is known. Here the single-channel function of single type 2 InsP3R channel is defined for the first time. The type 2 InsP3R forms channels with permeation properties similar to that of the type 1 receptor. The InsP3 regulation and Ca2+ regulation of type 1 and type 2 InsP3R channels are strikingly different. Both InsP3 and Ca2+ are more effective at activating single type 2 InsP3R, indicating that single type 2 channels mobilize substantially more Ca2+ than single type 1 channels in cells. Furthermore, high cytoplasmic Ca2+ concentrations inactivate type 1, but not type 2, InsP3R channels. This indicates that type 2 InsP3R channel is different from the type 1 channel in that its activity will not be inherently self-limiting, because Ca2+ passing through an active type 2 channel cannot feed back and turn the channel off. Thus the InsP3R identity will help define the spatial and temporal nature of local Ca2+ signaling events and may contribute to the segregation of parallel InsP3 signaling cascades in mammalian cells.     

Type 3 inositol 1,4,5-trisphosphate receptor modulates cell death.

Mechanisms accounting for the cellular entry of calcium that mediates cellular proliferation and apoptosis have been obscure. Previously we reported selective augmentation of type 3 inositol (1,4,5) trisphosphate receptors (IP(3)R3) in lymphocytes undergoing programmed cell death, which was prevented by antisense constructs to IP(3)R3. We now report increases in mRNA and protein levels for IP(3)R3 associated with cell death in several apoptotic paradigms in diverse tissues. Elevations of IP(3)R3 occur during developmental apoptosis in early postnatal cerebellar granule cells, dorsal root ganglia, embryonic hair follicles, and intestinal villi. Neurotoxic damage elicited by the glutamate agonist kainate is also associated with IP(3)R3 augmentation. In chick dorsal root ganglia neurons undergoing apoptosis due to deprivation of nerve growth factor, levels of IP(3)R3 are selectively increased and cell death is selectively prevented by antisense oligonucleotides to IP(3)R3. Thus, IP(3)R3 appears to participate actively in cell death in a diversity of tissues.     

Type 3 inositol 1,4,5-trisphosphate receptor: A calcium channel for all seasons

Inositol 1,4,5 trisphosphate receptors (ITPRs) are a family of endoplasmic reticulum Ca²⁺ channels essential for the control of intracellular Ca²⁺ levels in virtually every mammalian cell type. The three isoforms (ITPR1, ITPR2 and ITPR3) are highly homologous in amino acid sequence, but they differ considerably in terms of biophysical properties, subcellular localization, and tissue distribution. Such differences underscore the variety of cellular responses triggered by each isoform and suggest that the expression/activity of specific isoforms might be linked to particular pathophysiological states. Indeed, recent findings demonstrate that changes in expression of ITPR isoforms are associated with a number of human diseases ranging from fatty liver disease to cancer. ITPR3 is emerging as the isoform that is particularly important in the pathogenesis of various human diseases. Here we review the physiological and pathophysiological roles of ITPR3 in various tissues and the mechanisms by which the expression of this isoform is modulated in health and disease.     

Studying isoform-specific inositol 1,4,5-trisphosphate receptor function and regulation

Inositol 1,4,5-trisphosphate receptors (InsP3R) are a family of ubiquitously expressed intracellular Ca2+ channels. Isoform-specific properties of the three family members may play a prominent role in defining the rich diversity of the spatial and temporal characteristics of intracellular Ca2+ signals. Studying the properties of the particular family members is complicated because individual receptor isoforms are typically never expressed in isolation. In this article, we discuss strategies for studying Ca2+ release through individual InsP3R family members with particular reference to methods applicable following expression of recombinant InsP3R and mutant constructs in the DT40-3KO cell line, an unambiguously null InsP3R expression system.     

Autophosphorylation of inositol 1,4,5-trisphosphate receptors.

Inositol 1,4,5-trisphosphate (IP3) releases internal stores of calcium by binding to a specific membrane receptor which includes both the IP3 recognition site as well as the associated calcium channel. The IP3 receptor is regulated by ATP, calcium, and phosphorylation by protein kinase A, protein kinase C, and calcium/calmodulin-dependent protein kinase II. Its cDNA sequence predicts at least two consensus sequences where nucleotides might bind, and direct binding of ATP to the IP3 receptor has been demonstrated. In the present study, we demonstrate autophosphorylation of the purified and reconstituted IP3 receptor on serine and find serine protein kinase activity of the IP3 receptor toward a specific peptide substrate. Several independent purification procedures do not separate the IP3 receptor protein from the phosphorylating activity, and many different protein kinase activators and inhibitors do not identify protein kinases as contaminants. Also, renaturation experiments reveal autophosphorylation of the monomeric receptor on polyvinylidene difluoride membranes.     

The inositol 1,4,5-trisphosphate (InsP3) receptor

Conclusion In this review, we have described the functional properties and regulation of the InsP₃R. Not all aspects of InsP₃R function and regulation were covered, the main focus was on the most recent and physiologically important data. Information about the structure, heterogeneity, functional properties, and regulation of the InsP₃R is useful for understanding the spatiotemporal aspects of Ca signaling. The combination of biochemical, biophysical and molecular biological techniques has revealed the intricacies of the InsP₃R over the past decade. However, questions about the functional differences between various isoforms and splice variants of the InsP₃R, the structural determinants responsible for regulation of InsP₃R by Ca and ATP, the functional effects of InsP₃R phosphorylation and many others remain to be elucidated. Future investigations can be expected to provide answers to these important questions.We thank S. Bezprozvannaya for expert technical assistance. This work was supported by National Institutes of Health grants HL 33026 and GM 39029, and a Grant-in-Aid from the Patrick and Catherine Weldon Donaghue Medical Research Foundation.     

Three-dimensional rearrangements within inositol 1,4,5-trisphosphate receptor by calcium

Allosteric binding of calcium ion (Ca2+) to inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) controls channel gating within IP3R. Here, we present biochemical and electron microscopic evidence of Ca2+-sensitive structural changes in the three-dimensional structure of type 1 IP3R (IP3R1). Low concentrations of Ca2+ and high concentrations of Sr2+ and Ba2+ were shown to be effective for the limited proteolysis of IP3R1, but Mg2+ had no effect on the proteolysis. The electron microscopy and the limited proteolysis consistently demonstrated that the effective concentration of Ca2+ for conformational changes in IP3R1 was <10(-7) m and that the IP3 scarcely affected the conformational states. The structure of IP3R1 without Ca2+, as reconstructed by three-dimensional electron microscopy, had a "mushroom-like" appearance consisting of a large square-shaped head and a small channel domain linked by four thin bridges. The projection image of the "head-to-head" assembly comprising two particles confirmed the mushroom-like side view. The "windmill-like" form of IP3R1 with Ca2+ also contains the four bridges connecting from the IP3-binding domain toward the channel domain. These data suggest that the Ca2+-specific conformational change structurally regulates the IP3-triggered channel opening within IP3R1.