©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Kinetics of Calcium Release by Immunoaffinity-purified Inositol 1,4,5-Trisphosphate Receptor in Reconstituted Lipid Vesicles (*)

(Received for publication, February 7, 1995; and in revised form, June 6, 1995)

Junji Hirota (1) (2),  (§),   Takayuki Michikawa (1) Atsushi Miyawaki (1) Teiichi Furuichi (1) Ichiro Okura (2) Katsuhiko Mikoshiba (1) (3)

From the (1)Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, the (2)Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226, the (3)Molecular Neurobiology Laboratory, The Institute of Physical and Chemical Research (RIKEN), Tsukuba Life Science Center, 3-1-1 Koyadai, Tsukuba-shi, Ibaragi 305, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSIONS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The kinetics of inositol 1,4,5-trisphosphate (IP(3))-induced Ca release of the immunoaffinity-purified IP(3) receptor (IP(3)R), reconstituted into lipid vesicles, was investigated using the fluorescent Ca indicator fluo-3. IP(3)R was purified from mouse cerebellar microsomal fraction by using an immunoaffinity column conjugated with an anti-IP(3)R type 1 (IP(3)R1) antibody. The immunoblotting analysis using monoclonal antibodies against each IP(3)R type showed that the purified IP(3)R is almost homogeneous, composed of IP(3)R1. Ca efflux from the proteoliposomes was monitored as fluorescence changes of 10 µM fluo-3, whose concentration was high enough to buffer released Ca and to keep deviations of extravesicular free Ca concentration within 30 nM, excluding the possibility of Ca-mediated regulation of IP(3)-induced Ca release. We also examined IP(3)-induced Ca release using 1 µM fluo-3, where the deviations of free Ca concentration were within 300 nM. At both fluo-3 concentrations, IP(3)-induced Ca release showed similar kinetic properties, i.e. little Ca regulation of Ca release was observed in this system. IP(3)-induced Ca release of the purified IP(3)R exhibited positive cooperativity; the Hill coefficient was 1.8 ± 0.1. The half-maximal initial rate for Ca release occurred at 100 nM IP(3). At the submaximal concentrations of IP(3), the purified IP(3)R showed quantal Ca release, indicating that a single type of IP(3)R is capable of producing the phenomenon of quantal Ca release. The profiles of the IP(3)-induced Ca release of the purified IP(3)R were found to be biexponential with the fast and slow rate constants (k = 0.3 0.7 s, k = 0.03 0.07 s), indicating that IP(3)R has two states to release Ca. The amount of released Ca by the slow phase was constant over the range of 10-5000 nM IP(3) concentrations, whereas that by the fast phase increased in proportion to added IP(3). This provides evidence to support the view that the fast phase of Ca release is mediated by the low affinity state and the slow phase by the high affinity state of the IP(3)R. This also suggests that the fast component of Ca release is responsible for the process of quantal Ca release.


INTRODUCTION

Inositol 1,4,5-trisphosphate (IP(3)) (^1)is the second messenger derived from the hydrolysis of phosphatidylinositol bisphosphate via activation of phospholipase C, phospholipase C activity is enhanced by the activation of G protein-linked and tyrosine kinase-linked cell surface membrane receptors by various extracellular stimuli, such as hormones, growth factors, neurotransmitters, odorants, lights, etc. (1) . The IP(3) signal is converted into a Ca signal by binding to its specific receptor, i.e. the IP(3) receptor (IP(3)R), which is an IP(3)-induced Ca-releasing channel located on intracellular Ca stores such as the endoplasmic reticulum. This IP(3)-mediated Ca signaling plays a critical role in a variety of cell functions, including fertilization, cell proliferation, metabolism, secretion, contraction of smooth muscle, and neural signals(1) . For these multiple cell signaling, the mechanisms of transducing the IP(3) signal into a Ca signal, i.e. IP(3)-induced Ca release (IICR), may be diverse in each cell type.

There have been many studies on the kinetics of IICR; they describe the channel-opening mechanism of the IP(3)R(2, 3, 4, 5, 6) , the regulation of IICR by modulators such as protein kinase A(7, 8, 9) , ATP (10, 11) , GTP(12, 13) , and Ca(4, 14, 15, 16, 17, 18, 19, 20) , and the phenomenon of ``quantal Ca release'' originally described by Muallem et al.(21) , where submaximal concentrations of IP(3) cause the partial release of Ca from intracellular stores. There are often discrepancies in the properties of channel opening and the effects of modulators on IICR among the reports, because, in these studies, the arguments on some critical points of IICR have often been complicated due to the different experimental systems used, i.e. different micro-environments surrounding the IP(3)R and Ca pools assayed (e.g. different constitution of phospholipase C, IP(3)-metabolizing enzymes, Ca-binding proteins, Ca pumps, and modulators in each cell type). For example, the degree of the cooperativity of Ca release, which is an important issue for understanding the channel opening mechanism, differed among the reports. Some reports show no cooperativity of IICR(4, 15) , others show positive cooperativity (n(H) = 2)(2, 5) (n(H) = 4)(3) . In addition, recent molecular cloning studies have revealed that there are at least three types of the IP(3)R from distinct genes(22, 23, 24) . One of the major arguments on IICR derives from the fact that multiple IP(3)R types can coexist within a single cell type(25, 26) . To further characterize the channel opening mechanism, the kinetic study of IICR should be examined using a purified single type of the IP(3)R.

