©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Inositol 1,4,5-Trisphosphate Slowly Converts Its Receptor to a State of Higher Affinity in Sheep Cerebellum Membranes (*)

(Received for publication, April 20, 1995; and in revised form, December 4, 1995)

Jean-François Coquil (§) Jean-Pierre Mauger Michel Claret

From the Unité de Recherche U.274, INSERM, Université Paris-Sud, 91405 Orsay Cedex, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Incubation of cerebellar microsomes with D-myo-inositol 1,4,5-trisphosphate (InsP(3)) (0.01-1 µM), at 4 or 20 °C in a cytosolic-like medium devoid of Ca and Mg, followed by InsP(3) removal, induced an increase in InsP(3) binding determined with 1 nM [^3H]InsP(3). At 20 °C, and pH 7.1, maximal stimulation (1.5-2.5-fold) was obtained with 1 µM InsP(3), and the EC was 60 ± 5 nM. Several lines of evidence suggested that the activating site is identical with the InsP(3) binding site: (i) activation and binding exhibited the same inositol phosphate specificity; (ii) addition of decavanadate, a competitive inhibitor of [^3H]InsP(3) binding, to the preincubation mixture, prevented the activating effect of InsP(3); (iii) the concentration of InsP(3) giving half-maximal activation was close to that giving half-maximal InsP(3) binding. The time course of activation was found to be much slower than that of binding. While a t less than 0.4 s has been measured recently at neutral pH and 20 °C for binding of 0.5 nM [^3H]InsP(3) (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 µM InsP(3) was required to half-maximally stimulate binding. Under the present conditions, the InsP(3)-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 InsP(3) binding and InsP(3)-dependent activation, we performed binding experiments on a short period (3 s) to determine the effect of InsP(3) 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: K = 107 nM, B(max) = 28 pmol/mg of protein; after preincubation with 1 µM InsP(3): K = 53 nM, B(max) = 32 pmol/mg of protein). The two states of the receptor bound InsP(3) with a Hill coefficient close to 1 on a 3-s scale. In agreement with the effect of InsP(3) pretreatment, equilibrium binding experiments performed on 10-min incubations revealed an apparent positive cooperative behavior (apparent Hill coefficient = 1.6; apparent K = 66 nM). These results report a new regulatory process of the InsP(3) receptor in cerebellum occurring independently of Ca and on a relatively long time scale.


INTRODUCTION

The second messenger D-myo-inositol 1,4,5-trisphosphate (InsP(3)) (^1)mobilizes intracellular Ca by activating a receptor/channel located in the endoplasmic reticulum (Taylor and Richardson, 1991; Ferris and Snyder, 1992; Berridge, 1993). This process may be crucial in the genesis of repetitive calcium spikes which characterize the cellular calcium response to many stimuli (Berridge, 1993). Several genes encoding a family of InsP(3) receptors have been identified, and the molecular diversity of the receptor is amplified further by alternative splicing. In many tissues, diverse isoforms of the receptor are produced in different amounts (Südhof et al., 1991; Nakagawa et al., 1991; Ross et al., 1992; Blondel et al., 1993; De Smedt et al., 1994). In the cerebellum, most InsP(3) receptor mRNAs encode type I receptor (Furuichi et al., 1989; De Smedt et al., 1994). The high concentration of type I receptor in Purkinje cells of cerebellum, has facilitated its purification, and the receptor has been proposed to be an homotetramer (Supattapone et al., 1988; Maeda et al., 1990, 1991). Analysis of its cDNA sequence has suggested overall structural organization in three basic domains: an amino-terminal InsP(3) binding domain, a carboxyl-terminal Ca channel domain, and a linking domain containing sites for regulatory processes. This structure has been subsequently confirmed for other isoforms (Furuichi et al., 1989; Mignery and Südhof, 1990; Miyawaki et al., 1991; Südhof et al., 1991; Maranto, 1994).

