Developmental expression of the calcium release channels during early neurogenesis of the mouse cerebral cortex.

The developmental changes of intracellular calcium release channels of mouse neocortex were studied at the onset of neurogenesis, which occurs between embryonic days E11 and E17. The three main isoforms of the two families of intracellular calcium release channels, namely the inositol trisphosphate receptors (IP3R) and the ryanodine receptors (RyR), were detected by their transcripts in the cerebral hemispheres, as early as stage E11. The major isoforms of each family, IP3R-1 and RyR-2, were found at the protein level by Western blot analysis. Expression of these proteins increases progressively throughout brain development. Their localization in coronal sections of cortex has been observed by immunodetection from E12, and compared to the TuJ1 (anti-class III beta-tubulin antibody) neuronal specific labelling. The expression of both channels is greatly enhanced after E12, and both were seen to be present in most of the proliferative and neuronal cells of the slice. Between E12 and E13, there is a striking transition in the pattern of calcium release elicited by specific agonists of these channels, thimerosal for IP3R and caffeine for RyR. The signals induced by thimerosal were not zone-specific, while the observed calcium release signals induced by caffeine were predominantly restricted out of the ventricular zone. This zone-specific caffeine sensitivity is consistent with the main RyR localization immunodetected at E13. Our results indicate that there is a time lag of several days between the molecular detection of calcium release channels and their functional expression, around the time of neuronal differentiation. Altogether, they provide a molecular basis for analyzing the developmental modulation of calcium signals useful for neurogenesis progression.

Expression of intracellular calcium channels and pumps after partial hepatectomy in rat.

Ca(2+) signals regulate many cellular functions, including proliferation. They are governed by the inositol 1,4,5-trisphosphate receptor (IP(3)R), the only intracellular hepatic Ca(2+) channel and by the endoplasmic reticulum Ca(2+) pumps, SERCA. To characterise their role in regeneration, expression of their isoforms was studied after 2/3 hepatectomy by real-time quantitative PCR, Western blot and binding studies. We found an early increase in the expression of the IP(3)R isoform 1 which contrasted with the decrease of the expression of the IP(3)R isoforms 2 and 3 and of SERCA3. This results in a transient switch between IP(3)R isoforms 1 and 2, IP(3)R isoform 1 becoming predominant before the first round of mitosis. Binding studies detected a 30% diminution of the IP(3)R population at 24 h. In conclusion, the Ca(2+) signalling machinery is regulated, after hepatectomy, by changes in expression of the IP(3)R and SERCA isoforms to adapt Ca(2+) signals to the regenerative state.

Differential expression of inositol 1,4,5-trisphosphate receptor types 1, 2, and 3 in rat myometrium and endometrium during gestation.

The regulation of the phospholipase C (PLC) and the expression of inositol 1,4,5-trisphosphate receptors (IP(3)Rs) in terms of mRNA, proteins, and binding capacity were examined in the rat myometrium and endometrium at midgestation (Day 12) and at term (Day 21) comparatively to the estrogen-treated tissues (Day 0). In both uterine tissues, the production of inositol phosphates mediated by carbachol as well as by AlF(4)(-) was enhanced with advancing gestation. (3)[H]IP(3) binding sites in membranes also increased during pregnancy (Day 21 > Day 12 > Day 0). The mRNAs encoding for three isoforms of IP(3)R as well as their corresponding proteins, IP(3)R-1, IP(3)R-2, and IP(3)R-3 were coexpressed, albeit to different extents, in the myometrium and endometrium. The expression of IP(3)Rs increased with advancing gestation, except for IP(3)R-2 that increased only in the endometrium at term. Thus, the pregnancy-related upregulation of the PLC cascade coincided with an increase in the expression of IP(3)Rs. The difference noted between the two uterine tissues suggests that IP(3)Rs may have cell-specific functions.

Ligand-binding affinity of the type 1 and 2 inositol 1,4,5-trisphosphate receptors: effect of the membrane environment.

