InsP3 Binding Sites Research Articles

Differential change of Ins-P 3-Ca 2+ signaling during culture of rat hepatocytes

Decrease of α-adrenergic responses during primary culture of rat hepatocytes was studied. Activation of glycogen phosphorylase by phenylephrine was decreased in the early stage of the culture (within 6 h), however, Ins-P 3 production was almost intact until 12 h of the culture and then declined. α-Adrenoceptor-mediated Ca 2+-mobilization and Ins-P 3-induced Ca 2+ release from microsomal fractions were decreased in the early stage of the culture, similar to the above change of phosphorylase activation. We found that decrease of Ins-P 3-binding sites in the early stage of the culture was the cause of differential change of Ins-P 3-Ca 2+ signaling during the culture of hepatocytes. Similar changes described above were also observed in vasopressin-induced responses. However, the changes of Ins-P 3-Ca 2+ signaling did not occur in a high-cell density culture of rat hepatocytes. In conclusion, the loss of phenylephrine- and vasopressin-induced responses in cultured liver cells appear to be due to change of Ins-P 3-binding sites as well as decreased Ins-P 3 production due to reduction of receptor numbers.

Novel model of calcium and inositol 1,4,5-trisphosphate regulation of InsP3 receptor channel gating in native endoplasmic reticulum

The InsP3R Ca(2+)-release channel has biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). InsP3 activates gating primarily by reducing high [Ca2+]i inhibition. To determine whether relieving Ca2+ inhibition is sufficient for activation, we examined single-channels in low [Ca2+]i in the absence of InsP3 by patch clamping isolated Xenopus oocyte nuclei. For both endogenous Xenopus type 1 and recombinant rat type 3 InsP3R channels, spontaneous InsP3-independent activities with low open probability Po (approximately 0.03) were observed in [Ca2+]i < 5 nM, whereas none were observed in 25 nM Ca2+. These results establish the half-maximal inhibitory [Ca2+]i in the absence of InsP3 and demonstrate that the channel can be active when all of its ligand-binding sites are unoccupied. In the simplest allosteric model that fits all observations in nuclear patch-clamp studies, the tetrameric channel can adopt six conformations, the equilibria among which are controlled by two inhibitory, one activating Ca(2+)-binding, and one InsP3-binding sites in a manner similar to the Monod-Wyman-Changeux model. InsP3 binding activates gating by affecting the relative affinity for Ca2+ of one of the inhibitory sites in different channel conformations, transforming it into an activating site. Ca2+ inhibition of InsP3-liganded channels is mediated by an InsP3-independent second inhibitory site.

Spontaneous channel activity of the inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R). Application of allosteric modeling to calcium and InsP3 regulation of InsP3R single-channel gating.

The InsP3R Ca2+ release channel has a biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). InsP3 activates gating primarily by reducing the sensitivity of the channel to inhibition by high [Ca2+]i. To determine if relieving Ca2+ inhibition is sufficient for channel activation, we examined single-channel activities in low [Ca2+]i in the absence of InsP3, by patch clamping isolated Xenopus oocyte nuclei. For both endogenous Xenopus type 1 and recombinant rat type 3 InsP3R channels, spontaneous InsP3-independent channel activities with low open probability P o (∼0.03) were observed in [Ca2+]i < 5 nM with the same frequency as in the presence of InsP3, whereas no activities were observed in 25 nM Ca2+. These results establish the half-maximal inhibitory [Ca2+]i of the channel to be 1.2–4.0 nM in the absence of InsP3, and demonstrate that the channel can be active when all of its ligand-binding sites (including InsP3) are unoccupied. In the simplest allosteric model that fits all observations in nuclear patch-clamp studies of [Ca2+]i and InsP3 regulation of steady-state channel gating behavior of types 1 and 3 InsP3R isoforms, including spontaneous InsP3-independent channel activities, the tetrameric channel can adopt six different conformations, the equilibria among which are controlled by two inhibitory and one activating Ca2+-binding and one InsP3-binding sites in a manner outlined in the Monod-Wyman-Changeux model. InsP3 binding activates gating by affecting the Ca2+ affinities of the high-affinity inhibitory sites in different conformations, transforming it into an activating site. Ca2+ inhibition of InsP3-liganded channels is mediated by an InsP3-independent low-affinity inhibitory site. The model also suggests that besides the ligand-regulated gating mechanism, the channel has a ligand-independent gating mechanism responsible for maximum channel P o being less than unity. The validity of this model was established by its successful quantitative prediction of channel behavior after it had been exposed to ultra-low bath [Ca2+].