Studies of InsP(3) binding and InsP(3)-induced Ca release (IICR) with permeabilized cells or diverse receptor preparations including microsomes have allowed the identification of a variety of regulatory mechanisms for the InsP(3) receptor. Ca is centrally involved in the control of IICR: submicromolar concentrations of Ca activate, whereas higher concentrations desensitize the channel (Iino, 1990; Finch et al., 1991; Bezprozvanny et al., 1991; Combettes et al., 1994). In central nervous tissue, Ca inhibits InsP(3) binding through a Ca-binding protein, calmedin (Danoff et al., 1988), whereas, in peripheral tissues, high Ca concentrations transform the receptor into an inactive form which displays a high affinity for InsP(3) (Pietri et al., 1990; Rouxel et al., 1992; Watras et al., 1994). ATP has been shown to stimulate channel activity through specific sites on the receptor (Ferris et al., 1990; Maeda et al., 1991; Bezprozvanny et al., 1991; Combettes et al., 1994). Several protein kinases, including A, C, G, and Ca/calmodulin-dependent enzymes, phosphorylate the receptor (Ferris and Snyder, 1992; Joseph and Ryan, 1993; Komalavilas and Lincoln, 1994; Koga et al., 1994). Oxidizing sulfhydryl reagents such as thimerosal have been shown to increase the sensitivity of the Ca store by increasing the affinity of the InsP(3) receptor (Missiaen et al., 1991; Renard et al., 1992; Hilly et al., 1993). In addition, recent observations suggest that InsP(3) exerts effects on its receptor different from the mere rapid opening of the Ca channel. It has been shown that IICR activation in permeabilized hepatocytes is followed by a period of inactivity dependent on the duration of exposure to InsP(3) (Hajnóczky and Thomas, 1994). Furthermore, it was also observed that preincubation with InsP(3) potentiates the stimulation by thimerosal of the Ca channel activity of the purified cerebellum receptor reconstituted in phospholipid vesicles (Kaplin et al., 1994). The present studies report that occupancy of InsP(3) binding sites in sheep cerebellum microsomes induces a time-dependent increase in the affinity of InsP(3) receptor for its ligand. In contrast with results by Hajnóczky and Thomas(1994) on permeabilized hepatocytes, this InsP(3) effect was independent of Ca, suggesting that it represents a previously undescribed process.


EXPERIMENTAL PROCEDURES

Sheep cerebellar microsomes were prepared as described previously (Hannaert-Merah et al., 1994). The microsomal preparation was resuspended in homogenization buffer (5 mM Hepes, 250 mM sucrose, 10 mM KCl, 1 mM beta-mercaptoethanol, 10 µg/ml leupeptin, 10 µM pepstatin A, 0.2 mM phenylmethylsulfonyl fluoride, pH 7.4 at 4 °C) and then frozen in liquid N(2) and stored at -80 °C. Membranes were thawed and diluted in an ice-cold cytosolic-like medium (MI) containing 110 mM KCl, 20 mM NaCl, 1 mM NaH(2)PO(4), 1 mM EDTA, 25 mM Hepes/KOH (pH 7.1), and 10 µg/ml leupeptin. Where indicated, membranes were washed and resuspended in the same medium. Preincubation of membranes (0.2-1.0 mg/ml protein) with InsP(3) (1 nM to 1 µM) was also conducted in MI, supplemented with other agents as indicated. In the first series of experiments, InsP(3) was removed by centrifuging the preincubation mixture at 36,000 times g for 1 h and washing membranes twice with ice-cold MI. A more rapid washing procedure was used in subsequent experiments. The membrane suspension preincubated with or without InsP(3) was layered onto a Whatman GF/C glass fiber filter and then washed with 10 ml of MI. The vacuum pump was adjusted to give an outflow rate of 1 ml/s, except where indicated. The binding activity of membranes was then measured directly on the filter, as described below.

[^3H]InsP(3) binding was measured by mixing microsomes with an appropriate volume of a binding medium consisting of MI supplemented with 1 nM [^3H]InsP(3), 0.1 mg/ml bovine serum albumin, and the indicated concentrations of unlabeled InsP(3). Nonspecific binding was determined in the presence of 10 µM InsP(3). Binding was performed either in a test tube or on a GF/C glass fiber filter. In the former case, the binding mixture (final volume: 0.5 ml) was incubated for 10 min at 4 or 20 °C. Four hundred microliters of the mixture were transferred onto a GF/C glass fiber filter which was then rinsed with 1 ml of an ice-cold washing medium (25 mM Hepes, 250 mM sucrose, 1 mM NaH(2)PO(4), 1 mM EDTA, pH 7.1). To measure [^3H]InsP(3) binding to membranes absorbed onto the GF/C filter, 0.5 ml of the binding medium was added to the filter, the vacuum pump being either off or running according to the desired contact time with [^3H]InsP(3). Except where indicated, the filter was shortly rinsed with 1 ml of the ice-cold washing medium. As previously reported (Rouxel et al., 1992; Hannaert-Merah et al., 1994), InsP(3) dissociates very quickly from its receptor in a cytosolic-like medium at neutral pH and 20 °C (t < 0.4 s). Therefore, the rinsing step was not performed with MI but with a Hepes buffer containing 250 mM sucrose and cooled to 4 °C. In addition, the perfusion rate of outflow was adjusted so that the washing lasted less than 0.5 s. This procedure removed almost all [^3H]InsP(3) trapped in the GF/C glass fiber filter. An adsorption of [^3H]InsP(3) to this type of filter is possible, but was prevented by the addition of 1 mM EDTA to the binding medium. Excess fluid was removed from the filter under vacuum before transfer into a counting vial. Radioactivity was measured in a scintillation counter. Total binding and nonspecific binding were determined at least in triplicate, and the results were expressed as means ± S.E. The competitive binding curves and the dose-response curve for InsP(3)-dependent binding increase were fitted according to a one-site model, using Inplot Software (GraphPAD). Kinetics of InsP(3)-dependent binding increase were fitted to a simple exponential behavior.