The inositol 1,4,5-trisphosphate (InsP3) receptor is essential for Ca2+ release from intracellular stores. There are three InsP3 receptor types which are targets for several types of regulation. Ca2+, phosphorylation, and protein-protein interactions may contribute to the complex pattern of the Ca2+ signal in stimulated cells. Furthermore, the 3 receptor types could have different affinities for InsP3. We compared the affinities of the type 1 receptor from the cerebellum with the liver type 2 receptor both in their membrane environment and after isolation by immunoprecipitation. Measurements of [3H]InsP3 binding in a cytosol-like medium revealed that the Kd of the liver receptor (45 +/- 5 nM, N = 14) was higher than the Kd of the cerebellar receptor (28 +/- 3 nM, N = 9). Solubilization and immunopurification of the liver InsP3 receptor resulted in a 10-fold increase in its affinity for InsP3. The affinity of the cerebellar receptor did not change under these conditions. Therefore, the extraction of the liver and the cerebellar receptors from their membrane environments induced an inversion of their relative affinities. Treatment of liver membranes with low concentrations of detergents also increased the affinity for InsP3 binding. These data indicate that the type 1 and the type 2 InsP3 receptors have different affinities for InsP3 and that the properties of the type 2 receptor are strongly regulated by hydrophobic interactions within its membrane environment.

Regulation of cerebellar Ins(1,4,5)P3 receptor by interaction between Ins(1,4,5)P3 and Ca2+.

We have characterized in detail the Ca(2+)-dependent inhibition of [(3)H]Ins(1,4,5)P(3) ([(3)H]InsP(3)) binding to sheep cerebellar microsomes, over a short duration (3 s), with the use of a perfusion protocol. This procedure prevented artifacts previously identified in studies of this Ca(2+) effect. In a cytosol-like medium at pH 7.1 and 20 degrees C, a maximal inhibition of approx. 50% was measured. Both inhibition and its reversal were complete within 3 s. Ca(2+) decreased the affinity of the receptor for InsP(3) by approx. 50% (K(d) 146+/-24 nM at pCa 9 and 321+/-56 nM at pCa 5.3), without changing the total number of binding sites. Conversely, increasing the [(3)H]InsP(3) concentration from 30 to 400 nM tripled the IC(50) for Ca(2+) and decreased the maximal inhibition by 63%. This is similar to a partial competitive inhibition between InsP(3) binding and inhibitory Ca(2+) binding and is consistent with InsP(3) and Ca(2+) converting InsP(3) receptor into two different states with different affinities for these ligands. Mn(2+) and Sr(2+) also inhibited [(3)H]InsP(3) binding but were respectively only 1/10 and 1/200 as effective as Ca(2+). No inhibition was observed with Ba(2+). This selectivity is the same as that previously reported for the inhibitory Ca(2+) site of InsP(3)-induced Ca(2+) flux, suggesting that the same site is used by Ca(2+) to convert cerebellar InsP(3) receptor to a low-affinity state and to inhibit its channel activity. Our results also suggest a mechanism by which InsP(3) counteracts this Ca(2+)-dependent inhibition.

Multiple mechanisms of regulation of the inositol 1,4,5-trisphosphate receptor by calcium.

Ca2+ mobilisation by inositol 1,4,5-trisphosphate (InsP3) is a complex phenomenon which involves positive and negative feedback regulation by cytosolic Ca2+. It has been shown that Ca2+ increased the affinity of [3H]-InsP3 binding to liver membranes and inhibited [3H]-InsP3 binding to cerebellar membranes. We investigated the effects of Ca2+ on the [3H]-InsP3 binding to receptor solubilised and rapidly purified by immunoprecipitation. The InsP3 binding to the purified liver receptor was insensitive to the addition of Ca2+, indicating that Ca2+ did not interact directly with the receptor. The loss of the Ca2+ effect on liver receptor affinity was reproduced by alkaline treatment of liver membranes, which is known to extract the peripheral membrane proteins. This suggests that Ca2+ regulates the liver InsP3 receptor by interacting with a membrane-associated protein. Ca2+ inhibited the binding of [3H]-InsP3 to purified cerebellar receptors as was found with the membrane fraction. The treatment of the purified cerebellar receptor with media of high ionic strength or at alkaline pH did not abolish the effect of Ca2+ on the receptor. This indicates that the inhibitory effect of Ca2+ on [3H]-InsP3 binding to cerebellar membranes occurs either via direct interaction with the receptor or via an integral protein strongly associated with the receptor. In conclusion, the mechanisms of regulation of InsP3-induced Ca2+ release by Ca2+ involve different molecular support in cerebellum and in liver. This may reflect different regulation dependent on the receptor type.