The cerebellum is known to be the richest source of IP(3)R type 1 (IP(3)R1) among rodent tissues tested. A recent immunohistochemical study indicated that rat cerebellum contains three IP(3)R types whose expressing cell types are quite distinct; IP(3)R1 is well known to be enriched in Purkinje cells, IP(3)R type 3 (IP(3)R3) is present in Bergmann glia and astrocytes, and IP(3)R type 2 (IP(3)R2) is also present, but not in neurons and astrocytes(27) . The differential localization of each IP(3)R type in cerebellar cell types indicates that most IP(3)Rbulletchannel complexes in the cerebellum are homotetramers within single cells. In the present study, we have purified the cerebellar IP(3)R using an immunoaffinity column coupled with an anti-IP(3)R1 antibody as described previously(9) . The population of the purified IP(3)R has been found to be almost homogeneous, containing little IP(3)R2 and IP(3)R3. Therefore the results derived from the analysis of this purified IP(3)R reflect the properties of IP(3)R1. In this study, we have investigated the kinetics of IICR mediated by the purified and reconstituted IP(3)R using the fluorescent Ca indicator fluo-3 and have defined the cooperativity, quantal Ca release, and biphasic nature of IICR.


EXPERIMENTAL PROCEDURES

Materials

The following reagents were purchased: IP(3), CHAPS, and fluo-3 from Dojindo Laboratories (Kumamoto, Japan); Chelex 100 from Bio-Rad; diethylenetriamine-N,N,N`,N",N"-pentaacetic acid-conjugated polymetal sponge from Molecular Probes; phosphatidylcholine, phosphatidylserine, and cholesterol from Avanti Polar Lipids, Inc. All of other reagents were of analytical grade or the highest grade available.

Removal of CaContamination

Removal of Ca contamination is necessary to measure the calcium release and to improve the sensitivity of the fluorometric measurements. We removed the Ca contamination according to the method of Meyer et al.(3) . Briefly, all solutions used in fluorometric measurements were passed over a polymetal sponge, and all labwares were successively washed with detergent, 0.1 N HCl, distilled water, and the buffer to be used. Ca contamination in all solutions, cuvettes, and stir bars was checked using the Ca indicator fluo-3 before the measurements. IP(3) stock solution was also passed over the polymetal sponge to remove Ca. Passing IP(3) stock solution over the polymetal sponge did not cause any changes in IP(3) concentrations, which was checked using IP(3)^3H radio receptor assay kit (DuPont NEN).

Purification of IP(3)R

IP(3)R was purified from mouse cerebellar microsomal fraction by using an immunoaffinity column conjugated with a polyclonal antibody against IP(3)R1 by the method reported previously(9) .

Monoclonal Antibody

Monoclonal antibodies, 18A10, KM1083, and KM1082 against IP(3)R type 1, IP(3)R type 2, and IP(3)R type 3, respectively, were prepared as described elsewhere(26, 28, 29) .

Reconstitution of the Immunoaffinity-purified IP(3)R into Liposomes

Phosphatidylcholine, phosphatidylserine, and cholesterol dissolved in chloroform were mixed to give a concentration of 3, 1, and 0.8 mg/ml, respectively. The lipid mixture was dried to a thin film under a stream of nitrogen gas and then under vacuum. The lipid film was suspended at 2 mg/ml in buffer A (100 mM KCl, 1 mM 2-mercaptoethanol, 10 mM HEPES-KOH (pH 7.4), and 4 mM CaCl(2)) containing 1% CHAPS. The immunoaffinity-purified IP(3)R was concentrated by using Centriprep 100 (Amicon) to give a protein concentration of 100 µg/ml. The concentrated IP(3)R solution was mixed with buffer A containing lipids and detergent to give final IP(3)R, lipids, and CHAPS of 50 µg/ml, 0.5 mg/ml, and 1%, respectively. After 20-min incubation on ice with occasional gentle stirring, the IP(3)R/lipid mixtures were dialyzed for 72 h against eight changes of a 500-fold volume excess of buffer A at 4 °C. The resulting proteoliposomes (IP(3)R in lipid vesicles) were pelleted by centrifugation at 100,000 g for 30 min at 2 °C, washed with buffer B (buffer A without Ca + 10 or 1 µM of fluo-3) twice, and resuspended with buffer B in the same volume used before dialysis. After incubation for 10 min at 25 °C, the resuspended proteoliposomes were passed over Chelex 100 to remove Ca and were used for IICR assay.