[^3H]InsP(3) (17-21 Ci/mmol) was purchased from DuPont NEN. Nonradioactive InsP(3) was obtained from Calbiochem. Decavanadate was prepared as described previously (Föhr et al., 1989). All other products were from Sigma or Boehringer Mannheim.


RESULTS

Preincubation of Cerebellar Microsomes with InsP(3) Increases [^3H]InsP(3) Binding

When a microsomal fraction of sheep cerebellum was preincubated with 1 µM InsP(3) in a cytosolic-like medium (MI) at 4 °C and then extensively washed by centrifugation in the same medium, its ability to subsequently bind [^3H]InsP(3) at 1 nM was doubled (Table 1). As this centrifugation procedure was very time-consuming, another method was developed to remove the initially added unlabeled InsP(3), this being washing the membranes with MI on a GF/C glass fiber filter. Following the washing step, [^3H]InsP(3) binding was directly measured on the filter by adding 0.5 ml of MI containing 1 nM [^3H]InsP(3). The InsP(3)-dependent increase in [^3H]InsP(3) binding measured with this filtration method was the same as that with the centrifugation method (Table 1). In initial studies, several characteristics of the effect of InsP(3) pretreatment were examined at 4 °C. It was found that maximal activation was achieved with 0.1-0.3 µM InsP(3) and that the activating effect of InsP(3) was much slower to develop than InsP(3) binding. While the binding of 1 nM [^3H]InsP(3) to cerebellar microsomes layered on the filter reached equilibrium within 15 s, the activation due to preincubation with 1 µM InsP(3) was only maximal after 1-2 min of preincubation at 4 °C in MI. The activation remained unchanged for at least 2 h. In subsequent experiments, the pretreatment of cerebellar membranes with InsP(3) was generally performed for 10 min. The fact that the same level of activation was measured when membranes were washed free of InsP(3) by either filtration or by centrifugation, a much longer procedure, suggests slow reversibility of the activation under these conditions. Indeed, at 4 °C, the activation persisted for days. When washed membranes were placed in MI at 37 °C instead of being stored in this medium at 4 °C, the same qualitative results were obtained; however, within 5 min, a reduction to 48% of the initial activation occurred and remained at this level for at least 40 min. The degree of reversibility was not improved by the addition of an antiprotease mixture (10 µg/ml leupeptin, 0.2 mM phenylmethylsulfonyl fluoride, 10 µM pepstatin A, 2 mM benzamidine, 1 µg/ml O-phenanthroline, 50 µg/ml trypsin inhibitor) to the preincubation medium, suggesting that the InsP(3)-induced binding increase did not involve proteolysis.