The properties of a subtype of the inositol 1,4,5-trisphosphate receptor resulting from alternative splicing of the mRNA in the ligand-binding domain.

Subtypes of the type-1 inositol 1,4,5-trisphosphate (InsP3) receptor differ at the mRNA level in two small variably spliced segments. One segment (SI) encodes for a sequence within the InsP3-binding domain, thus its presence or absence could affect the functions of the receptor. We have used anti-peptide antibodies to confirm the existence of different subtypes of the InsP3 receptor (InsP3R) protein. The antibody against residues 322-332, within the SI region, recognized a 260 kDa polypeptide in membranes prepared from rat cerebellum or cerebral cortex. The cerebellum contained a few percent of the InsP3R protein having the SI region, whereas the cerebral cortex contained a high proportion of receptors with the SI region. These two tissues were representative of both isoforms, SI- or SI+, and displayed the same [3H]InsP3-binding characteristics. Thus, the SI region was not involved in the basic properties of the receptor. Deletion of the peptide 316-352 containing the SI segment greatly reduced InsP3 binding [Miyawaki, Furuichi, Ryou, Yoshikawa, Nakagawa, Saitoh and Mikoshiba (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4911-4915]. The antibodies against the SI region or against residues 337-349 did not modify the binding of [3H]InsP3 in the cortical membranes rich in the SI+ isoform or in cerebellar membranes. These results suggested that the SI region was not part of the binding site. The subcellular distribution of these two isoforms was then investigated in rat liver. The two isoforms were identified in different membrane fractions and they followed the same subcellular distribution. We suggest that the domain with the SI region may be involved in a function other than InsP3-induced Ca2+ release.

Intracellular calcium stores and inositol 1,4,5-trisphosphate receptor in rat liver cells.

The D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptor was localized by immunofluorescence experiments in situ in liver cryosections. Two anti-Ins(1,4,5)P3 receptor antibodies (against the 14 C-terminal residues of the type 1 receptor or against the entire cerebellar receptor) weakly decorated the whole cytoplasm, and a more intense labelling was observed at the periphery of the hepatocytes, particularly beneath the canalicular and the sinusoidal domains of the plasma membrane (PM). Antibodies against calreticulin, the Ca2+ pump (SERCA2b) or endoplasmic reticulum (ER) membranes homogeneously labelled the cytoplasm and the subplasmalemmal area. These data indicate that the ER can be divided into at least two specialized subregions: one is located throughout most of the cytoplasm and contains markers of the rough ER (RER), calreticulin, SERCA2b and a low density of Ins(1,4,5)P3 receptor, and the other is confined to the periphery of the cells and contains calreticulin, Ca2+ pump, RER markers and a high density of Ins(1,4,5)P3 receptor. A membrane fraction enriched in Ins(1,4,5)P3 receptor and in markers of the PM was immuno-adsorbed with the antibody against the C-terminal end of the Ins(1,4,5)P3 receptor and pelleted with Sepharose protein A. The immuno-isolated material was enriched in Ins(1,4,5)P3 receptor, but none of the markers of the ER or of the PM could be detected. This suggests that the Ins(1,4,5)P3 receptor is localized on discrete domains of the ER membrane beneath the canalicular and the sinusoidal membranes, where it was found at higher densities than the other markers.

Inositol 1,4,5-trisphosphate slowly converts its receptor to a state of higher affinity in sheep cerebellum membranes.

