Pharmacology of inositol trisphosphate receptors.

In almost all cells, cytosolic Ca(2+) is a crucial intracellular messenger, regulating many cellular processes. In non-excitable as well as in some excitable cells, Ca(2+) release from the intracellular stores into the cytoplasm is primarily initiated by the second messenger inositol 1,4,5-trisphosphate (IP(3)), which interacts with the IP(3) receptor (IP(3)R), a tetrameric intracellular Ca(2+)-release channel. This review focuses on the pharmacological modulation of the various functionally important sub-domains of the IP(3)R, including the IP(3)-binding domain, calmodulin-binding sites, adenine nucleotide-binding sites and the sites for interaction for FK506-binding proteins and other regulators. We will particularly focus on the pharmacological tools that interfere with these domains and discuss their relative specificity for the IP(3)R, thereby indicating their potential usefulness for unraveling the complex functional regulation of the IP(3)R.

Mapping of the ATP-binding sites on inositol 1,4,5-trisphosphate receptor type 1 and type 3 homotetramers by controlled proteolysis and photoaffinity labeling.

Submillimolar ATP concentrations strongly enhance the inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release, by binding specifically to ATP-binding sites on the IP(3) receptor (IP(3)R). To locate those ATP-binding sites on IP(3)R1 and IP(3)R3, both proteins were expressed in Sf9 insect cells and covalently labeled with 8-azido-[alpha-(32)P]ATP. IP(3)R1 and IP(3)R3 were then purified and subjected to a controlled proteolysis, and the labeled proteolytic fragments were identified by site-specific antibodies. Two fragments of IP(3)R1 were labeled, each containing one of the previously proposed ATP-binding sites with amino acid sequence GXGXXG (amino acids 1773-1780 and 2016-2021, respectively). In IP(3)R3, only one fragment was labeled. This fragment contained the GXGXXG sequence (amino acids 1920-1925), which is conserved in the three IP(3)R isoforms. The presence of multiple interaction sites for ATP was also evident from the IP(3)-induced Ca(2+) release in permeabilized A7r5 cells, which depended on ATP over a very broad concentration range from micromolar to millimolar.

Basic properties of an inositol 1,4,5-trisphosphate-gated channel in carp olfactory cilia.

In addition to the activation of cAMP-dependent pathways, odorant binding to its receptor can lead to inositol 1,4,5-trisphosphate (InsP3) production that may induce the opening of plasma membrane channels. We therefore investigated the presence and nature of such channels in carp olfactory cilia. Functional analysis was performed by reconstitution of the olfactory cilia in planar lipid bilayers (tip-dip method). In the presence of InsP3 (10 microM) and Ca2+ (100 nM), a current of 1.6 +/- 0.1 pA (mean +/- SEM, n = 4) was measured, using Ba2+ as charge carrier. The I/V curve displayed a slope conductance of 45 +/- 5 pS and a reversal potential of -29 mV indicating a higher selectivity for divalent cations. This current was characterized by two mean open times (3.0 +/- 0.4 ms and 42.0 +/- 2.6 ms, n = 4) and was strongly inhibited by ruthenium red (30 microM) or heparin (10 microg/mL). Importantly, the channel activity was closely dependent on the Ca2+ concentration, with the highest open probability (Po) at 100 nM Ca2+ (Po = 0.50 +/- 0.02, n = 4). Po is lower at both higher and lower Ca2+ concentrations. A structural identification of the channel was attempted by using a large panel of antibodies, raised against several InsP3 receptor (InsP3R)/Ca2+ release channel isoforms. The type 1 InsP3R was detected in carp cerebellum and whole brain, while a lower molecular mass InsP3R, which may correspond to type 2 or 3, was detected in heart, whole brain and the soma of the olfactory neurons. None of the antibodies, however, cross-reacted with olfactory cilia. Taken together, these results indicate that in carp olfactory cilia an InsP3-dependent channel is present, distinct from the classical InsP3Rs localized on intracellular membranes.

Ca2+-calmodulin inhibits Ca2+ release mediated by type-1, -2 and -3 inositol trisphosphate receptors.