IP(3)-induced CaRelease Measurements

Ca efflux from the proteoliposomes was measured by monitoring the fluorescence changes of fluo-3. Fluorometric measurements of IICR were performed by using an F-2000 fluorometer (Hitachi, Inc.) interfaced to a PC9801-VX computer (NEC, Inc.). The excitation and emission wavelength were 500 and 525 nm, respectively, with 10 nm bandpass. Fluorescence signals were corrected for fluctuations in excitation light intensity. Measurements were made at 25 °C in a 0.5 0.5-cm quartz cuvette containing 0.4 ml of the proteoliposome solution with continuous stirring by a Teflon stir bar. IICR was monitored after addition of 2 µl of IP(3) to give the desired IP(3) concentration. The data were acquired every 200 ms. The fluorescent intensities of fluo-3 were calibrated to free Ca concentrations using Ca-EGTA buffering system(30) . The calibration curve gave the dissociation constant of fluo-3 for Ca of 170 nM, which was used to estimate the free and total Ca concentrations. To exclude the possibility of Ca regulation of IICR, we used 10 µM fluo-3, whose concentration was high enough to buffer the released Ca and to keep deviations of extravesicular free Ca concentration within 10-30 nM. We also examined IICR using 1 µM fluo-3, where the deviations of free Ca concentration were 150-300 nM, to compare the effects of changes in free Ca concentration on IICR.


RESULTS

Immunoaffinity Purification of IP(3)R

We purified the IP(3)R by the immunoaffinity method using the anti-IP(3)R1 antibody as described previously(9) . To investigate the homogeneity of the IP(3)R1, existence of IP(3)R type 2 (IP(3)R2) and type 3 (IP(3)R3) in the purified IP(3)R was analyzed by immunoblotting with monoclonal antibodies to each type of IP(3)R. The same amount of [^3H]IP(3) binding activity of cerebellar microsomal fraction and the purified IP(3)R (1.5 pmol of IP(3)R/lane) were applied to the gel, followed by immunoblotting with the monoclonal antibodies (Fig.1). The cerebellar microsomal fraction showed strong immunoreactivity with mAb 18A10 against IP(3)R1 and little with mAbs KM1083 and KM1082 against IP(3)R2 and IP(3)R3, respectively. The purified IP(3)R also showed strong immunoreactivity with mAb 18A10 and little with mAbs KM1083 and KM1082, and the contents of IP(3)R2 and IP(3)R3 in the purified receptors which might form heterotetramer with IP(3)R1 (31) were very small and decreased after the immunoaffinity purification in comparison with the cerebellar microsomal fraction. These results showed that the purified IP(3)R was chiefly composed of homotetramers of IP(3)R1. A recent immunohistochemical study also indicated that rat cerebellum contains three IP(3)R types whose expressing cell types are quite distinct; IP(3)R1 is well known to be enriched in Purkinje cells, IP(3)R type 3 (IP(3)R3) is in Bergmann glia and astrocytes, and IP(3)R type 2 (IP(3)R2) is also present, but not in neurons and astrocytes(27) . The differential localization of each IP(3)R type in cerebellar cell types indicate that most IP(3)Rbulletchannel complexes in the cerebellum are homotetramers within single cells.


Figure 1: Immunoblots of the immunoaffinity-purified IP(3)R. The purified IP(3)R was analyzed by Western blotting to investigate its homogeneity. The same amounts of [^3H]IP(3) binding activity of cerebellar microsomal fraction and the purified IP(3)R (1.5 pmol of IP(3)R/lane) were applied to the gel, followed by immunoblotting with monoclonal antibodies 18A10, KM1083, and KM1082 against IP(3)R1, IP(3)R2, and IP(3)R3, respectively. Lanes 1, 3, and 5, the solubilized cerebellar membrane fraction with 1% of CHAPS. Lanes 2, 4, and 6, the immunopurified IP(3)R. The arrow indicates the position of IP(3)R.



Reconstitution of the Immunoaffinity-purified IP(3)R

The immunoaffinity-purified IP(3)R was reconstituted into lipid vesicles by the dialysis method described previously(9) . The liposomes were observed using electron microscopy. The average diameter of the liposome was 170 ± 50 nm (n = 300), and the distribution of the size was represented in single peak (data not shown). IP(3)-induced Ca efflux from the proteoliposomes was monitored as fluorescence changes of fluo-3, whose values were used to calculate total Ca concentrations outside the proteoliposomes. The profiles of IICR were highly reproducible. Free Ca concentrations prior to addition of IP(3) were approximately 100 and 200 nM using 10 and 1 µM of fluo-3, respectively, throughout the experiments. Following the addition of maximal concentrations of IP(3), 10 µM of Ca ionophore Br-A23187 was added to estimate the fraction of liposomes with the purified IP(3)R. About 6% of the total released Ca by Br-A23187 responded to IP(3), indicating 6% of the liposome were reconstituted with the purified IP(3)R.