Incubation of cerebellar microsomes with d-myo-inositol 1,4,5-trisphosphate (InsP3) (0.01 1 microM), at 4 or 20 degrees C in a cytosolic-like medium devoid of Ca2+ and Mg2+, followed by InsP3 removal, induced an increase in InsP3 binding determined with 1 nm [3H]InsP3. At 20 degrees C, and pH 7.1, maximal stimulation (1.5 2. 5-fold) was obtained with 1 mum InsP3, and the EC50 was 60 +/- 5 nm. Several lines of evidence suggested that the activating site is identical with the InsP3 binding site: (i) activation and binding exhibited the same inositol phosphate specificity; (ii) addition of decavanadate, a competitive inhibitor of [3H]InsP3 binding, to the preincubation mixture, prevented the activating effect of InsP3; (iii) the concentration of InsP3 giving half-maximal activation was close to that giving half-maximal InsP3 binding. The time course of activation was found to be much slower than that of binding. While a t1/2 less than 0.4 s has been measured recently at neutral pH and 20 degrees C for binding of 0.5 nm [3H]InsP3 (Hannaert-Merah, Z., Coquil, J.-F., Combettes, L., Claret, M., Mauger, J.-P., and Champeil, P. (1994) J. Biol. Chem. 269, 29642-29649), a 20-s preincubation with 1 microM InsP3 was required to half-maximally stimulate binding. Under the present conditions, the InsP3-induced binding increase was only partially reversible. However, this effect was not blocked by antiproteases suggesting that it did not involve proteolysis. Taking advantage of the marked difference in the kinetics of InsP3 binding and InsP3-dependent activation, we performed binding experiments on a short period (3 s) to determine the effect of InsP3 pretreatment on the binding parameters. The data showed that this treatment increased the affinity of the receptor without changing the number of binding sites (control: KD = 107 nm, Bmax = 28 pmol/mg of protein; after preincubation with 1 microM InsP3: KD = 53 nm, Bmax = 32 pmol/mg of protein). The two states of the receptor bound InsP3 with a Hill coefficient close to 1 on a 3-s scale. In agreement with the effect of InsP3 pretreatment, equilibrium binding experiments performed on 10-min incubations revealed an apparent positive cooperative behavior (apparent Hill coefficient = 1.6; apparent KD = 66 nm). These results report a new regulatory process of the InsP3 receptor in cerebellum occurring independently of Ca2+ and on a relatively long time scale.

Characterization of the co-agonist effects of strontium and calcium on myo-inositol trisphosphate-dependent ion fluxes in cerebellar microsomes.

Using sheep cerebellum microsomes previously loaded with 45Ca2+ or 90Sr2+, we measured the dependence of inositol 1,4,5-trisphosphate (InsP3)-induced efflux of these ions on Ca2+ or Sr2+ on the cytosolic side. At a low InsP3 concentration, Ca2+ in the submicromolar range only poorly activated 45Ca2+ or 90Sr2+ efflux, and higher Ca2+ concentrations were inhibitory. In contrast, Sr2+ in the micromolar range activated release efficiently, while only very high Sr2+ concentrations were inhibitory. Experiments were repeated in the presence of a high InsP3 concentration, which allowed increasing free Ca2+ to micromolar concentrations without inducing complete inhibition of the InsP3-dependent efflux. Under these conditions, micromolar Ca2+ was found to activate efflux to a large extent, similar to that previously found with Sr2+. Optimal activation by Ca2+ of the InsP3-dependent channel occurs at micromolar rather than submicromolar free Ca2+ concentrations, but at too low an InsP3 concentration, Ca(2+)-induced activation is counteracted by Ca(2+)-induced inactivation. Separate measurements of [3H]-InsP3 binding at a low concentration showed that Sr2+ and Ca2+ did not enhance the amount of bound [3H]-InsP3, implying that the activating effect of Sr2+ and Ca2+ in cerebellar microsomes is mediated by an increase in the channel opening probability and not by an increase in the receptor's affinity for InsP3. A similar relationship also holds in the case of the activating effect of nucleotides.

Rapid kinetics of myo-inositol trisphosphate binding and dissociation in cerebellar microsomes.

Using sheep cerebellum microsomes adsorbed on a filter, we measured the kinetics of [3H]inositol 1,4,5-trisphosphate (InsP3) binding and dissociation on the subsecond time scale during rapid perfusion of the filter with [3H]InsP3-containing or InsP3-free media. At 20 degrees C and pH 7.1, in a cytosol-like medium containing MgCl2, the half-time for InsP3 dissociation was as short as 125 ms. The receptor behaved as a simple target for binding of its ligand, with the rate constant for InsP3 binding increasing linearly with InsP3 concentration. Various modulators of InsP3 binding (KCl, NaCl, pH, Mg2+, and Ca2+) were found to affect the receptor's apparent affinity for InsP3 mainly by altering the rate constant for [3H]InsP3 dissociation. ATP (but not InsP3) also accelerated [3H]InsP3 dissociation. In contrast to these modulators, luminal Ca2+ was found to have no effect on the amount of microsome-bound [3H]InsP3.