InsP(3) binding to type-1, but not type-3, InsP(3) receptors is inhibited by calmodulin in a Ca(2+)-independent fashion [Cardy and Taylor (1998) Biochem. J. 334, 447-455], and Ca(2+) mobilization by type-1 InsP(3) receptors of cerebellum is inhibited by calmodulin [Patel, Morris, Adkins, O'Beirne and Taylor (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 11627-11632]. Using cell types expressing predominantly type-1, -2 or -3 InsP(3) receptors, we show that InsP(3)-evoked Ca(2+) mobilization from each is similarly inhibited by calmodulin. In SH-SY5Y cells, which express largely type-1 receptors, calmodulin (IC(50) approximately 15 microM) inhibited InsP(3)-evoked Ca(2+) release only in the presence of Ca(2+). The inhibition was unaffected by calcineurin inhibitors. The effect of calmodulin did not result from enhanced metabolism of InsP(3) because calmodulin also decreased the sensitivity of the Ca(2+) stores to adenophostin A, a non-metabolizable InsP(3)-receptor agonist. Protein kinase A-catalysed phosphorylation of type-1 InsP(3) receptors was unaffected by Ca(2+)-calmodulin. Using a scintillation proximity assay to measure (125)I-calmodulin binding to glutathione S-transferase-fusion proteins, we identified two regions of the type-1 InsP(3) receptor (cyt1, residues -6 to 159; and cyt11, residues 1499-1649) that bound (125)I-calmodulin. The higher-affinity site (cyt11) was also photoaffinity labelled with N-hydroxysuccinimidyl-4-azidobenzoate (HSAB)-calmodulin. We speculate that Ca(2+)-independent binding of calmodulin to a site within the first 159 residues of the type-1 InsP(3) receptor inhibits InsP(3) binding and may thereby regulate the kinetics of Ca(2+) release. Ca(2+)-dependent inhibition of Ca(2+) release by calmodulin is mediated by a different site: it may reside on an accessory protein that associates with all three receptor subtypes, or Ca(2+)-calmodulin binding to a site lying between residues 1499 and 1649 of the type-1 receptor may inhibit Ca(2+) release from any tetrameric receptor that includes a type-1 subunit.

Adenine-nucleotide binding sites on the inositol 1,4,5-trisphosphate receptor bind caffeine, but not adenophostin A or cyclic ADP-ribose.

Binding of ATP to the inositol 1,4,5-trisphosphate receptor (IP3R) results in a more pronounced Ca2+ release in the presence of inositol 1,4,5-trisphosphate (IP3). We have expressed the cDNAs encoding two putative adenine-nucleotide binding sites of the neuronal form of IP3R-1 as glutathione S-transferase (GST)-fusion proteins in bacteria. Specific [alpha-32P]ATP binding was observed for the two GST-fusion proteins, representing aa 1710-1850 and aa 1944-2040 of IP3R-1. The ATP-binding sites in both fusion proteins had the same nucleotide specificity as found for the intact IP3R (ATP > ADP > AMP > GTP). Smaller GST-fusion proteins (aa 1745-1792 and aa 2005-2023) displayed a much weaker ATP-binding activity. CoA, which also potentiated IP3-induced Ca2+ release in A7r5 cells, interacted with the ATP-binding sites on the fusion proteins. Such interaction was not observed for 1,N6-etheno CoA and 3'-dephospho-CoA, which are much less effective in potentiating IP3-induced Ca2+ release. Since the adenine-containing compounds adenophostin A, caffeine and cyclic ADP-ribose modulate IP3-induced Ca2+ release, a possible effect of these compounds on the ATP-binding sites was examined. ATP stimulated adenophostin A- and IP3-induced Ca2+ release in A7r5 cells with an EC50 of respectively 21 and 20 microM. Also the threshold concentration of ATP for stimulating the release was similar for the two agonists. Adenophostin A (100 microM) and cyclic ADP-ribose (100 microM) were ineffective in displacing [alpha-32P]ATP from the binding sites of both GST-fusion proteins. Caffeine (50 mM), however, inhibited [alpha-32P]ATP binding to both fusion proteins by more than 50%. These data provide evidence for a direct interaction of caffeine but not of adenophostin A or cyclic ADP-ribose with the adenine-nucleotide binding sites of the IP3R.

Modulation of inositol 1,4,5-trisphosphate binding to the recombinant ligand-binding site of the type-1 inositol 1,4, 5-trisphosphate receptor by Ca2+ and calmodulin.