The inhibition of [3H] inositol 1,4,5-trisphosphate binding by Ca2+is modified after long-term ethanol treatment and ethanol withdrawal

We have analysed the influence of long-term ethanol exposure on the effect exerted by Ca²⁺ on the binding of tritiated inositol 1,4,5-trisphosphate to its receptors in rat cerebellar membranes. After 21 days of ethanol treatment the binding of the agonist was reduced in the absence of Ca²⁺. The decrease was due to reduction in B max without any alteration of K d. In membranes from control animals Ca²⁺ inhibited the binding of InsP₃ in a dose-dependent manner by altering the affinity of the protein for the ligand. However, the inhibitory effect of Ca²⁺ was abolished following chronic ethanol exposure. Five days after withdrawing ethanol, the B max recovered to control values, but the inhibitory effect of Ca²⁺ was recovered at only 10 days after withdrawal. The results indicate that long-term ethanol exposure may have differential effects on the InsP₃binding site and on the Ca²⁺ binding site, or alternatively on a Ca²⁺ -related regulatory cycle.

Association of the Inositol (1,4,5)-Trisphosphate Receptor Ligand Binding Site with Phosphatidylinositol (4,5)-Bisphosphate and Adenophostin A

The inositol 1,4,5-trisphosphate receptor (InsP3R) is activated by InsP3 binding to amino-terminal ligand binding domain (InsP3R-N). Recently we reported functional coupling of phosphatidylinositol (4,5)-bisphosphate (PIP2) to the InsP3R. Specific binding of PIP2 to InsP3R-N domain was postulated as a part of the InsP3R-PIP2 functional coupling model. Here we utilized bacterially expressed and purified InsP3R-N domain to characterize its binding specificity for InsP3, Adenophostin A (AdA) and the water-soluble PIP2 analog dioctanoyl-(4,5)PIP2 (ShPIP2). Obtained data led us to conclude that specific InsP3, AdA, and ShPIP2 binding sites are located within the InsP3R-N domain, that the extra receptor binding element responsible for enhanced binding of AdA is an integral part of the InsP3R-N domain, that ShPIP2 is able to displace InsP3 from the InsP3R-N, but InsP3 or AdA is unable to completely displace ShPIP2. These results support the InsP3R-PIP2 functional coupling model and provide novel insights into InsP3R ligand specificity.

Regulation of type 1 inositol 1,4,5-trisphosphate-gated calcium channels by InsP3 and calcium: Simulation of single channel kinetics based on ligand binding and electrophysiological analysis.

Cytosolic calcium acts as both a coagonist and an inhibitor of the type 1 inositol 1,4,5-trisphosphate (InsP3)-gated Ca channel, resulting in a bell-shaped Ca dependence of channel activity (Bezprozvanny, I., J. Watras, and B.E. Ehrlich. 1991. Nature. 351:751-754; Finch, E.A., T.J. Turner, and S.M. Goldin. 1991. Science. 252: 443-446; Iino, M. 1990. J. Gen. Physiol. 95:1103-1122). The ability of Ca to inhibit channel activity, however, varies dramatically depending on InsP3 concentration (Combettes, L., Z. Hannaert-Merah, J.F. Coquil, C. Rousseau, M. Claret, S. Swillens, and P. Champeil. 1994. J. Biol. Chem. 269:17561-17571; Kaftan, E.J., B.E. Ehrlich, and J. Watras. 1997. J. Gen. Physiol. 110:529-538). In the present report, we have extended the characterization of the effect of cytosolic Ca on both InsP3 binding and InsP3-gated channel kinetics, and incorporated these data into a mathematical model capable of simulating channel kinetics. We found that cytosolic Ca increased the Kd of InsP3 binding approximately 3.5-fold, but did not influence the maximal number of binding sites. The ability of Ca to decrease InsP3 binding is consistent with the rightward shift in the bell-shaped Ca dependence of InsP3-gated Ca channel activity. High InsP3 concentrations are able to overcome the Ca-dependent inhibition of channel activity, apparently due to a low affinity InsP3 binding site (Kaftan, E.J., B.E. Ehrlich, and J. Watras. 1997. J. Gen. Physiol. 110:529-538). Constants from binding analyses and channel activity determinations were used to develop a mathematical model that fits the complex Ca-dependent regulation of the type 1 InsP3-gated Ca channel. This model accurately simulated both steady state data (channel open probability and InsP3 binding) and kinetic data (channel activity and open time distributions), and yielded testable predictions with regard to the regulation of this intracellular Ca channel. Information gained from these analyses, and our current molecular model of this Ca channel, will be important for understanding the basis and regulation of intracellular Ca waves and oscillations in intact cells.