The inositol 1,4,5-trisphosphate receptor is localized on specialized sub-regions of the endoplasmic reticulum in rat liver.

Inositol 1,4,5-trisphosphate (InsP3) is involved in the mobilization of Ca2+ from intracellular non-mitochondrial stores. In rat liver, it has been shown that the InsP3-binding site co-purifies with the plasma membrane. This suggests that in the liver the InsP3 receptor (InsP3R) associates with plasma membrane. We studied the subcellular distribution of the liver InsP3R by measuring the maximal binding capacity of [3H]InsP3 and using antibodies against the 14 C-terminal residues of the type 1 InsP3R. The antibodies recognized a large amount of an InsP3R protein of 260 kDa in a membrane fraction which is also enriched with [3H]InsP3-binding sites and with markers of the basal, the lateral and the bile-canalicular membrane and the plasma-membrane Ca2+ pump (PMCA). The fractions enriched in markers of the endoplasmic reticulum (ER) and the Ca2+ pump of the ER (SERCA2b) contained low levels of InsP3 receptors. The immunofluorescent labelling of cultured hepatocytes with anti-InsP3R antibodies indicated that the receptor is concentrated in the perinuclear area and in some regions near the plasma membrane. The fraction enriched with InsP3R is also contaminated with markers of the ER and with SERCA2b. It was exposed to alkaline medium (pH 10.5) to extract endogenous actin and membrane-associated proteins before being subfractionated by Percoll-gradient centrifugation. The alkaline treatment allowed partial separation of the markers of the ER from the markers of the plasma membrane. The InsP3R was recovered in the heavy subfraction, which was also enriched with markers for the ER and with the SERCA2b and contained low levels of markers of the plasma membrane. These data indicate that the InsP3R is neither localized on the plasma membrane itself nor homogeneously distributed on the ER membrane. This supports the view that part of the receptor is localized on a specialized sub-region of the ER which interacts with the plasma membrane.

The inositol 1,4,5-trisphosphate receptor: kinetic properties and regulation.

Inositol 1,4,5-triphosphate (InsP3) is a second messenger responsible for the mobilization of intracellular Ca2+ after receptor-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate. InsP3 binds to a specific receptor located on the membrane of an intracellular compartment and opens a Ca2+ channel causing the cytosolic Ca2+ concentration to increase. Measurement of radiolabelled InsP3 binding and InsP3-induced Ca2+ release in parallel experiments indicated that the liver InsP3 receptor exists in two main states: an active state (A) and an inactive one (I). The "I" form of the receptor is found in the presence of high Ca2+ concentrations (above 1 microM). The binding properties of the "A" and the "I" states of the receptor have been characterized by analysing a membrane fraction enriched in InsP3 receptors. The inactive "I" state displays a high affinity (Kd = 2 nM) and slow rates of association and dissociation. The active state "A" of the receptor displays complex kinetic properties. The rate of association and the rate of dissociation of labelled InsP3 are rapid phenomena probably involving several components. The apparent Kd for the InsP3 binding is about 40 nM in a low Ca2+ medium. The affinity of the "A" state of the receptor is increased by Ca2+ (at concentrations lower than 0.5 microM) and by thiol reagents. The increase of the affinity of the receptor is due to a decrease of the dissociation rate constants. This lowers the threshold such that Ca2+ is released at lower concentrations of InsP3. These data indicate that the binding of InsP3 to its receptor is a complex phenomenon involving the transition among several states.(ABSTRACT TRUNCATED AT 250 WORDS)

Thiol reagents increase the affinity of the inositol 1,4,5-trisphosphate receptor.

