Ilse Sienaert

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). Strong45Ca2+ 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 InsP3concentrations, 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+.

Rat basophilic leukemia cells as model system for inositol 1,4,5-trisphosphate receptor IV, a receptor of the type II family: functional comparison and immunological detection.

This study concerns the detection and analysis of the highly homologous type II-like inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3R-II, -IV and -V). We have particularly investigated RBL-2H3 cells, which at the mRNA level predominantly expressed InsP3R-IV [De Smedt H. Missiaen L. Parys JB. et al. (1994) Determination of relative amounts of inositol trisphosphate receptor mRNA isoforms by ratio polymerase chain reaction. J. Biol. Chem., 269, 21691-21698]. When measured in identical experimental conditions, microsomes from RBL-2H3 cells were characterized by a much higher InsP3 binding affinity (Kd 3.8 +/- 0.8 nM, Bmax 0.40 +/- 0.08 pmol/mg protein) than microsomes from A7r5 cells (Kd 65 +/- 7 nM, Bmax 0.65 +/- 0.08 pmol/mg protein) or from cerebellum (Kd 135 +/- 14 nM, Bmax 7.35 +/- 1.13 pmol/mg protein). An affinity-purified antibody against the C-terminus of type II-like InsP3Rs detected, after SDS-PAGE and immunoblotting, a 250 kD protein in RBL-2H3 and C3H10T1/2 cells, but not in other cell types. An isoform-specific antibody against the C-terminus of InsP3R-I was used to determine the presence of the various InsP3R-I splice isoforms at the protein level. The 273 kD (brain), 261 kD (peripheral tissues) and 256 kD (Xenopus oocytes) isoforms were recognized. Expression of InsP3R-I in RBL-2H3 cells was very low. Taken together, our results support the hypothesis that InsP3R isoforms may differ to a large extent in their affinity for InsP3 and suggest that RBL-2H3 cells are a useful model for the study of InsP3R-IV.

Localization and function of a calmodulin-apocalmodulin-binding domain in the N-terminal part of the type 1 inositol 1,4,5-trisphosphate receptor.

Calmodulin (CaM) is a ubiquitous protein that plays a critical role in regulating cellular functions by altering the activity of a large number of proteins, including the d-myo-inositol 1,4,5-trisphosphate (IP3) receptor (IP3R). CaM inhibits IP3 binding in both the presence and absence of Ca2+ and IP3-induced Ca2+ release in the presence of Ca2+. We have now mapped and characterized a Ca2+-independent CaM-binding site in the N-terminal part of the type 1 IP3R (IP3R1). This site could be responsible for the inhibitory effects of CaM on IP3 binding. We therefore expressed the N-terminal 581 amino acids of IP3R1 as a His-tagged recombinant protein, containing the functional IP3-binding pocket. We showed that CaM, both in the presence and absence of Ca2+, inhibited IP3 binding to this recombinant protein with an IC50 of approx. 2 microM. Deletion of the N-terminal 225 amino acids completely abolished the effects of both Ca2+ and CaM on IP3 binding. We mapped the Ca2+-independent CaM-binding site to a recombinant glutathione S-transferase fusion protein containing the first 159 amino acids of IP3R1 and then made different synthetic peptides overlapping this region. We demonstrated that two synthetic peptides matching amino acids 49-81 and 106-128 bound CaM independently of Ca2+ and could reverse the inhibition of IP3 binding caused by CaM. This suggests that these sequences are components of a discontinuous Ca2+-independent CaM-binding domain, which is probably involved in the inhibition of IP3 binding by CaM.

The role of calmodulin for inositol 1,4,5-trisphosphate receptor function

Intracellular calcium release is a fundamental signaling mechanism in all eukaryotic cells. The ryanodine receptor (RyR) and inositol 1,4,5-trisphosphate receptor (IP(3)R) are intracellular calcium release channels. Both channels can be regulated by calcium and calmodulin (CaM). In this review we will first discuss the role of calcium as an activator and inactivator of the IP(3)R, concluding that calcium is the most important regulator of the IP(3)R. In the second part we will further focus on the role of CaM as modulator of the IP(3)R, using results of the voltage-dependent Ca(2+) channels and the RyR as reference material. Here we conclude that despite the fact that different CaM-binding sites have been characterized, their function for the IP(3)R remains elusive. In the third part we will discuss the possible functional role of CaM in IP(3)-induced Ca(2+) release (IICR) by direct and indirect mechanisms. Special attention will be given to the Ca(2+)-binding proteins (CaBPs) that were shown to activate the IP(3)R in the absence of IP(3).