A crucial point in this type of experiment was to remove carefully the nonradioactive InsP(3) present in the preincubation mixture, as [^3H]InsP(3) binding might be reduced by residual InsP(3) leading to an underestimation of the InsP(3)-dependent activation. An experiment was therefore performed in which the volume of the washing medium was varied. When membranes were preincubated with 1 µM InsP(3), the lowest subsequent binding of [^3H]InsP(3) was observed in the absence of washing; that is, the condition for which the highest contamination of the filter with unlabeled InsP(3) was expected. Washing the filter with MI increased the binding of [^3H]InsP(3) to the membranes, the maximal binding being attained at about 3-5 ml of washing medium. Increasing the volume of the washing medium to 30 ml did not change [^3H]InsP(3) binding. When membranes were preincubated without InsP(3), washing the filter with up to 30 ml of MI had no effect on [^3H]InsP(3) binding. These results indicate that unlabeled InsP(3) carried over from the preincubation mixture could be removed from the filter easily and the InsP(3) remaining after 5 ml of washing does not reduce the subsequent [^3H]InsP(3) binding when measured with 1 nM [^3H]InsP(3). An additional experiment to determine the amount of residual InsP(3) after a washing with 10 ml of MI (the standard protocol) was performed. In this experiment, the preincubation mixture containing both cerebellar membranes and 1 µM unlabeled InsP(3) was supplemented with 10 nM [^3H]InsP(3). The results indicate that a two-thousandth of the initial [^3H]InsP(3) remained on the filter, corresponding to 1 pmol of InsP(3) (final concentration of 2 nM after addition of binding mixture on the filter). From the [^3H]InsP(3) displacement curve by nonradioactive InsP(3), we conclude that this residual InsP(3) was about 10-fold lower than the InsP(3) required to significantly reduce [^3H]InsP(3) binding measured with 1 nM [^3H]InsP(3). Thus, 10 ml of MI were adequate to wash membranes.

Specificity of the Activating Effect of InsP(3)

The data reported above show that InsP(3) is both able to bind to cerebellar microsomes and to increase, by preincubation, its own binding. Therefore, we investigated whether these two processes involved the same binding site or two distinct types of sites on the cerebellar membranes. Firstly, we used several different inositol phosphates to compare the specificity of [^3H]InsP(3) binding site with that of the site responsible for the activating effect of InsP(3). To do this, we examined the ability of inositol phosphates to inhibit the binding of [^3H]InsP(3) to cerebellar membranes by simultaneous incubation, and their ability to increase binding of [^3H]InsP(3) by preincubation with membranes. The results in Table 2show that the increase in [^3H]InsP(3) binding after preincubation with inositol phosphates is obtained with agonists of InsP(3) receptor, but by different degrees. The order of potency of these agents was the same for the two effects: Ins(1,4,5)P(3) > Ins(2,4,5)P(3) GroPIns(4,5)P(2) > Ins(1,3,4,5)P(4) > Ins(1,3,4)P(3). This specificity corresponds to that previously described for particulate or purified InsP(3) receptor preparations from cerebellum and peripheral tissues (Nahorski and Potter, 1989; Mourey et al., 1990; Südhof et al., 1991; Maeda et al., 1991; Rouxel et al., 1992).



Secondly, as heparin is a potent inhibitor of InsP(3) binding to its receptor (Taylor and Richardson, 1991), we examined whether this agent was able to block the activating effect of InsP(3). Unfortunately, pre-exposure of the cerebellar membranes to heparin led to an irreversible inhibition of InsP(3) binding activity. A similar effect was reported previously for microsomes from bovine adrenal cortex at 4 °C in InsP(3) binding assays (Guillemette et al., 1989). However, in contrast with these studies, we were unable to recover more than 30% of [^3H]InsP(3) binding by diluting and washing cerebellar microsomes at 37 °C. Therefore, we repeated the same type of experiments with decavanadate, another agent described as a competitive antagonist of InsP(3) receptor (Föhr et al., 1989; Taylor and Richardson, 1991). As illustrated in Fig. 1, A and B, 3 µM decavanadate inhibited InsP(3) binding measured with 0.1 µM [^3H]InsP(3) by 91% (A) and greatly reduced the activation by 0.1 µM InsP(3) (B). Cerebellar membranes preincubated with 3 µM decavanadate alone did not exhibit a modified binding activity. Thus, decavanadate was entirely removed by washing membranes with MI and was unable to mimic the activating effect of InsP(3).


Figure 1: Blockade by decavanadate of the increase in binding induced by InsP(3). Experiments were performed at 4 °C. Microsomes were prepared as described under ``Experimental Procedures'' and washed with ice-cold MI as indicated in Table 1. A, [^3H]InsP(3) binding was performed in a test tube by incubating microsomes for 10 min in binding medium containing 20 nCi/ml [^3H]InsP(3), 0.1 µM InsP(3), and decavanadate (DV) as specified. Bound [^3H]InsP(3) was measured by filtering 0.4 ml of the incubation mixture. Results are expressed as percent of [^3H]InsP(3) binding measured in the absence of decavanadate. B, microsomes were preincubated for 10 min in MI with or without 0.1 µM InsP(3) and 3 µM decavanadate as indicated. Five hundred-microliter aliquots of the preincubation mixtures were transferred onto GF/C glass fiber filters, membranes were washed, and InsP(3) binding was measured on a filter with 1 nM [^3H]InsP(3). Data are means of one experiment representative of two.