Distribution of inositol 1,4,5-trisphosphate receptor isoforms, SERCA isoforms and Ca2+ binding proteins in RBLm2H3 rat basophilic leukemia cells

RBL-2H3 rat basophilic leukemia cells were homogenized and fractionated. A fraction F3 obtained by differential centrifugation was 6-fold enriched in [3H]-inositol 1,4,5-trisphosphate (InsP3) binding activity, while the NADH-cytochrome c oxidoreductase and sulphatase-C activities were only 3.8- and 2.9-fold enriched, respectively. Furthermore, the three InsP3 receptor (InsP3R) isoforms, two sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) isoforms (2b and 3) as well as four Ca 2+ binding proteins (calreticulin, calnexin, protein disulfide isomerase (PDI) and BiP), were present in this fraction. Fraction F3 was, therefore, further purified on a discontinuous sucrose density gradient, and the 3 resulting fractions were analyzed. The InsP3 binding sites were distributed over the gradient and did not co-migrate with the RNA. We examined the relative content of the three InsP3R isoforms, of both SERCA2b and 3, as well as that of the four Ca 2+ binding proteins in fraction F3 and the sucrose density gradient fractions. Ins P3R-1 and InsP3R-2 showed a similar distribution, with the highest level in the light and intermediate density fractions. InsP3R-3 distributed differently, with the highest level in the intermediate density fraction. Both SERCA isoforms distributed similarly to InsP3R-1 and InsP3R-2. SERCA3 was present at a very low level in the high density fraction. Calreticulin and BiP showed a pattern similar to that of InsP3R-1 and InsP3R-2 and the SERCs. PDI was clearly enriched in the light density fraction while calnexin was broadly distributed. These results indicate a heterogeneous distribution of the three InsP3R isoforms, the two SERCA isoforms and the four Ca2+ binding proteins investigated. This heterogeneity may underlie specialization of the Ca2+ stores and the subsequent initiation of intracellular Ca2+ signals.

Inositol 1,4,5-trisphosphate (InsP3) and calcium interact to increase the dynamic range of InsP3 receptor-dependent calcium signaling.