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 IP3binding 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 K d increased from 115 ± 15 nm in the absence to 196 ± 18 nm in the presence of 5 μm Ca2+. The maximal effect of Ca2+ on Lbs-1 (5 μm 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 μm. Maximal inhibition (10 μmcalmodulin, 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 μmCa2+ (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.

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 Ca efflux technique in which 60 cumulative doses of inositol 1,4,5-trisphosphate (InsP), each lasting 6 s, were subsequently added to permeabilized A7r5 cells. This technique allowed an accurate determination of the threshold for InsP action, which was around 32 nM InsP under control conditions. The InsP-induced Ca 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 InsP concentrations. Stimulatory concentrations of thimerosal (10 μM) shifted the threshold to 4.5 nM InsP and increased both the cooperativity and the maximal normalized rate of Ca release. This low threshold was maintained when the thimerosal concentration was increased to inhibitory levels (100 μM) but then the effects on the cooperativity and on the normalized rate of Ca 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 InsP concentrations. Luminal Ca did not affect the threshold for InsP action but stimulated the normalized release at each InsP concentration. The inhibitory effect of 10 μM free cytosolic Ca was associated with a shift in threshold to higher InsP concentrations and a decreased cooperativity of the release process. We conclude that this novel technique of accurately measuring the threshold for InsP action under various experimental conditions has allowed us to refine the analysis of the kinetic parameters involved in the regulation of the InsP receptor.

Regulation of Inositol 1,4,5-Trisphosphate-Induced Ca2+ Release by Ca2+

Activation of cells by extracellular stimuli like hormones, growth factors or neurotransmitters leads to a controlled release of Ca2+ ions from intracellular Ca2+ stores into the cytosol. This release can take place locally or produce a global cellular response whereby the release takes the form of complex spatio-temporal signals, such as Ca2+ oscillations and Ca2+ waves (Berridge, 1997). The intracellular Ca2+ stores are part of the endoplasmic reticulum and their function is governed by three major types of proteins: (1) the ATP-driven Ca2+ pumps responsible for store filling, (2) intraluminal Ca2+-binding proteins (see Michalak et al., this book), and (3) one or both of the two major types of intracellular Ca2+-release channels, the inositol 1,4,5-trisphosphate receptors (IP3Rs) and the ryanodine receptors (RyRs) (see Rossi et al., this book; Macrez and Mironneau, this book).

Pharmacology of inositol trisphosphate receptors

Abstract. In almost all cells, cytosolic Ca2+ is a crucial intracellular messenger, regulating many cellular processes. In non-excitable as well as in some excitable cells, Ca2+ release from the intracellular stores into the cytoplasm is primarily initiated by the second messenger inositol 1,4,5-trisphosphate (IP3), which interacts with the IP3 receptor (IP3R), a tetrameric intracellular Ca2+-release channel. This review focuses on the pharmacological modulation of the various functionally important sub-domains of the IP3R, including the IP3-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 IP3R, thereby indicating their potential usefulness for unraveling the complex functional regulation of the IP3R.

Inhibition of the Inositol Trisphosphate Receptor of Mouse Eggs and A7r5 Cells by KN-93 via a Mechanism Unrelated to Ca2+/Calmodulin-dependent Protein Kinase II Antagonism

KN-93, a Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor, concentration-dependently and reversibly inhibited inositol 1,4,5-trisphosphate receptor (IP3R)-mediated [Ca2+] i signaling in mouse eggs and permeabilized A7r5 smooth muscle cells, two cell types predominantly expressing type-1 IP3R (IP3R-1). KN-92, an inactive analog, was ineffective. The inhibitory action of KN-93 on Ca2+ signaling depended neither on effects on IP3 metabolism nor on the filling grade of Ca2+stores, suggesting a direct action on the IP3R. Inhibition was independent of CaMKII, since in identical conditions other CaMKII inhibitors (KN-62, peptide 281–309, and autocamtide-related inhibitory peptide) were ineffective and since CaMKII activation was precluded in permeabilized cells. Moreover, KN-93 was most effective in the absence of Ca2+. Analysis of Ca2+ release in A7r5 cells at varying [IP3], of IP3R-1 degradation in eggs, and of [3H]IP3 binding in Sf9 microsomes all indicated that KN-93 did not affect IP3binding. Comparison of the inhibition of Ca2+ release and of [3H]IP3 binding by KN-93 and calmodulin (CaM), either separately or combined, was compatible with a specific interaction of KN-93 with a CaM-binding site on IP3R-1. This was also consistent with the much smaller effect of KN-93 in permeabilized 16HBE14o− cells that predominantly express type 3 IP3R, which lacks the high affinity CaM-binding site. These findings indicate that KN-93 inhibits IP3R-1 directly and may therefore be a useful tool in the study of IP3R functional regulation.