Thiol reagents have been shown to increase cytosolic Ca2+ in several cell types. In non-muscle cells, these agents induce Ca2+ spikes by increasing the sensitivity of the intracellular Ca2+ stores to D-myo-inositol-1,4,5-trisphosphate (InsP3). We have investigated the effects of thimerosal and oxidized glutathione on the binding properties of the InsP3 receptor in permeabilized hepatocytes and liver and cerebellar membranes. Thimerosal, at the maximal concentration of 100 microM, decreased the KD for the InsP3 binding to permeabilized hepatocytes and cerebellar membranes from 16 to 3 nM and from 25 to 8 nM, respectively, without affecting the maximal binding capacities. On liver membranes, both thimerosal and high Ca2+ concentrations increased the affinity for InsP3 binding. The Ca2+ and the thimerosal effects were differentiated by kinetic experiments. In low Ca2+ media, two kinetic components were identified and thimerosal decreased the rate of dissociation from both these components without affecting the rate of association. In the high Ca2+ medium, a single kinetic component was found with a very slow rate of dissociation. These data suggest that the InsP3 receptor exists in different states. The high-affinity inactive state induced by high Ca2+ concentrations displays slow rates of association and dissociation. The binding properties of the receptor in its active state can be regulated by thiol reagents which increase the affinity by decreasing the dissociation rate constants. At a resting concentration of 100-200 nM, Ca2+ has two effects: it increases the affinity of the active state of the receptor as thiol reagents do and transforms part of the receptors into the inactive high-affinity state.

Characterization of a rapidly dissociating inositol 1,4,5-trisphosphate-binding site in liver membranes.

The binding of inositol 1,4,5-trisphosphate (InsP3) to a specific receptor induces the release of Ca2+ from an intracellular store. In the liver, the KD of a low affinity state of the receptor (RL) found at low Ca2+ concentration ([Ca2+]) is in close agreement with the EC50 of the InsP3-induced Ca2+ release. We have developed an experimental procedure for measuring the rate of dissociation of this low affinity [32P]InsP3-receptor complex in less than 1 s. When the receptor was in the RL state, two kinetic components, RL1 and RL2, were identified with respective rate constants (k(off)) of 1-2 s-1 and 0.03-0.06 s-1. Increasing the [Ca2+] up to 1 microM transformed the receptor into the high affinity state (RH) and decreased the dissociation rate constant to 2 x 10(-2) min-1. We also investigated the time course of the transformation of the receptor from the high affinity (RH) to the low affinity state (RL) after decreasing the [Ca2+] to less than 10 nM. This reversion was dramatically dependent on temperature: at 4 degrees C, the receptor was locked in the RH state, whereas at 37 degrees C the receptor reverted to the RL state with a half-time of less than 1 s. The reversion from the RH state to the RL one is associated to a recovery of InsP3-induced 45Ca2+ release on permeabilized hepatocytes. The rapid and reversible transformation of the InsP3 receptor from an active to an inactive state may be a key event in the Ca2+ release process in intact cells.

Cortical alveoli of Paramecium: a vast submembranous calcium storage compartment.

The plasma membrane of Paramecium is underlain by a continuous layer of membrane vesicles known as cortical alveoli, whose function was unknown but whose organization had suggested some resemblance with muscle sarcoplasmic reticulum. The occurrence of antimonate precipitates within the alveoli first indicated to us that they may indeed correspond to a vast calcium storage site. To analyze the possible involvement of this compartment in calcium sequestration more directly, we have developed a new fractionation method, involving a Percoll gradient, that allows rapid purification of the surface layer (cortex) of Paramecium in good yield and purity and in which the alveoli retain their in vivo topological orientation. This fraction pumped calcium very actively in a closed membrane compartment, with strict dependence on ATP and Mg2+. The pumping activity was affected by anti-calmodulin drugs but no Triton-soluble calmodulin binding protein could be identified, using gel overlay procedures. The high affinity of the pump for calcium (Km = 0.5 microM) suggests that it plays an important role in the normal physiological environment of the cytosol. This may be related to at least three calcium-regulated processes that take place in the immediate vicinity of alveoli: trichocyst exocytosis, ciliary beating and cytoskeletal elements dynamics during division.

Calcium mediates the interconversion between two states of the liver inositol 1,4,5-trisphosphate receptor.