The Activating Effect of Preincubation with InsP(3) Occurs at 20 °C

All studies described above were performed at 4 °C, the temperature most often used in InsP(3) binding assays. However, it was important to know whether activation by InsP(3) could also occur at higher temperatures. Therefore, we then performed a series of experiments in which membrane preincubation, membrane washing, and InsP(3) binding assay were done at 20 °C. This temperature has been found to be suitable for the study of IICR (Combettes et al., 1994) and InsP(3) binding (Hannaert-Merah et al., 1994) in cerebellar microsomes. In this latter study, equilibrium for InsP(3) binding was shown to be attained more quickly at 20 °C than at 4 °C. Fig. 2exhibits the dependence of [^3H]InsP(3) binding on the concentration of InsP(3) in the preincubation mixture at 20 °C. The maximal amplitude of binding activation was obtained at about 1 µM InsP(3) and was identical with that measured at 4 °C. Stimulation of 82 ± 5% was determined in 21 experiments at 20 °C and of 83 ± 7% at 4 °C (results from Table 1). The half-maximal response was observed at 60 ± 5 nM InsP(3).


Figure 2: Effect of increasing InsP(3) concentrations in the preincubation mixture on subsequent [^3H]InsP(3) binding. Experiments were performed at 20 °C. Microsomes were preincubated for 10 min in MI in the absence or presence of InsP(3) at the specified concentration. Five hundred-microliter aliquots of the preincubation mixture were used to measure the InsP(3) binding on GF/C glass fiber filters with 1 nM [^3H]InsP(3), as described under ``Experimental Procedures'' and in Table 1. Results are expressed as percent of [^3H]InsP(3) binding of microsomes preincubated without InsP(3). Data points are means of three different experiments.



Time Course of the InsP(3)-dependent Activation

In order to compare further the characteristics of [^3H]InsP(3) binding and the activating effect of InsP(3), we determined the kinetics of activation by preincubation with 1 µM InsP(3) at 20 °C. For technical reasons, the method of InsP(3) pretreatment differed according to its duration. For the shortest times (2 and 5 s), membranes were adsorbed onto the filter and perfused with an appropriate volume of MI with or without 1 µM InsP(3). The rate of outflow through the filter was adjusted to 0.5 ml/s. For 10-s to 2-min periods, membranes were also treated with InsP(3) on the filter, except incubations were performed instead of perfusions. At the longer time of 5 min, preincubations were conducted within test tubes. In all cases, InsP(3) pretreatment was stopped by washing the membranes with 10 ml of MI. Fig. 3illustrates the results of these experiments. The maximal increase of [^3H]InsP(3) binding activity was reached within 2 min and remained unchanged up to 5 min. The experimental data points were fitted to a simple exponential with a half-time (t) of 20 s. Additional experiments at 20 °C showed that binding activation remained unchanged for at least a 20-min preincubation (data not shown). Furthermore, we observed that the activation was also completed within 1-3 min with lower InsP(3) concentrations even if the maximal extent of activation was not greater than 15%. Thus, the observed activation by InsP(3) is a much slower process than InsP(3) binding itself, the latter having a half-time shorter than 0.4 s under the same experimental conditions as employed here (Hannaert-Merah et al., 1994).


Figure 3: Time course of activation of [^3H]InsP(3) binding during preincubation of microsomes with InsP(3). The experiment was carried out at 20 °C. For 2-s and 5-s pretreatments, 1-ml aliquots of the diluted microsome preparation were layered on GF/C glass fiber filters, and adsorbed microsomes were perfused manually at 0.5 ml/s with 1 ml or 2.5 ml of MI containing 1 µM InsP(3). Pretreatments of 10 s to 2 min were performed by applying 1 ml of the InsP(3) solution onto the filters with the vacuum pump being off. Five-minute pretreatments were conducted in test tubes. After the 5-min incubation, 1 ml of preincubation mixture was transferred onto GF/C filters. In all cases, microsome pretreatment was stopped by passing 10 ml of MI through the filter. [^3H]InsP(3) binding to microsomes was measured on the filters as described under ``Experimental Procedures'' and in Table 1. Controls were performed according to the same protocols except that InsP(3) was omitted from the preincubation medium. Results are expressed as percent increase of [^3H]InsP(3) binding as compared to the corresponding control. Data points are means of one experiment representative of two.