A recombinant protein (Lbs-1) containing the N-terminal 581 amino acids of the mouse type 1 inositol 1,4,5-trisphosphate receptor (IP3R-1), including the complete IP3-binding site, was expressed in the soluble fraction of E. coli. The characteristics of IP3 binding to this protein were similar as observed previously for the intact IP3R-1. Ca2+ dose-dependently inhibited IP3 binding to Lbs-1 with an IC50 of about 200 nM. This effect represented a decrease in the affinity of Lbs-1 for IP3, because the Kd increased from 115 +/- 15 nM in the absence to 196 +/- 18 nM in the presence of 5 microM Ca2+. The maximal effect of Ca2+ on Lbs-1 (5 microM Ca2+, 42.0 +/- 6.4% inhibition) was similar to the maximal inhibition observed for microsomes of insect Sf9 cells expressing full-length IP3R-1 (33.8 +/- 10.2%). Conceivably, the two contiguous Ca2+-binding sites (residues 304-450 of mouse IP3R-1) previously found by us (Sienaert, I., Missiaen, L., De Smedt, H., Parys, J.B., Sipma, H., and Casteels, R. (1997) J. Biol. Chem. 272, 25899-25906) mediate the effect of Ca2+ on IP3 binding to IP3R-1. Calmodulin also dose-dependently inhibited IP3 binding to Lbs-1 with an IC50 of about 3 microM. Maximal inhibition (10 microM calmodulin, 43.1 +/- 5.9%) was similar as observed for Sf9-IP3R-1 microsomes (35.8 +/- 8.7%). Inhibition by calmodulin occurred independently of Ca2+ and was additive to the inhibitory effect of 5 microM Ca2+ (together 74.5 +/- 5.1%). These results suggest that the N-terminal ligand-binding region of IP3R-1 contains a calmodulin-binding domain that binds calmodulin independently of Ca2+ and that mediates the inhibition of IP3 binding to IP3R-1.

ATP-induced Ca2+ signals in bronchial epithelial cells.

Ca2+-dependent Cl- secretion in the respiratory tract occurs physiologically or under pathophysiological conditions when inflammatory mediators are released. The mechanism of intracellular Ca2+ release was investigated in the immortalized bronchial epithelial cell line 16HBE14o-. Experiments on both intact and permeabilized cells revealed that only inositol 1,4,5-trisphosphate (InsP3) receptors and not ryanodine receptors are involved in intracellular Ca2+ release. The expression pattern of the three InsP3 receptor isoforms was assessed both at the mRNA and at the protein level. The level of expression at the mRNA level was type 3 (92.5%) >> type 2 (5.4%) > type 1 (2.1%) and this rank order was also observed at the protein level. The ATP-induced Ca2+ signals in the intact cell, consisting of abortive Ca2+ spikes or fully developed [Ca2+] rises and intracellular Ca2+ waves, were indicative of positive feedback of Ca2+ on the InsP3 receptors. Low Ca2+ concentrations stimulated and high Ca2+ concentrations inhibited InsP3-induced Ca2+ release in permeabilized 16HBE14o- cells. We localized a cytosolic Ca2+-binding site between amino acid residues 2077 and 2101 in the type-2 InsP3 receptor and between amino acids 2030 and 2050 in the type-3 InsP3 receptor by expressing the respective parts of these receptors as glutathione S-transferase fusion proteins in bacteria. We conclude that the InsP3 receptor isoforms expressed in 16HBE14o- cells (mainly type-3 and type-2) are stimulated by Ca2+ and that this phenomenon contributes to the ATP-induced Ca2+ signals in intact 16HBE14o- cells.

Functional properties of the type-3 InsP3 receptor in 16HBE14o- bronchial mucosal cells.