The inositol 1,4,5-trisphosphate (InsP3)-gated Ca channel in cerebellum is tightly regulated by Ca (Bezprozvanny, I., J. Watras, and B.E. Ehrlich. 1991. Nature (Lond.). 351:751-754; Finch, E.A., T. J. Turner, and S.M. Goldin. 1991. Science (Wash. DC). 252:443-446; Hannaert-Merah, Z., J.F. Coquil, L. Combettes, M. Claret, J.P. Mauger, and P. Champeil. 1994. J. Biol. Chem. 269:29642-29649; Iino, M. 1990. J. Gen. Physiol. 95:1103-1122; Marshall, I., and C. Taylor. 1994. Biochem. J. 301:591-598). In previous single channel studies, the Ca dependence of channel activity, monitored at 2 microM InsP3, was described by a bell-shaped curve (Bezprozvanny, I., J. Watras, and B.E. Ehrlich. 1991. Nature (Lond.). 351:751-754). We report here that, when we used lower InsP3 concentrations, the peak of the Ca-dependence curve shifted to lower Ca concentrations. Unexpectedly, when we used high InsP3 concentrations, channel activity persisted at Ca concentrations as high as 30 microM. To explore this unexpected response of the channel, we measured InsP3 binding over a broad range of InsP3 concentrations. We found the well-characterized high affinity InsP3 binding sites (with Kd < 1 and 50 nM) (Maeda, N., M. Niinobe, and K. Mikoshiba. 1990. EMBO (Eur. Mol. Biol. Organ.) J. 9:61-67; Mignery, G., T.C. Sudhof, K. Takei, and P. De Camilli. 1989. Nature (Lond.). 342:192-195; Ross, C.A., J. Meldolesi, T.A. Milner, T. Satoh, S. Supattapone, and S.H. Snyder. 1989. Nature (Lond.). 339:468-470) and a low affinity InsP3 binding site (Kd = 10 microM). Using these InsP3 binding sites, we developed a new model that accounts for the shift in the Ca-dependence curve at low InsP3 levels and the maintained channel activity at high Ca and InsP3 levels. The observed Ca dependence of the InsP3-gated Ca channel allows the cell to abbreviate the rise of intracellular Ca in the presence of low levels of InsP3, but also provides a means of maintaining high intracellular Ca during periods of prolonged stimulation.

Does the nuclear envelope contain two types of ligand-gated Ca 2+ release channels?

The nuclear envelope is composed of two membranes deliminating a perinuclear space which displays functional properties similar to those of a Ca 2+-storing compartment. ATP-driven Ca 2+ uptake and InsP 3-induced Ca 2+ release processes have been described in isolated nuclei. Recently, it was reported that cADP-ribose and InsP 3 can trigger a nucleoplasmic Ca 2+ increase. It was hypothesized that the inner nuclear membrane possesses Ca 2+ channels that are regulated by ryanodine or InsP 3. Radio-ligand binding assays and Western blot experiments were performed in order to investigate their presence in sheep cardiac and rat liver nuclear envelopes. Ryanodine receptors (RyR) were not detected in liver nuclear envelopes by either binding assay or Western blot analysis. However, cardiac nuclear envelopes were found to retain a very low level of specific ryanodine binding, which was not detected on immuno-blots obtained with three types of isoform-specific RyR antibodies. In contrast, nuclear InsP 3-binding sites were consistently detected in both cardiac and liver nuclear envelopes. Altogether, these results provide evidence for the major contributor InsP 3-gated Ca 2+ channels in control of Ca 2+ release from the perinuclear space in liver and cardiac cells.

Ligand-gated Ca2+ channels and Ca2+ signalling in higher plants

Ligand-gated Ca(2+) channels provide a possible mechanism for linking perception of stimuli to intracellular Ca(2+) mobilization. Evidence for ligand-gated Ca(2+) release in plant cells arises from radiolabeled ligand binding, microsomal ion flux, and electrophysiological approaches. Results from these diverse approaches demonstrate that two classes of ligand-gated channels are present at the plant cell vacuolar membrane. One class of channel is gated by inositol 1,4,5 trisphosphate (InsP(3)) and the second is gated by cyclic adenosine 5'-diphosphoribose (cADPR). Previous biochemical studies on plant InsP(3) binding sites have been hampered by low density of specific binding. The present work reports optimization of yield for solubilized InsP(3) binding sites with respect to detergent type and concentration, and the originating tissue. Further studies reveal a pharmacological similarity between cADPR-activated Ca(2+) release in plant and animal cells and demonstrate that the extent of cADPR-induced Ca(2+) release is dependent on the plant tissue type. In animal cells cADPR releases Ca(2+) through activation of at least one isoform of the so-called ryanodine receptor. It is shown here that ryanodine itself is able to activate single channel currents in vacuolar membranes. These observations are integrated into current models for ligand-gated Ca(2+) release in plant and animal cells and their role in Ca(2+)-based cell signalling.