Determination of the InsP(3) Binding Characteristic Modified by InsP(3) Pretreatment

We then examined whether the pre-exposure of microsomes to InsP(3) affected the affinity or the number of binding sites for [^3H]InsP(3). As the determination of K(D) and B(max) requires an equilibrium binding experiment with increasing InsP(3) concentrations, it was a prerequisite to measure the level of [^3H]InsP(3) binding within a sufficiently short time to avoid InsP(3)-dependent activation. This condition is satisfied in MI at 20 °C, a medium in which equilibrium for [^3H]InsP(3) binding is attained in less than 2 s (Hannaert-Merah et al., 1994). Competitive binding experiments were performed by perfusing membranes adsorbed onto the filter for periods of 2-3 s with the binding medium containing 1 nM [^3H]InsP(3) and increasing concentrations of unlabeled ligand (Fig. 4A). Nonlinear regression analysis for a one-site model gave a Hill coefficient close to 1 for membranes preincubated with or without 1 µM InsP(3) (n(H) = 1.07 and 0.95, respectively). Assuming n(H) = 1, a K(D) of 107 nM was determined for control membranes and 53 nM for InsP(3)-pretreated membranes. Corresponding B(max) values of 28 and 32 pmol/mg of protein were calculated as suggested by Swillens(1992). Therefore, it appears that, during preincubation, InsP(3) increases the affinity of the receptor without changing the number of binding sites. Scatchard plots constructed from the same data are shown in Fig. 4B.


Figure 4: Effect of pre-exposure of microsomes to InsP(3) on [^3H]InsP(3) binding parameters. A, the experiment was carried out at 20 °C. Microsomes were preincubated for 10 min in MI with (bullet) or without (circle) 1 µM InsP(3). One-ml aliquots of the preincubation mixtures were layered onto GF/C glass fiber filters. Adsorbed microsomes were washed with 10 ml of MI and then perfused for 3 s with 0.5 ml of binding mixture containing 1 nM [^3H]InsP(3) (20 nCi/ml) and the indicated unlabeled InsP(3) concentrations. The filters were not rinsed with ice-cold Hepes/sucrose buffer to remove free ligand; however, the data were corrected for nonspecific binding determined with 10 µM InsP(3). Results are expressed as percent of [^3H]InsP(3) binding determined for control membranes without unlabeled InsP(3). Simulated curves were constructed for a one-site model and n(H) = 1. B, Scatchard plots obtained from the same data as in A. Data points are means of one experiment representative of two.



From the data reported above, we anticipated that measurement of [^3H]InsP(3) binding in the presence of increasing InsP(3) concentrations on a time long enough to allow activation by InsP(3) should express positive cooperativity. As illustrated by the squares in Fig. 5, binding values (B) measured at InsP(3) concentrations, shown previously to trigger the activation process during preincubation, were higher than that determined with 1 nM [^3H]InsP(3) alone (B(0)). These results proved such positive cooperativity directly. When transformed into a direct coordinate system and fitted to the Hill equation, half-maximal binding was calculated to be 66 nM InsP(3) and the apparent Hill coefficient to be 1.6 (data not shown). In 5 different experiments performed at 4 or 20 °C, we measured a maximal B/B(0) value of 1.31 ± 0.06.


Figure 5: [^3H]InsP(3) displacement curve by unlabeled InsP(3) obtained from 10-min incubations. Comparison with displacement curves obtained from 3-s incubations of microsomes pretreated with or without 1 µM InsP(3). Experiments were performed at 20 °C. For 10-min incubations (), cerebellar microsomes were added to a binding medium (final volume, 0.5 ml), consisting of MI supplemented with 1 nM [^3H]InsP(3) and increasing concentrations of unlabeled InsP(3). Four hundred microliters of the incubation mixture was then transferred on GF/C glass fiber filters to measure the amount of bound [^3H]InsP(3). Results are expressed as percent of [^3H]InsP(3) binding determined without the addition of unlabeled ligand. Displacement curves obtained from 3-s perfusions of microsomes pretreated for 10 min with (solid line) or without (dashed line) InsP(3) were determined from data presented in Fig. 4. Data points are means of one representative experiment.