The type-3 inositol 1,4,5-trisphosphate (InsP3) receptor is the major isoform expressed in 16HBE14o- cells from bronchial mucosa, representing 93% at the mRNA level as determined by ratio reverse transcription-polymerase chain reaction and about 81% at the protein level as determined with isoform-specific antibodies (Sienaert, I., Huyghe, S., Parys, J. B., Malfait, M., Kunzelmann, K., De Smedt, H., Verleden, G. M., and Missiaen, L., Pflügers Arch. Eur. Y. Physiol., in press). The present 45Ca2+ efflux experiments indicate that these InsP3 receptors were 3 times less sensitive to InsP3 and 11 times less sensitive to ATP than those in A7r5 cells, where the type-1 InsP3 receptor is the main isoform. ATP did not increase the cooperativity of the InsP3-induced Ca2+ release in 16HBE14o- cells, in contrast to its effect in A7r5 cells. The sulfhydryl reagent thimerosal also did not stimulate InsP3-induced Ca2+ release in 16HBE14o- cells, again in contrast to its effect in A7r5 cells. Adenophostin A was more potent than InsP3 in stimulating the release in both cell types. The biphasic activation of the InsP3 receptor by cytosolic Ca2+ occurred in both cell types. We conclude that Ca2+ release mediated by the type-3 InsP3 receptor mainly differs from that mediated by the type-1 InsP3 receptor by its lack of stimulation by sulfhydryl oxidation and its lower ATP and InsP3 sensitivity. The predominant expression of the type-3 InsP3 receptor in the bronchial mucosa may be part of a mechanism coping with oxidative stress in that tissue.

Inhibition of inositol trisphosphate-induced calcium release by cyclic ADP-ribose in A7r5 smooth-muscle cells and in 16HBE14o- bronchial mucosal cells.

Ca2+ release from intracellular stores occurs via two families of intracellular channels, each with their own specific agonist: Ins(1, 4,5)P3 for the Ins(1,4,5)P3 receptor and cyclic ADP-ribose (cADPR) for the ryanodine receptor. We now report that cADPR inhibited Ins(1, 4,5)P3-induced Ca2+ release in permeabilized A7r5 cells with an IC50 of 20 microM, and in permeabilized 16HBE14o- bronchial mucosal cells with an IC50 of 35 microM. This inhibition was accompanied by an increase in specific [3H]Ins(1,4,5)P3 binding. 8-Amino-cADPR, but not 8-bromo-cADPR, antagonized this effect of cADPR. The inhibition was prevented by a whole series of inositol phosphates (10 microM) that did not affect Ins(1,4,5)P3-induced Ca2+ release, and by micromolar concentrations of PPi and various nucleotide di- or triphosphates. We propose that cADPR must interact with a novel regulatory site on the Ins(1,4,5)P3 receptor or on an associated protein. This site is neither the Ins(1,4,5)P3-binding domain, which prefers Ins(1,4,5)P3 and only binds nucleotides and PPi in the millimolar range, nor the stimulatory adenine nucleotide binding site.

Molecular and functional evidence for multiple Ca2+-binding domains in the type 1 inositol 1,4,5-trisphosphate receptor.

Structural and functional analyses were used to investigate the regulation of the inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) by Ca2+. To define the structural determinants for Ca2+ binding, cDNAs encoding GST fusion proteins that covered the complete linear cytosolic sequence of the InsP3R-1 were expressed in bacteria. The fusion proteins were screened for Ca2+ and ruthenium red binding through the use of 45Ca2+ and ruthenium red overlay procedures. Six new cytosolic Ca2+-binding regions were detected on the InsP3R in addition to the one described earlier (Sienaert, I., De Smedt, H., Parys, J. B., Missiaen, L., Vanlingen, S., Sipma, H., and Casteels, R. (1996) J. Biol. Chem. 271, 27005-27012). Strong 45Ca2+ and ruthenium red binding domains were localized in the N-terminal region of the InsP3R as follows: two Ca2+-binding domains were located within the InsP3-binding domain, and three Ca2+ binding stretches were localized in a 500-amino acid region just downstream of the InsP3-binding domain. A sixth Ca2+-binding stretch was detected in the proximity of the calmodulin-binding domain. Evidence for the involvement of multiple Ca2+-binding sites in the regulation of the InsP3R was obtained from functional studies on permeabilized A7r5 cells, in which we characterized the effects of Ca2+ and Sr2+ on the EC50 and cooperativity of the InsP3-induced Ca2+ release. The activation by cytosolic Ca2+ was due to a shift in EC50 toward lower InsP3 concentrations, and this effect was mimicked by Sr2+. The inhibition by cytosolic Ca2+ was caused by a decrease in cooperativity and by a shift in EC50 toward higher InsP3 concentrations. The effect on the cooperativity occurred at lower Ca2+ concentrations than the inhibitory effect on the EC50. In addition, Sr2+ mimicked the effect of Ca2+ on the cooperativity but not the inhibitory effect on the EC50. The different [Ca2+] and [Sr2+] dependencies suggest that three different cytosolic interaction sites were involved. Luminal Ca2+ stimulated the release without affecting the Hill coefficient or the EC50, excluding the involvement of one of the cytosolic Ca2+-binding sites. We conclude that multiple Ca2+-binding sites are localized on the InsP3R-1 and that at least four different Ca2+-interaction sites may be involved in the complex feedback regulation of the release by Ca2+.