Multiple Inositol 1,4,5-Trisphosphate Receptor Isoforms Are Present in Platelets

Platelets are activated by an increase in cytosolic Ca2+, and a portion of this increase is derived from inositol 1,4,5-trisphosphate (InsP3)-mediated Ca2+release from internal stores via the InsP3receptor. There is some uncertainty concerning the localization of the InsP3receptor within platelets, and experiments were designed to help resolve this question. [3H]InsP3binding to unphosphorylated and phosphorylated platelet internal membranes revealed both low and high affinity InsP3binding sites, indicating the presence of more than one isoform of InsP3receptor within the internal membranes. Phosphorylation did not significantly affect InsP3binding. In contrast, a single class of high affinity sites was observed in plasma membranes indicating only one type of InsP3receptor. Western blotting of platelet internal and plasma membranes with antibodies against the three major InsP3receptor isoforms revealed that the internal membranes contain both type 1 and type 2 InsP3receptors while the plasma membrane contains only InsP3receptor type 2.

Isolation and characterization of vascular smooth muscle inositol 1,4,5-trisphosphate receptor

myo-Inositol 1,4,5-trisphosphate (InsP3) receptor of porcine aorta was purified to near homogeneity and its biochemical properties were compared with those of cerebellar InsP3 receptor of the same animal species. The aortic InsP3 receptor consisted of equal amounts of two polypeptides with slightly differing molecular masses of around 240 kDa and was found to possess a single population of InsP3-binding site (Kd of 1.2 nM). The InsP3 receptor purified from porcine cerebellum was also comprised of two polypeptides. However, the molecular mass was slightly but definitely larger, being 250 kDa, and the amounts of the two polypeptides were not equal. The aortic InsP3 receptor cross-reacted with polyclonal antibody specific to type 1 InsP3 receptor as did the cerebellar InsP3 receptor. The aortic InsP3 receptor bound to calmodulin-Sepharose in a Ca(2+)-dependent manner, while the cerebellar InsP3 receptor did not. Reverse transcriptase-PCR analysis revealed two splicing variants of the type 1 InsP3 receptor in porcine aortic smooth muscle distinct from those of the type 1 InsP3 receptor of porcine cerebellum. The possible relevance of this difference to difference in calmodulin-binding property was discussed.

Identification and Preliminary Characterization of a Ca2+- Dependent High-Affinity Binding Site for Inositol-1,4,5-Trisphosphate from Chenopodium rubrum.

Using a radioligand-binding assay we have identified a Ca2+- dependent high-affinity D-myo-inositol-1,4,5-trisphosphate (InsP3) binding site in a membrane vesicle preparation from Chenopodium rubrum. Millimolar concentrations of Ca2+ were required to observe specific binding of [3H]InsP3. A stable equilibrium between bound and free ligand was established within 5 min and bound [3H]InsP3 could be completely displaced by InsP3 in a time- and concentration-dependent manner. Displacement assays indicated a single class of binding sites with an estimated dissociation constant of 142 [plus or minus] 17 nM. Other inositol phosphates bound to the receptor with much lower affinity. The glycosaminoglycan heparin was an effective competitor for the binding site (inhibitor concentration for 50% displacement = 534 nM). ATP at higher, although physiologically relevant, concentrations (inhibitor concentration for 50% displacement = 241 [mu]M) also displaced [3H]InsP3 from the receptor. Recent studies in animals have highlighted the importance of Ca2+ regulation of InsP3-induced Ca2+ release. The potential for the operation of similar regulatory mechanisms in plants is discussed.

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.