Time Course of IICR by the Immunoaffinity-purified IP(3)R

Fig.2shows a typical profile of IICR by the immunoaffinity-purified IP(3)R reconstituted into lipid vesicles. Five-hundred nanomolar IP(3)-induced Ca release from the liposomes followed a constant leakage of Ca (Fig.2A), which was linear over the time range of the experiments. The rate of leak from the liposomes was calculated to be about 1.5 nM/s. The net IICR (Fig.2B) was obtained by extrapolating and subtracting the constant Ca leakage (Fig.2A, the solid line) from the profile. The net IICR could not be fitted by a single exponential but was found to be a biexponential () (Fig.2C, the solid line) with the fast and slow rate constants (k = 0.51 ± 0.01 s (71 ± 1%), k = 0.042 ± 0.001 s (29 ± 1%)), indicating that the purified IP(3)R has two states for IICR.


Figure 2: Typical profile of IP(3)-induced Ca release from proteoliposomes reconstituted with the purified IP(3)R. Changes of fluorescence of the Ca indicator fluo-3 ([fluo-3] = 10 µM) were recorded after injection of IP(3) (500 nM). The total Ca concentration was estimated from the fluorescent intensity as described in the text. A, IP(3)-induced Ca release from the liposomes was followed by a constant leakage of Ca (the solid line). B, the net IICR was obtained by extrapolating and subtracting the constant Ca leakage from the profile. C, the net IICR was found to be well fitted by a biexponential (the solid line) with the fast and slow rate constants.



where T represents a total amount of released Ca, A is the amplitude of the fast and slow components (percent) (A + A = 100%), k is the rate constant (s), and t is time (s).

Kinetic Analysis of IICR

Different concentrations of IP(3) were added to obtain dose-response curves. Fig.3shows typical time courses of IICR observed using the same batch of proteoliposomes. Submaximal concentrations of IP(3) caused partial Ca releases, and rates of Ca release were dependent on the IP(3) concentration. Each profile of IICR consisted of the sum of two single exponentials as described in Fig.2C and Fig. 6.


Figure 3: Time course of IP(3)-induced Ca release following the injection of different IP(3) concentrations. IP(3)-induced Ca release at different concentrations of IP(3) was performed on a single batch of the proteoliposomes ([fluo-3] = 10 µM). 5 µM (a), 200 nM (b), 70 nM (c), 40 nM (d) and 20 nM (e) of IP(3).




Figure 6: Biexponential analysis of IP(3)-induced Ca release: IP(3) dependence of the rate constants (A and B) and the amplitudes (C and D). All profiles of IICR was found to be biexponential, with the fast and slow rate constants as described in the legend to Fig.1and in the text (). The fast (squares) and slow (circles) rate constants (A and B) and the amplitudes of the fast (squares) and slow (circles) (C and D) were plotted as a function of the concentration of IP(3). A and C were measured at 10 µM fluo-3 (values are mean ± S.D., n = 3-4; initial free Ca concentration = 100 nM; deviations of free Ca concentration by the released Ca = 10-30 nM) and B and D at 1 µM fluo-3 (values are mean ± S.D., n = 2-5; initial free Ca concentration = 200 nM; deviations of free Ca concentration by the released Ca = 150-300 nM).



Relative amounts of released Ca at various concentration of IP(3) are shown in Fig.4, A (n = 3-4) and B (n = 2-5). The amount of released Ca increased as a function of IP(3) concentration, indicating that the single type of IP(3)R is capable of producing the quantal response of Ca release.


Figure 4: The amounts of released Ca plotted as a function of IP(3) concentration. The amounts of released Ca were plotted as a function of IP(3) concentration. The data were normalized to the amplitude for 5.0 µM IP(3). A, 10 µM fluo-3 (values are mean ± S.D., n = 3-4, initial free Ca concentration = 100 nM, deviations of free Ca concentration by the released Ca = 10-30 nM). B, 1 µM fluo-3 (values are mean ± S.D., n = 2-5, initial free Ca concentration = 200 nM, deviations of free Ca concentration by the released Ca = 150-300 nM).



The initial rates of Ca release varied with IP(3) concentrations and saturated above 1 µM IP(3) at both fluo-3 concentrations of 10 µM (Fig.5A, n = 3-4; deviations of [Ca] = 10-30 nM) and 1 µM (Fig.5B, n = 2-5; deviations of [Ca] = 150-300 nM). Both half-maximal initial rates of IICR in the presence of 10 and 1 µM fluo-3 occurred at 100 nM. We determined the degree of cooperativity of IICR by Hill plotting (Fig.5C, n = 3-4 and 5D, n = 2-5). The slopes in the Hill plot over the range of submaximal concentrations of IP(3) (20-200 nM) were calculated to be 1.8 ± 0.1 (Fig.5, C and D), indicating that the IICR of the purified IP(3)R exhibited positive cooperativity. As the EC value and the Hill coefficient of IICR at both concentrations of fluo-3 were calculated to be the same, the changes of free Ca concentration by the released Ca had no significant effect on the sensitivity for IP(3) and the cooperativity of IICR.


Figure 5: Analysis of IP(3)-induced Ca release. Initial rates were measured from the initial and fast slope of IICR. A and B, normalized initial rates of Ca release were plotted as a function of the concentration of IP(3). C and D, analysis of initial rates by a Hill plot shows the positively cooperativity of IICR. A and C were measured at 10 µM fluo-3 (values are mean ± S.D., n = 3-4; initial free Ca concentration = 100 nM; deviations of free Ca concentration by the released Ca = 10-30 nM), B and D at 1 µM fluo-3 (values are mean ± S.D., n = 2-5, initial free Ca concentration = 200 nM; deviations of free Ca concentration by the released Ca = 150-300 nM).