DISCUSSION

The present studies show that in sheep cerebellar microsomes the affinity for InsP(3) of its receptor was markedly increased during exposure to InsP(3) over a period of 2 s to 2 min. As the total number of InsP(3) binding sites was not changed by this treatment, it appears that these sites were converted to a state of higher affinity. Several lines of evidence indicate that the activating effect of InsP(3) resulted from an interaction of InsP(3) with the same receptor. Firstly, the selectivity of the activating site was the same as that of the [^3H]InsP(3) binding site and that previously reported for the InsP(3) receptor in cerebellum from other species and peripheral tissues (Nahorski and Potter, 1989; Mourey et al., 1990; Südhof et al., 1991; Maeda et al., 1991; Rouxel et al., 1992). Secondly, decavanadate, a competitive inhibitor of InsP(3) receptor (Föhr et al., 1989; Taylor and Richardson, 1991), prevented the activation by InsP(3). Thirdly, the EC value for activation at 20 °C (60 nM) was close to the apparent K(D) value determined for [^3H]InsP(3) binding in a 10-min incubation (66 nM). Whereas the sites for activation and binding appear to be identical, the former effect developed much more slowly than the latter. We conclude from these observations that the activation process results from prolonged occupancy of the InsP(3) binding site.

Most of the experiments in the present work were performed with a 10-min preincubation of cerebellar microsomal membranes with nonradioactive InsP(3), followed by washing and determination of [^3H]InsP(3) binding after a short incubation period. The resulting increase of affinity observed in these two-step experiments was confirmed in binding measurements performed after longer (10 min) incubation periods, which revealed an apparent positive cooperative behavior (Fig. 5) and hence also indicate that InsP(3) stimulates its own binding. Consistent with the slow activation by preincubation with InsP(3) (Fig. 3), this behavior was not observed in experiments in which this incubation was only 2-3 s at 20 °C, as indicated by the Hill coefficient value close to 1 determined with untreated membranes (Fig. 4, A and B). Considered together, these characteristics of InsP(3) binding to its cerebellar receptor are reminiscent of properties of a hysteretic protein for which the apparent cooperative behavior results from slow conformational transition upon binding of its ligand. Positive cooperative behavior has not been reported previously for InsP(3) binding to cerebellum receptor. However, it has been proposed that upon interaction with its binding site, InsP(3) elicits a large conformational change in its receptor (Mignery and Südhof, 1990). Recently, this conformational change has been suggested to alter accessibility of thimerosal to certain sulfhydryl groups (Kaplin et al., 1994).

In peripheral tissues, elevation of cytosolic Ca above its resting concentration (100-200 nM) increases the affinity of the InsP(3) receptor for its ligand (Hilly et al., 1993; Marshall and Taylor, 1993) by reducing the dissociation rate constant (Hilly et al., 1993). When the free Ca concentration reaches 0.5-1 µM, these receptors are converted into a high affinity inactive state characterized by low rates of association and dissociation of InsP(3) (Pietri et al., 1990; Hilly et al., 1993; Watras et al., 1994). In contrast, the InsP(3)-dependent conversion of sheep cerebellum InsP(3) receptor to a higher affinity state was observed in the presence of 1 mM EDTA, that is, at nanomolar free Ca concentrations. In this tissue, Ca inhibits InsP(3) binding (Worley et al., 1987; Hannaert-Merah et al., 1994), an effect which has been proposed to be mediated by the Ca-binding protein, calmedin (Danoff et al., 1988). However, the InsP(3) activating effect cannot be due to removal of inhibitory influence by calmedin, since 1 mM EDTA has been reported to prevent and reverse the inhibition by this protein (Worley et al., 1987; Joseph et al., 1989). Furthermore, we found that sheep cerebellar microsomes preincubated with free Ca concentrations up to 100 µM and then washed with 10 ml of MI containing 1 mM EDTA did not exhibit a lower [^3H]InsP(3) binding as compared with membranes exposed to nanomolar Ca concentrations (data not shown). Therefore, we conclude that the activation by InsP(3) occurs independently of Ca. However, this does not preclude any regulatory influence of Ca on this process.