Control of inositol 1,4,5-trisphosphate-induced Ca2+ release by cytosolic Ca2+

The synergistic action of cytosolic Ca2+ and inositol 1,4,5-trisphosphate (InsP3) in releasing intracellular Ca2+ stores has been suggested to be responsible for the complex intracellular Ca2 signals observed during hormonal stimulation of many cell types. However, the ability of cytosolic Ca2+ to potentiate Ca2+ release has recently been questioned because of the observed inhibitory effects of Ca2+ chelators used in previous studies. In the present study, EGTA and BAPTA [1,2-bis-(2-amino-phenoxy)ethane- NNN'N'-tetra-acetic acid] poorly inhibited InsP3-induced Ca2+ release from permeabilized A7r5 smooth-muscle cells. Additionally, stimulatory effects of cytosolic and luminal Ca2+ were observed either in the complete absence of Ca2+ chelator or at constant Ca(2+)-free chelator concentration. These data suggest that potentiation of InsP3-induced Ca2+ release by Ca2+ in A7r5 cells reflects an interaction between Ca2+ and InsP3 receptors, rather than a decrease in chelator-dependent inhibition. The EC50 for activation of InsP3-induced Ca2+ release by cytosolic Ca2+ was unaffected by ATP, or by changing InsP3 concentration, although InsP3-induced Ca2+ release became less sensitive to the inhibitory effects of cytosolic Ca2+ as the InsP3 concentration was elevated. Increasing H+ or Mg2+ concentration shifted the Ca(2+)-activation curve towards higher Ca2+ concentrations. These data suggest that, in addition to the InsP3-binding site, the affinity of the Ca(2+)-binding site(s) on InsP3 receptors can be modulated by intracellular cations.

Two inositol 1,4,5-trisphosphate binding sites in rat basophilic leukemia cells: relationship between receptor occupancy and calcium release.

Quantal calcium release is a novel paradigm for second messenger signal transduction which provides spatial and temporal control of calcium release from intracellular stores by inositol 1,4,5-trisphosphate (InsP3). We have proposed a mechanism to account for this phenomenon [Kindman, L. A., & Meyer, T. (1993) Biochemistry 32, 1270-1277], which hypothesized the existence of five channels, each with a different affinity for InsP3. As a direct test of this hypothesis, InsP3 binding to microsomes from RBL cells was examined under conditions similar to those used for calcium release. Scatchard analyses performed under a variety of conditions indicates the presence of high affinity (KD = 0.9 +/- 0.3 nM) and low affinity (KD = 47 +/- 5 nM) InsP3 binding sites. The low affinity sites are more prevalent, constituting 82 +/- 5% of the total. Both sites are identified in the presence and absence of MgATP. Moreover, both sites are selective for InsP3 over InsP4, through high concentrations of InsP4 displace InsP3 from each site (with inhibition constants of 16 and 267 nM InsP4, respectively). The relative abundance of the two InsP3 binding sites is Ca2+ dependent. An increase in Ca2+ from 0.1 to 0.5 microM results in the apparent conversion of a portion of the low affinity sites into high affinity sites into high affinity sites. Ca2+ (0.5 microM) also increased the KD of the low affinity InsP3 binding site. Given the presence of both high and low affinity InsP3 binding sites, two simple mathematical models describing both the kinetics of calcium release and quantal calcium release from RBL cells were developed.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacologic Characterization of the Inositol Trisphosphate Receptor in Rat Pancreas

Inositol 1,4,5-trisphosphate (InsP3) is an intracellular second messenger, produced upon stimulation of the phosphoinositide system, capable of mobilizing calcium from intracellular stores. The properties of InsP3 receptor sites in the rat pancreas were evaluated by binding studies with InsP3 labeled with 3H. Specific binding was very sensitive to pH variations between 7 and 9. Kinetic studies showed that specific binding of InsP3 at 0 degrees C was half-maximal in about 10 min and reached a plateau within 60 min with a K+1 = 3.37 x 10(9) mol-1min-1. Binding was reversible as addition of 10(-6) M unlabeled InsP3 was followed by dissociation of the bound ligand with a K-1 = 0.016 min-1. Scatchard analysis of the binding data was consistent with a single set of high-affinity sites with KD of 9.9 +/- 2.47 nM and a maximal binding capacity of 210 +/- 55 fmol/mg of protein (n = 7). The specificity of [3H]InsP3 binding to these sites was illustrated by the much weaker affinity for structural analogs such as inositol 1,3,4,5-tetrakisphosphate, phytic acid, fructose 1,6-bisphosphate, and heparin. To assess the functional relevance of the InsP3 binding sites, the Ca(2+)-releasing activity of InsP3 was measured in permeabilized acinar cell preparations. In the presence of oligomycin (10 micrograms/ml), Ca2+ movements were monitored with the fluorescent indicator fura-2 (free acid). Under these conditions, 4 mM ATP caused rapid uptake of Ca2+ by the vesicular component of the acinar cells. Addition of InsP3 (0.1-15 microM) caused a dose-dependent release of Ca2+ with half-maximal effect at 3.2 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Micromolar calcium decreases affinity of inositol trisphosphate receptor in vascular smooth muscle.