Analysis of Biphasic Nature of IICR and Quantal CaRelease

To analyze the kinetic features of IICR in detail, we attempted to curve fit the profiles of IICR. As mentioned above, the profile of IICR could not be fitted by a single exponential but could be fitted to a biexponential with the fast and slow rate constants () at both concentrations of fluo-3. The rate constants of the fast and slow components differed by a factor of about 10 (Fig.6, A (n = 3-4) and B (n = 2-5)). Both the fast and slow rate constants were influenced by the concentration of IP(3). The amplitudes of both states (A and A) were plotted as a function of the concentrations of IP(3) (Fig.6, C (n = 3-4) and D (n = 2-5)). A increased as the concentration of IP(3) increased, whereas A decreased. Considering these amplitudes with the amount of total released Ca (Fig.4), the amounts of released Ca by the fast and slow phases were then calculated. The amounts of released Ca by the fast and slow components relative to the total released Ca at 5 µM IP(3) were plotted as a function of the concentrations of IP(3) (Fig.7, A (n = 3-4) and B (n = 2-5)). The amount of released Ca by the fast component increased as a function of the concentration of IP(3), whereas the amount by the slow component remained almost constant over the range of 10-5000 nM IP(3) at both concentrations of fluo-3. This result revealed that the fast phase of IICR, with the time constants of 0.3-0.7 s, was mainly responsible for the quantal Ca release.


Figure 7: The amounts of released Ca by the fast and slow components of IP(3)-induced Ca release. The amounts of total released Ca in Fig.3and the amplitude of the two components of IICR allowed us to calculate the amounts of released Ca by the fast (squares) and slow (circles) components. A, 10 µM fluo-3 (values are mean ± S.D., n = 3-4; initial free Ca concentration = 100 nM; deviations of free Ca concentration by the released Ca = 10-30 nM). B, 1 µM fluo-3 (values are mean ± S.D., n = 2-5; initial free Ca concentration = 200 nM; deviations of free Ca concentration by the released Ca = 150-300 nM).




DISCUSSIONS

Measurements of IP(3)-induced CaRelease by the Immunoaffinity-purified IPR

There are many reports characterizing the properties of IICR using permeabilized cells and microsomal preparations, which have been complicated by the following factors. (i) Composition of subtypes of IP(3)Rs: the presence of multiple IP(3)R types in single cells may affect the kinetics of IICR. (ii) Metabolism of IP(3): IP(3) could easily be metabolized by specific kinases and phosphatases which may be present in crude systems. The concentration of ligand during experiments is known to be one of the critical factors for IICR, since most IICR properties (multiple affinity sites on single IP(3)Rs, quantal release by submaximal doses, inactivation by IP(3) itself) are dependent on IP(3) doses. (iii) Ca pump: the activity of the ATP-driven Ca pump affects IICR by refilling Ca stores following Ca release. This prevents us from evaluating the cooperativity of IICR by reducing the net IICR to a great extent at low concentrations of IP(3) than at high concentrations(2) . (iv) Molecules sensing changes in Ca concentration: dynamic changes in cytosolic and luminal Ca concentrations have been argued to be involved in functional regulation of IICR properties by modifying the function of the IP(3)R itself and by activating IP(3)R modulator proteins (e.g. protein kinases (e.g. Ca-calmodulin-dependent protein kinase and protein kinase C) and phosphatases (e.g. calcineurin) and Ca-binding proteins (e.g. calmodulin) and IP(3)-metabolizing enzymes (e.g. IP(3) kinase)). (v) Heterogeneity in IICR-Ca pools: there is a subcellular heterogeneity in IP(3) sensitive Ca stores, e.g. subsurface cisternae, calciosomes, nuclear membranes, etc., which may have different IICR properties. Artificial effects on IP(3)-sensitive Ca stores by experimental conditions must be considered, e.g. fusion of cisternae membranes by excess treatment with saponin (32) and induction of formation of cisternal stacks mediated by IP(3)Rs by nonphysiological treatment(33) .