D-myo-Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) regulates intracellular Ca2+ by mobilizing Ca2+ from a non-mitochondrial store. We have investigated the effects of Ca2+ on the binding of [32P]Ins (1,4,5)P3 to permeabilized rat hepatocytes and a liver plasma membrane-enriched fraction. Increasing the free Ca2+ concentration in the medium from 0.1 nM to 0.7 microM increased the capacity of a high affinity binding component (KD = 2-3 nM) in permeabilized cells by a factor of 10. If the membrane fraction was preincubated at 37 degrees C before binding was measured at 4 degrees C, all of the Ins(1,4,5)P3 receptors were transformed to a low affinity state (KD = 65 +/- 12 nM, Bmax = 3.1 +/- 0.1 fmol/mg, n = 4). When 0.7 microM of Ca2+ was added, the receptors were totally transformed to a high affinity state (KD = 2.8 +/- 0.4 nM, Bmax = 2.7 +/- 0.4 fmol/mg, n = 4). The EC50 of the Ca2(+)-induced interconversion of the Ins(1,4,5)P3 receptor was 140 nM. This Ca2(+)-induced transformation of the Ins(1,4,5)P3 receptor from a low affinity to a high affinity state was associated with an inhibition of the Ins(1,4,5)P3-induced Ca2+ release in permeabilized hepatocytes. These data suggest that the Ins(1,4,5)P3-dependent hormones, by increasing the intracellular Ca2+ concentration, induce a reversible transformation of the receptor from its low affinity state, coupled to the Ca2+ release, to a desensitized high affinity state. Transformation of the receptor may play a role in the oscillatory release of Ca2+ observed in single isolated hepatocytes.

[Characterisation of 2 reversible states of the inositol 1,4,5-trisphosphate receptor in rat hepatocytes]. / Caractérisation de deux états interconvertibles du récepteur de l'inositol 1,4,5-trisphosphate dans les hépatocytes de rat.

Myo-inositol-1,4,5-trisphosphate or (I(1,4,5)P3) is generated in liver cells after hormonal stimulation. It bind to specific receptors and induces the release of Ca2+ from an intracellular store. This receptor has been found in permeabilized hepatocytes and showed two states of low- and high-affinity with KD of 1-2 and 40-50 nmol/l, respectively. Measurements of 45Ca2+ release, mediated by different analogues, revealed that the low-affinity site was coupled to the Ca2+ channel open state. The pretreatment of cells with vasopressin, an I(1,4,5)P3-dependent agonist, induced an 60 percent increase of the binding capacity of the high-affinity sites. Incubation with 1 mumol/l Ca2+ increased the number of high-affinity sites from 5 to 65 fmol/10(6) cells. This effect was associated with a decrease in the number of low-affinity sites from 130 to 80 fmol/10(6) cells. Our results suggest that the intracellular Ca2+ concentration rise mediated by I(1,4,5)P3-dependent agonists, induced a conversion of the low-affinity form of the I(1,4,5)P3 receptor, coupled to Ca2+ release, to a desensitized high-affinity one. This process could explain the oscillations of the intracellular Ca2+ concentration observed in hormone-treated single cells.

Characterization of two forms of inositol 1,4,5-trisphosphate receptor in rat liver.

Two binding sites for [32P]myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) were detected in a crude particulate fraction prepared from rat liver homogenate and in permeabilized hepatocytes. The same high- and low-affinity sites with KDs of 1.8-2.6 nM and 35-71 nM, respectively, were detected in subcellular fractions enriched in plasma membranes, mitochondria and microsomes, with relative proportions close to those found in the crude membrane fraction. The order of potency of three inositol phosphates in inhibiting [32P]Ins(1,4,5)P3 binding to the two sites, i.e. Ins(1,4,5)P3 greater than Ins(2,4,5)P3] greater than Ins(1,3,4,5)P4, and the inhibition by heparin, strongly suggest that neither of the binding sites reflected components due to the 3-kinase or the 5-phosphatase. A close correlation was observed between the dose-response curves for Ca2+ release by Ins(1,4,5)P3 and Ins(2,4,5)P3 and the occupancy of the low-affinity binding site by these agonists. These results support the view that the two [32P]Ins(1,4,5)P3 binding sites are two forms of the same receptor.