The mechanism by which Ca2+ inhibits InsP3-induced Ca2+ release from sarcoplasmic reticulum of vascular smooth muscle was investigated. InsP3 binding to sarcoplasmic-reticulum vesicles from dog aortic smooth muscle was inhibited by 51 +/- 6% by 2 microM Ca2+ in the presence of 10 nM [3H]InsP3. Scatchard analysis indicated the presence of two InsP3-binding sites in the absence of Ca2+ (Kd = 2.5 +/- 0.9 and 49 +/- 8 nM InsP3), though the low-affinity site was more prevalent (representing 92 +/- 3% of the total number of binding sites). Ca2+ (2 microM) did not alter InsP3 binding to the high-affinity site (P > 0.05), but increased the Kd of the low-affinity site 3-fold (Kd = 155 +/- 4 nM InsP3; P < 0.001). The possibility that the apparent decrease in InsP3 affinity was caused by Ca(2+)-dependent activation of an endogenous phospholipase C could be excluded, because the Ca(2+)-dependent inhibition of InsP3 binding was completely reversible and insensitive to an inhibitor of phospholipase C. Moreover, Ca2+ did not inhibit InsP3 binding to InsP3 receptor partially purified by heparin-Sepharose chromatography, though another fraction (devoid of InsP3 receptor) restored Ca(2+)-sensitivity of the partially purified InsP3 receptor. Thus Ca2+ binding to a Ca(2+)-sensitizing factor associated with the InsP3 receptor decreases the affinity of the receptor complex for InsP3. This Ca(2+)-sensitizing factor may provide a negative-feedback mechanism for regulating the rise in cytosolic Ca2+ concentration in vascular smooth muscle after hormone activation of the phosphoinositide cascade.

Inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate binding sites in smooth muscle.

1. We have previously demonstrated that activation of M3 muscarinic receptors increases inositol 1,4,5-trisphosphate (InsP3) and inositol 1,3,4,5-tetrakisphosphate (InsP4) accumulation in colonic smooth muscle. 2. In the present study, we demonstrate the existence of InsP3 and InsP4 binding sites in colonic circular smooth muscle by use of radioligand binding methods. Both [3H]-InsP3 and [3H]-InsP4 bound rapidly and reversibly to a single class of saturable sites in detergent-solubilized colonic membranes with affinities of 5.04 +/- 1.03 nM and 3.41 +/- 0.78 nM, respectively. The density of [3H]-InsP3 binding sites was 335.3 +/- 19.3 fmol mg-1 protein which was approximately 2.5 fold greater than that of [3H]-InsP4 sites (127.3 +/- 9.1 fmol mg-1 protein). 3. The two high affinity inositol phosphate binding sites exhibited markedly different pH optima for binding of each radioligand. At pH 9.0, specific [3H]-InsP3 binding was maximal, whereas [3H]-InsP4 binding was only 10% that of [3H]-InsP3. Conversely, at pH 5.0, [3H]-InsP4 binding was maximal, while [3H]-InsP3 binding was reduced to 15% of its binding at pH 9.0. 4. InsP3 was about 20 fold less potent (KI = 50.7 +/- 8.3 nM) than InsP4 in competing for [3H]-InsP4 binding sites and could compete for only 60% of [3H]-InsP4 specific binding. InsP4 was also capable of high affinity competition with [3H]-InsP3 binding (KI = 103.5 +/- 1.5 nM), and could compete for 100% of [3H]-InsP3 specific binding. 5. [3H]-InsP3 binding in subcellular fractions separated by discontinuous sucrose density gradients followed NADPH-cytochrome c reductase activity, suggesting an intracellular localization for the majority of InsP3 receptors in this tissue, whereas [3H]-InsP4 binding appeared to be equally distributed between plasma membrane and intracellular membrane populations.6. These results suggest the existence of distinct and specific InsP3 and InsP4 binding sites which may represent the physiological receptors for these second messengers; differences in the subcellular distribution of these receptors may contribute to differences in their putative physiological roles.