In this study, we have investigated the kinetics of IICR of the immunoaffinity-purified and reconstituted IP(3)R, excluding the possibility of modulation of IICR by factors other than Ca and IP(3) itself. Furthermore, as the immunoaffinity-purified IP(3)R showed very strong immunoreactivity with the monoclonal antibody against IP(3)R type 1 and little with the monoclonal antibodies against IP(3)R types 2 and 3, the population of the immunoaffinity-purified IP(3)R was almost homogeneous of IP(3)R1 but contained very small amounts of IP(3)R2 and IP(3)R3. Therefore the results derived from the analysis of this purified IP(3)R reflect mainly the properties of IP(3)R1. Due to the absence of IP(3) metabolizing enzymes, in our system, applied IP(3) doses should be constant throughout each experiment. However, we must consider the regulation of the IP(3)R by changes in free Ca concentration. Feedback regulations of IICR by the released Ca have been observed in permeabilized cells (14, 16) and microsomal systems(4, 15) . On the other hand, the high concentrations of Ca chelators and Ca indicators caused artificial effects on IICR in those experiments(18, 34) . In this study, to avoid problems concerning the regulation of IICR by the released Ca, we used high enough concentration of fluo-3 to keep extravesicular free Ca concentration within 100-130 nM. We also used 1 µM fluo-3, 200-500 nM free Ca concentration, to compare the effect of changes of extravesicular free Ca by IICR on the kinetics of Ca release. At both fluo-3 concentrations, where the extravesicular free Ca concentration changed from 100 to 130 nM (10 µM of fluo-3) and from 200 to 500 nM (1 µM of fluo-3), the kinetics of the Ca release was essentially the same, indicating little feedback regulation by the released Ca in our system. We also observed similar kinetics of Ca release at the initial extravesicular free Ca concentration of 300 nM (data not shown), where Ca release using cerebellar microsomes was shown to be inhibited(35) . Feedback regulation by the released Ca or the regulation by Ca outside of pools may be mediated by the action(s) of other molecules which can sense changes of Ca concentration.

Fundamental Properties of the Immunoaffinity-purified IP(3)R

The extent of cooperativity of Ca release is an important and fundamental issue for understanding the channel opening mechanism. In previous reports, there is a controversy about the cooperativity of IICR, i.e. no cooperativity (4, 15) or positive cooperativity (n(H) = 2) (2, 5) (n(H) = 4) (3) has been demonstrated. The Hill plots of the initial rates of Ca release by the purified IP(3)R (Fig.5, C and D) showed a positive cooperativity (n(H) = 1.8 ± 0.1) at submaximal concentrations of IP(3). This cooperativity was observed in the presence of both 10 µM and 1 M fluo-3, indicating that at least two molecules of IP(3) is needed for channel opening and that the positive cooperativity could not be due to sensitization of IICR by the rise in free Ca concentration, i.e. by the released Ca. This result shows that the positive cooperativity of IICR could be mediated by a single type of IP(3)R.

The dose-response curves of IICR mediated by the purified IP(3)R showed that the amount of released Ca increased as a function of IP(3) concentration (Fig.4) and provided evidence to suggest that the purified IP(3)R, which was chiefly composed of IP(3)R1, can exhibit the quantal response of Ca release. Ferris et al.(36) have also reported that conventionally purified and reconstituted cerebellar IP(3)Rs showed the quantal response and suggested that heterogeneity of IP(3)R types was a possible mechanism underlying quantal Ca release. However, it is likely that the quantal Ca release is an intrinsic property of IP(3)R1.

Detailed Kinetic Analysis of CaRelease

The profiles of IICR mediated by the purified IP(3)R did not obey a single exponential but were found to be biexponential with the fast and slow rate constants. The rate constants of the fast and slow components were calculated to be 0.3-0.7 and 0.03-0.07 s, respectively. We also analyzed the contribution of the fast and slow components to the total amounts of released Ca, which were estimated as described in . The amounts of released Ca by the fast component increased as a function of the concentration of IP(3), whereas those by the slow component were constant. These results suggest that the fast component is kinetically the state of low affinity for IP(3) and high permeability of Ca, and the slow component is of high affinity and low permeability. Consistent with this view, the studies of IP(3) binding in permeabilized hepatocytes and a liver plasma membrane-enriched fraction displayed the existence of two states with high and low affinity for IP(3)(37, 38) . Since our data show that the fast phase of Ca release increases with increasing IP(3) concentrations and the slow phase remains constant, it appears that the fast phase is the determinant of the amount of Ca release and is responsible for the quantal Ca release.

Recently, heterogeneity of IP(3)R densities in pools, which had equal sensitivity to IP(3), was reported to be responsible for biphasic Ca release(39) . If this is the reason for biphasic nature of IICR, the amplitudes of the fast and slow components in the curve fitting should be independent to the IP(3) concentrations, and the ratio of the amounts of released Ca by the fast and slow components must be constant. Because in such an assumption, the amplitudes and the ratio of the amounts of released Ca should reflect the distribution of such heterogeneity, i.e. proportion of IP(3)-sensitive Ca pools with high and low density of IP(3)R reflect the amplitudes and the ratio of amounts of the released Ca by the fast and slow phases, respectively. However, in our experiments, the amplitudes of the fast and slow components and the ratio of the total released Ca were dependent on IP(3) concentrations, indicating that the biphasic nature of Ca release was not due to such heterogeneity of receptor density. A possibility of heterogeneity in the size of individual Ca pools was also excluded by the same reasons and by the direct observation using electron microscopy as described under ``Results.'' The present study has demonstrated that the purified IP(3)R has two states with different affinity for IP(3), i.e. a low affinity and a high affinity state. This could arise from alternative splicing leading to the production of variants of IP(3)R1(40) . Alternatively, there may be two different states of a single IP(3)R due to an IP(3)-dependent inactivation or a Ca-dependent interconversion.