The ability of InsP(3) to increase its own binding in microsomes washed with MI, indicates that molecules involved in this process are tightly associated with membranes. Several membrane-associated proteins have been proposed to interact with the cerebellum InsP(3) receptor, including ankyrin (Joseph and Samanta, 1993; Bourguignon et al., 1993) and calmedin which, however, is not involved as discussed above. Another possibility is that the transition induced by InsP(3) involves changes in interaction between the InsP(3) receptor subunits. Evidence for an association between subunits of InsP(3) receptors of adjacent cisternae of smooth endoplasmic reticulum has been obtained in immunocytological studies of Purkinje cells (Satoh et al., 1990; Otsu et al., 1990; Villa et al., 1991; Takei et al., 1992, 1994). Alternatively, it may be possible that the activating effect of InsP(3) might involve a covalent modification of the InsP(3) receptor, e.g. a change in the phosphorylation state. Cerebellum InsP(3) receptor has been shown to be phosphorylated by several protein kinases (Ferris and Snyder, 1992; Koga et al., 1994).

A major characteristic of the affinity increase of the cerebellum InsP(3) receptor following InsP(3) pretreatment is the slowness of its kinetics (t = 20 s; Fig. 3) as compared with that of the InsP(3) binding (see above) and IICR (t = 0.2 s with 0.15 µM InsP(3); Combettes et al.(1994)). This difference implies that the increase in InsP(3) receptor affinity, resulting from InsP(3) binding, occurs after Ca efflux has been completed and thus affects subsequent events only. Recently, it has been reported that pre-exposure of permeabilized hepatocytes to InsP(3) is followed by a time-dependent inactivation of IICR (Hajnóczky and Thomas, 1994). The time course for [^3H]InsP(3) binding activation in the present studies (t = 20 s, Fig. 3) is very similar to that of the InsP(3)-induced inactivation described by Hajnóczky and Thomas (t = 15 s). However, in contrast to our results on cerebellar microsomes, inactivation by InsP(3) in permeabilized hepatocytes was dependent on the presence of Ca and was accelerated by increasing the Ca concentration up to 1 µM. With respect to the effect of InsP(3) pretreatment of cerebellar microsomes on the function of the InsP(3) receptor (IICR), further studies will be required. The slow kinetics of the increase in InsP(3) affinity suggests that it will be dependent on a prolonged increase in the level of InsP(3) in intact cells, and that, therefore, it reflects a long-term regulation process. The same effect might also be attained with repetitive increases in the cellular level of InsP(3) if the InsP(3) binding activation is slowly reversible in intact cells, as suggested by the present in vitro conditions. Interestingly, such a situation might be encountered in long-term potentiation and long-term depression, two important models of synaptic plasticity, induced by tetanic and/or repetitive or prolonged stimulation of synapses (Madison et al., 1991; Bliss and Collingridge, 1993; Artola and Singer, 1993). In cerebellum, long term depression is well known to occur at the parallel fiber-Purkinje cell synapses (Ito, 1989; Daniel et al., 1992; Conquet et al., 1994). Evidence has been obtained for involvement of the InsP(3)/Ca signaling system in these processes (Kato, 1993; Kasai and Petersen, 1994). Many different cell types respond by repetitive Ca spikes to sustained application of agonists acting through InsP(3). Characteristically, the agonist-stimulated accumulation of InsP(3) consists in a rapid peak followed by a much lower but sustained phase (Willars and Nahorski, 1995). We hypothesize that such a pattern of InsP(3) accumulation may lead to an increase in the affinity of the InsP(3) receptor for InsP(3) and therefore facilitate the generation of Ca oscillations while InsP(3) level is increased only slightly. Repetitive Ca spikes have been observed at basal InsP(3) concentration following intracellular injection of thimerosal, a thiol alkylating agent which sensitizes the Ca stores to InsP(3) (Bootman et al., 1992), by increasing the affinity of the InsP(3) receptor (Hilly et al., 1993). Consistent with our hypothesis, studies in intact cells have shown that InsP(3)-dependent Ca mobilization may also be sensitized by a prior InsP(3) injection or agonist activation (Parker and Miledi, 1989).

In summary, the present studies show that in sheep cerebellar microsomes a prolonged exposure of InsP(3) to its receptor, converts this protein to a state exhibiting higher affinity. This phenomenon indicates that, upon binding, InsP(3) not only opens the Ca channel (a rapid process) but also initiates a slower regulation of the protein.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

(^1)
The abbreviations used are: InsP(3), D-myo-inositol-1,4,5-trisphosphate; IICR, InsP(3)-induced Ca release.


ACKNOWLEDGEMENTS

We thank J. Simon for excellent technical help, D. Reuter for expert secretarial assistance, and Drs. P. Champeil and F. Crépel for helpful discussions and critical reading of the manuscript.


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