Synergism between hypotonically induced calcium release and fatty acyl-CoA esters induced calcium release from intracellular stores.

The non-mitochondrial Ca2+ stores in permeabilized A7r5 cells responded to a decrease in Mg-ATP concentration with a pronounced Ca2+ release if 20 microM CoA was present. This release was rather specific for the preincubation or removal of ATP. ATP gamma S was much less effective and AMP-PNP, GTP, ITP, CTP, UTP, ADP, AMP, adenosine and adenine had no effect. CoA activated with an EC50 of 6 microM. Dephospho-CoA was a less effective cofactor and desulfo-CoA was ineffective. The release induced by Mg-ATP removal did not occur in the presence of 2% fatty acid-free bovine serum albumin and did not develop at 4 degrees C. All these findings suggest that CoA had to be acylated by endogenous fatty-acyl-CoA synthetase to become effective. Myristoyl- and palmitoyl-CoA esters were identified as the most effective cofactors for the release. Ca2+ release induced by removing Mg-ATP did not occur if the osmolality of the medium was kept constant by addition of mannitol, sucrose, KCl, MgCl2 or Mg-GTP, indicating that the decrease in tonicity was the trigger for the release. Mg-ATP plus CoA also synergized with Ca2+ release induced by a hypotonic shock imposed by diluting the medium with H2O. Osmolality changes induced by decreasing the Mg-ATP concentration were more effective in releasing Ca2+ than equal decreases in concentration of all solutes. We conclude that fatty acyl-CoA esters sensitize the hypotonically induced Ca2+ release from the non-mitochondrial Ca2+ stores.

Effect of adenine nucleotides on myo-inositol-1,4,5-trisphosphate-induced calcium release.

The effects of a whole series of adenine nucleotides on Ins(1,4,5)P3-induced Ca2+ release were characterized in permeabilized A7r5 smooth-muscle cells. Several adenine nucleotides activated the Ins(1, 4,5)P3 receptor. It was observed that 3'-phosphoadenosine 5'-phosphoulphate, CoA, di(adenosine-5')tetraphosphate (Ap4A) and di(adenosine-5')pentaphosphate (Ap5A) were more effective than ATP. Ap4A and Ap5A also interacted with a lower EC50 than ATP. In order to find out how these adenine nucleotides affected Ins(1,4, 5)P3-induced Ca2+ release, we have measured their effect on the response of permeabilized A7r5 cells to a progressively increasing Ins(1,4,5)P3 concentration. Stimulatory ATP and Ap5A concentrations had no effect on the threshold Ins(1,4,5)P3 concentration for initiating Ca2+ release, but they stimulated Ca2+ release in the presence of supra-threshold Ins(1,4,5)P3 concentrations by increasing the co-operativity of the release process. Inhibition of the Ins(1,4,5)P3-induced Ca2+ release at higher ATP concentrations was associated with a further increase in co-operativity and also with a shift in threshold towards higher Ins(1,4,5)P3 concentrations. ATP had no effect on the non-specific Ca2+ leak in the absence of Ins(1,4,5)P3. We conclude that the adenine-nucleotide-binding site can be activated by many different adenine nucleotides. Binding of these compounds to the transducing domain of the Ins(1,4,5)P3 receptor increases the efficiency of transmitting Ins(1,4,5)P3 binding to channel opening. The inhibition by high ATP concentrations is exerted at a different site, related to Ins(1,4,5)P3 binding.

Slow kinetics of inositol 1,4,5-trisphosphate-induced Ca2+ release: is the release 'quantal' or 'non-quantal'?

Inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ release from intracellular stores is generally assumed to be a 'quantal' process because low InsP3 concentrations mobilize less Ca2+ than high concentrations and a submaximal concentration does not release all the InsP3-mobilizable Ca2+. However, some recent reports questioned the generally accepted view that a low dose of InsP3 is unable to empty the whole store. We have now challenged the stores of permeabilized A7r5 cells in InsP3 for much longer periods than previously reported, to assess directly whether the slow phase of the release would empty the whole store (a non-quantal response) or only a fraction of it (a quantal response). Addition of a maximal [InsP3] at the end of a prolonged (92 min) stimulation with a submaximal [InsP3] resulted in additional Ca2+ release. Experiments in which the stores were challenged with different submaximal InsP3 concentrations for long time periods revealed that a lower [InsP3] never released the same amount of Ca2+ as a higher [InsP3]. This quantal pattern of Ca2+ release occurred both at 25 degrees C and at 4 degrees C. There was a time-dependent increase in the fraction of Ca2+ recruited by the lower compared with the higher [InsP3]. This recruitment of Ca2+ persisted if the [InsP3] was decreased, but was largely prevented by palmitoyl-CoA, a potent blocker of the luminal Ca2+ translocation between individual store units. ATP, in the presence of InsP3, released Ca2+ under conditions permitting the recruitment of no additional InsP3 receptors, indicating that an all-or-none emptying of a fraction of the stores cannot be the only mechanism responsible for quantal Ca2+ release in A7r5 cells. We conclude that some of the previously published evidence for a non-quantal Ca2+ release pattern should be reinterpreted.

Isoform diversity of the inositol trisphosphate receptor in cell types of mouse origin.

Previous reports suggested the expression of four or five different Ins(1,4,5)P3 receptor [Ins(1,4,5)P3R] isoforms in mouse cells [Ross, Danoff, Schell, Snyder and Ullrich (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 4265-4269; De Smedt, Missiaen, Parys, Bootman, Mertens, Van Den Bosch and Casteels (1994) J. Biol. Chem. 269, 21691-21698]. To explore this diversity further, we have isolated and sequenced partial clones of two Ins(1,4,5)P3R mRNAs from the mouse embryonic C3H10T1/2 cell line. These clones showed between 94.2 and 94.9% sequence identity with the corresponding rat Ins(1,4,5)P3R-II and Ins(1,4,5)P3R-III isoforms. Based on these newly obtained sequences we have determined the relative expression of the different Ins(1,4,5)P3R mRNAs in cultured cells and in animal tissues of mouse origin by a ratio reverse transcriptase polymerase chain reaction (RT-PCR). Ins(1,4,5)P3R-I was very prominent in brain and cerebellum and Ins(1,4,5)P3R-II in epithelia such as kidney as well as in both cardiac and skeletal muscle. Ins(1,4,5)P3R-III was highly expressed in all cultured cell types and in tissues with high cell turnover, e.g. testis. The prominent expression of Ins(1,4,5)P3R-I and Ins(1,4,5)P3R-III in A7r5 and C3H10T1/2 cells respectively was confirmed by immunoblot analysis and was compatible with a lower threshold for Ins(1,4,5)P3-induced Ca2+ release in the former cell type. Screening of a large number of mouse cell lines and tissues revealed the presence of Ins(1,4,5)P3R-I as well as of the Ins(1,4,5)P3R-II and Ins(1,4,5)P3R-III isoforms which were identified in the present study, but in contrast with previous reports there was no evidence for more isoform diversity.

Characterization of a cytosolic and a luminal Ca2+ binding site in the type I inositol 1,4,5-trisphosphate receptor.

To study the Ca2+ regulation of the inositol 1,4,5-trisphosphate receptor (InsP3R) at the molecular level, we expressed various cytosolic and luminal regions of the mouse type I InsP3R as glutathione S-transferase fusion proteins. 45Ca2+ and ruthenium red overlay studies and Stains-all spectra and staining revealed both a cytosolic and a luminal Ca2+ binding site. The luminal Ca2+ binding site was mapped to the nonconserved acidic subregion of the luminal loop between amino acids 2463 and 2528. A K0.5 of 0.3 microM and a Hill coefficient of 1.1 were determined by 45Ca2+ overlay by quantification of 45Ca2+ binding on blots. The cytosolic Ca2+ binding site was localized in a region just preceding the transmembrane domain M1. The Ca2+ binding was mapped to a 23-amino acid stretch between amino acids 2124 and 2146. This cytosolic region showed a single high affinity site for Ca2+, with a K0.5 of 0. 8 microM and a Hill coefficient of 1.0. Neither of the identified Ca2+ binding regions contained an EF-hand motif. We conclude that the type I InsP3R has at least two quite distinct types of Ca2+ binding sites, which are localized in different structural regions of the protein.