In the absence and presence of changes in extravesicular free Ca concentrations, we observed the biphasic nature of IICR, indicating that the changes in the free Ca concentration may not be responsible for the biphasic kinetics. However, we cannot rule out the possibility that the released Ca causes an instantaneous and local rise in Ca concentrations near the channel pore, which cannot be promptly chelated by fluo-3, and would mediate the interconversion of the two states. If the released Ca instantaneously rises near the channel pore, in cooperation with IP(3), the inactivation of the IP(3)R reported by Hajnoczky et al.(41) may be caused by the interconversion of IP(3)R. This hypothesis could be supported by the observations in Fig.3of (41) , where the degree of inactivation (interconversion) varied with the cytosolic free Ca concentrations during the preincubation with IP(3) and IP(3)R, whereas no significant change in IICR at various cytosolic free Ca concentrations were observed without preincubation.

We demonstrated here the positive cooperativity of IICR and the quantal Ca release phenomenon of IICR by the purified IP(3)R, which was mainly composed of a single type of IP(3)R (IP(3)R1), and the biphasic nature of IICR, which had kinetically two states to release Ca. The purification and reconstitution of other types of IP(3)Rs may reveal new insights into IICR and may allow us to relate any differences in the kinetic properties of IICR to the differences in the structure of the different types of IP(3)R. Also, this will allow us to observe the effects of modulators, such as protein kinase A, ATP, Ca, etc. on type-specific IICR.


FOOTNOTES

*
This work was supported by grants from the Japanese Ministry of Education, Science and Culture, the Japan Society of the Promotion of Science, the Intractable Diseases Research Foundation, and the Human Frontier Science Program. 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: Dept. of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan. Tel.: 81-3-5449-5320; Fax: 81-3-5449-5420.

^1
The abbreviations used are: IP(3), D-myo-inositol 1,4,5-trisphosphate; IP(3)R, IP(3) receptor; IP(3)R1, IP(3)R type 1; IP(3)R2, IP(3)R type 2; IP(3)R3, IP(3)R type 3; IICR, IP(3)-induced Ca release; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; mAb, monoclonal antibody.


ACKNOWLEDGEMENTS

We thank Drs. Michio Niinobe and Shinji Nakade for their help in purification of IP(3)R and fruitful discussions, Dr. Eisaku Katayama for electron microscopic analysis, Dr. Lee G. Sayers for critical reading of the manuscript, and all the members of Mikoshiba Laboratory for their support.