Mechanisms responsible for quantal Ca2+ release from inositol trisphosphate-sensitive calcium stores.

Activation of cells by hormones, growth factors or neurotransmitters leads to an increased production of inositol trisphosphate (InsP3) and, after activation of the InsP3 receptor (InsP3R), to Ca2+ release from intracellular Ca2+ stores. The release of intracellular Ca2+ is characterised by a graded response when submaximal doses of agonists are used. The basic phenomenon, called "quantal Ca2+ release", is that even the maintained presence of a submaximal dose of agonist or of InsP3 for long time periods (up to 20 min) provokes only a partial release of Ca2+. This partial, or quantal, release phenomenon is due to the fact that the initially very rapid InsP3-induced Ca2+ release eventually develops into a much slower release phase. Physiologically, quantal release allows the Ca2+ stores to function as increment detectors and to induce local Ca2+ responses. The basic mechanism for quantal release of Ca2+ is presently not known. Possible mechanisms to explain the quantal behaviour of InsP3- induced Ca2+ release include the presence of InsP3Rs with varying sensitivities for InsP3, heterogeneous InsP3R distribution, intrinsic inactivation of the InsP3Rs, and regulation of the InsP3Rs by Ca2+ store content. This article reviews critically the evidence for the various mechanisms and evaluates their functional importance. A Ca2+-mediated conformational change of the InsP3R is most likely the key feature of the mechanism for quantal Ca2+ release, but the exact mode of operation remains unclear. It should also be pointed out that in intact cells more than one mechanism can be involved.

Threshold for inositol 1,4,5-trisphosphate action.

We developed a unidirectional 45Ca2+ efflux technique in which 60 cumulative doses of inositol 1,4,5-trisphosphate (InsP3), each lasting 6 s, were subsequently added to permeabilized A7r5 cells. This technique allowed an accurate determination of the threshold for InsP3 action, which was around 32 nM InsP3 under control conditions. The InsP3-induced Ca2+ release was characterized by an initial rapid phase, after which the normalized rate progressively decreased. The slowing of the release was associated with a shift of the threshold to higher InsP3 concentrations. Stimulatory concentrations of thimerosal (10 microM) shifted the threshold to 4.5 nM InsP3 and increased both the cooperativity and the maximal normalized rate of Ca2+ release. This low threshold was maintained when the thimerosal concentration was increased to inhibitory levels (100 microM) but then the effects on the cooperativity and on the normalized rate of Ca2+ release disappeared. Oxidized glutathione (5 mM) was much less effective in stimulating the release and did not have an effect on the threshold or on the cooperativity. ATP (5 mM) stimulated the release despite a shift in threshold toward higher InsP3 concentrations. Luminal Ca2+ did not affect the threshold for InsP3 action but stimulated the normalized release at each InsP3 concentration. The inhibitory effect of 10 microM free cytosolic Ca2+ was associated with a shift in threshold to higher InsP3 concentrations and a decreased cooperativity of the release process. We conclude that this novel technique of accurately measuring the threshold for InsP3 action under various experimental conditions has allowed us to refine the analysis of the kinetic parameters involved in the regulation of the InsP3 receptor.

Hypotonically induced calcium release from intracellular calcium stores.

Osmotic cell swelling induced by hypotonic stress is associated with a rise in intracellular Ca2+ concentration, which is at least partly due to a release of Ca2+ from internal stores. Since osmotic influx of water dilutes the cytoplasmic milieu, we have investigated how nonmitochondrial Ca2+ stores in permeabilized A7r5 cells respond to a reduction in cytoplasmic tonicity. We now present experimental evidence for a direct Ca2+ release from the stores when exposed to a hypotonic medium. The release is graded, but does not occur through the inositol trisphosphate or the ryanodine receptor. Ca2+ seems to be released through the passive leak pathway, and this phenomenon can be partially inhibited by divalent cations in the following order of potency: Ni2+ = Co2+ > Mn2+ > Mg2+ > Ba2+. This release also occurs in intact A7r5 cells. This novel mechanism of hypotonically induced Ca2+ release is therefore an inherent property of the stores, which can occur in the absence of second messengers. Intracellular stores can therefore act as osmosensors.