REFERENCES

  1. Berridge, M. J. (1993) Nature 361,315-325 [CrossRef][Medline] [Order article via Infotrieve]
  2. Champeil, P., Combettes, L., Berthon, B., Doucet, E., Orlowski, S., and Claret, M. (1989) J. Biol. Chem. 264,17665-17673 [Abstract/Free Full Text]
  3. Meyer, T., Wensel, T., and Stryer, L. (1990) Biochemistry 29,32-37 [Medline] [Order article via Infotrieve]
  4. Finch, E. A., Turner, T. J., and Goldin, S. M. (1991) Science 252,443-446 [Medline] [Order article via Infotrieve]
  5. Somlyo, A. V., Horiuti, K., Trentham, D. R., Kitazawa, T., and Somlyo, A. P. (1992) J. Biol. Chem. 267,22316-22322 [Abstract/Free Full Text]
  6. Kindman, L. A., and Meyer, T. (1993) Biochemistry 32,1270-1277 [Medline] [Order article via Infotrieve]
  7. Supattapone, S., Danoff, S. K., Theibert, A., Joseph, S. K., Steiner, J., and Snyder, S. H. (1988) Proc. Natl. Acad. Sci. U. S. A. 85,8747-8750 [Abstract]
  8. Hajnoczky, G., Gao, E., Nomura, T., Hoek, J. B., and Thomas, A. P. (1993) Biochem. J. 293,413-422 [Medline] [Order article via Infotrieve]
  9. Nakade, S., Rhee, S. K., Hamanaka, H., and Mikoshiba, K. (1994) J. Biol. Chem. 269,6735-6742 [Abstract/Free Full Text]
  10. Ferris, C. D., Huganir, R. L., and Snyder, S. H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87,2147-2151 [Abstract]
  11. Maeda, N., Kawasaki, T., Nakade, S., Yokota, N., Taguchi, T., Kasai, M., and Mikoshiba, K. (1991) J. Biol. Chem. 266,1109-1116 [Abstract/Free Full Text]
  12. Ghosh, T. K., Mullaney, J. M., Tarazi, F. I., and Gill, D. L. (1989) Nature 340,236-239 [Medline] [Order article via Infotrieve]
  13. Hajnoczky, G., Lin, C., and Thomas, A. P. (1994) J. Biol. Chem. 269,10280-10287 [Abstract/Free Full Text]
  14. Iino, M. (1990) J. Gen. Physiol. 95,1103-1122 [Abstract]
  15. Watras, J., Bezprozvanny, I., and Ehrlich, B. E. (1991) J. Neurosci. 11,3239-3245 [Abstract]
  16. Iino, M., and Endo, M. (1992) Nature 360,76-78 [CrossRef][Medline] [Order article via Infotrieve]
  17. Missiaen, L., De Smedt, H., Droogmans, G., and Casteels, R. (1992) J. Biol. Chem. 267,22961-22966 [Abstract/Free Full Text]
  18. Combettes, L., and Champeil, P. (1994) Science 265,813-815 [Medline] [Order article via Infotrieve]
  19. Missiaen, L., De Smedt, H., Parys, J. B., and Casteels, R. (1994) J. Biol. Chem. 269,7238-7242 [Abstract/Free Full Text]
  20. Missiaen, L., Parys, J. B., De Smedt, H., Himpens, B., and Casteels, R. (1994) Biochem. J. 300,81-84 [Medline] [Order article via Infotrieve]
  21. Muallem, S., Pandol, S. J., and Beeker, T. G. (1989) J. Biol. Chem. 264,205-212 [Abstract/Free Full Text]
  22. Furuichi, T., Yoshikawa, S., Miyawaki, A., Wada, K., Maeda, N., and Mikoshiba, K. (1989) Nature 342,32-38 [CrossRef][Medline] [Order article via Infotrieve]
  23. Sudhof, T. C., Newton, C. L., Archer, B. T., III, Ushkaryov, Y. A., and Mignery, G. A. (1991) EMBO J. 10,3199-3206 [Abstract]
  24. Blondel, O., Takeda, J., Janssen, H., Seino, S., and Bell, G. I. (1993) J. Biol. Chem. 268,11356-11363 [Abstract/Free Full Text]
  25. Sugiyama, T., Yamamoto-Hino, M., Miyawaki, A., Furuichi, T., Mikoshiba, K., and Hasegawa, M. (1994) FEBS Lett. 349,191-196 [CrossRef][Medline] [Order article via Infotrieve]
  26. Sugiyama, T., Furuya, A., Monkawa, T., Yamamoto-Hino, M., Satoh, S., Ohmori, K., Miyawaki, A., Hanai, N., Mikoshiba, K., and Hasegawa, M. (1994) FEBS Lett. 354,149-154 [CrossRef][Medline] [Order article via Infotrieve]
  27. Yamamoto-Hino, M., Miyawaki, A., Kawano, H., Sugiyama, T., Furuichi, T., Hasegawa, M., and Mikoshiba, K. (1995) Neuroreport 6,273-276 [Medline] [Order article via Infotrieve]
  28. Maeda, N., Niinobe, M., Nakahira, K., and Mikoshiba, K. (1988) J. Neurochem. 51,1724-1730 [Medline] [Order article via Infotrieve]
  29. Maeda, N., Niinobe, M., and Mikoshiba, K. (1990) EMBO J. 9,61-67 [Abstract]
  30. Tsien, R., and Pozzan, T. (1989) Methods Enzymol. 172,230-262 [Medline] [Order article via Infotrieve]
  31. Monkawa, T., Miyawaki, A., Sugiyama, T., Yoneshima, H., Yamamoto-Hino, M., Furuichi, T., Saruta, T., Hasegawa, M., and Mikoshiba, K. (1995) J. Biol. Chem. 270,14700-14704 [Abstract/Free Full Text]
  32. Renard-Rooney, D. C., Hajnoczky, G., Seitz, M. B., Schneider, T. G., and Thomas, A. P. (1993) J. Biol. Chem. 268,23601-23610 [Abstract/Free Full Text]
  33. Takei, K., Mignery, G. A., Mugnaini, E., Sudhof, T. C., and De Camilli, P. (1994) Neuron 12,327-342 [Medline] [Order article via Infotrieve]
  34. Richardson, A., and Taylor, C. W. (1993) J. Biol. Chem. 268,11528-11533 [Abstract/Free Full Text]
  35. Bezprozvanny, I., Watras, J., and Ehrlich, B. E. (1991) Nature 351,751-754 [CrossRef][Medline] [Order article via Infotrieve]
  36. Ferris, C. D., Cameron, A. M., Huganir, R. L., and Snyder, S. H. (1992) Nature 356,350-352 [CrossRef][Medline] [Order article via Infotrieve]
  37. Pietri, F., Hilly, M., and Mauger, J. P. (1990) J. Biol. Chem. 265,17478-17485 [Abstract/Free Full Text]
  38. Pietri, F., Hilly, M., Claret, M., and Mauger, J. P. (1990) Gastroenterol. Clin. Biol. 14,710-4 [Medline] [Order article via Infotrieve]
  39. Hirose, K., and Iino, M. (1994) Nature 372,791-794 [CrossRef][Medline] [Order article via Infotrieve]
  40. Nakagawa, T., Okano, H., Furuichi, T., Aruga, J., and Mikoshiba, K. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,6244-6248 [Abstract]
  41. Hajnoczky, G., and Thomas, A. P. (1994) Nature 370,474-477 [CrossRef][Medline] [Order article via Infotrieve]

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