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Kinetics of Calcium Release by Immunoaffinity-purified Inositol 1,4,5-Trisphosphate Receptor in Reconstituted Lipid Vesicles *

  • Junji Hirota
    Correspondence
    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
    Affiliations
    From the Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108

    Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226
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  • Takayuki Michikawa
    Affiliations
    From the Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108
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  • Atsushi Miyawaki
    Affiliations
    From the Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108
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  • Teiichi Furuichi
    Affiliations
    From the Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108
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  • Ichiro Okura
    Affiliations
    Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226
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  • Katsuhiko Mikoshiba
    Affiliations
    From the Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108

    Molecular Neurobiology Laboratory, The Institute of Physical and Chemical Research (RIKEN), Tsukuba Life Science Center, 3-1-1 Koyadai, Tsukuba-shi, Ibaragi 305, Japan
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  • Author 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.
Open AccessPublished:August 11, 1995DOI:https://doi.org/10.1074/jbc.270.32.19046
      The kinetics of inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release of the immunoaffinity-purified IP3 receptor (IP3R), reconstituted into lipid vesicles, was investigated using the fluorescent Ca2+ indicator fluo-3. IP3R was purified from mouse cerebellar microsomal fraction by using an immunoaffinity column conjugated with an anti-IP3R type 1 (IP3R1) antibody. The immunoblotting analysis using monoclonal antibodies against each IP3R type showed that the purified IP3R is almost homogeneous, composed of IP3R1. Ca2+ efflux from the proteoliposomes was monitored as fluorescence changes of 10 μM fluo-3, whose concentration was high enough to buffer released Ca2+ and to keep deviations of extravesicular free Ca2+ concentration within 30 nM, excluding the possibility of Ca2+-mediated regulation of IP3-induced Ca2+ release. We also examined IP3-induced Ca2+ release using 1 μM fluo-3, where the deviations of free Ca2+ concentration were within 300 nM. At both fluo-3 concentrations, IP3-induced Ca2+ release showed similar kinetic properties, i.e. little Ca2+ regulation of Ca2+ release was observed in this system. IP3-induced Ca2+ release of the purified IP3R exhibited positive cooperativity; the Hill coefficient was 1.8 ± 0.1. The half-maximal initial rate for Ca2+ release occurred at 100 nM IP3. At the submaximal concentrations of IP3, the purified IP3R showed quantal Ca2+ release, indicating that a single type of IP3R is capable of producing the phenomenon of quantal Ca2+ release. The profiles of the IP3-induced Ca2+ release of the purified IP3R were found to be biexponential with the fast and slow rate constants (kfast = 0.3 ∼ 0.7 s−1, kslow = 0.03 ∼ 0.07 s−1), indicating that IP3R has two states to release Ca2+. The amount of released Ca2+ by the slow phase was constant over the range of 10-5000 nM IP3 concentrations, whereas that by the fast phase increased in proportion to added IP3. This provides evidence to support the view that the fast phase of Ca2+ release is mediated by the low affinity state and the slow phase by the high affinity state of the IP3R. This also suggests that the fast component of Ca2+ release is responsible for the process of quantal Ca2+ release.
      Inositol 1,4,5-trisphosphate (IP3)
      The abbreviations used are: IP3
      D-myo-inositol 1,4,5-trisphosphate
      IP3R
      IP3 receptor
      IP3R1
      IP3R type 1
      IP3R2
      IP3R type 2
      IP3R3
      IP3R type 3
      IICR
      IP3-induced Ca2+ release
      CHAPS
      3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
      mAb
      monoclonal antibody.
      1The abbreviations used are: IP3
      D-myo-inositol 1,4,5-trisphosphate
      IP3R
      IP3 receptor
      IP3R1
      IP3R type 1
      IP3R2
      IP3R type 2
      IP3R3
      IP3R type 3
      IICR
      IP3-induced Ca2+ release
      CHAPS
      3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
      mAb
      monoclonal antibody.
      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. (
      • Berridge M.J.
      ). The IP3 signal is converted into a Ca2+ signal by binding to its specific receptor, i.e. the IP3 receptor (IP3R), which is an IP3-induced Ca2+-releasing channel located on intracellular Ca2+ stores such as the endoplasmic reticulum. This IP3-mediated Ca2+ signaling plays a critical role in a variety of cell functions, including fertilization, cell proliferation, metabolism, secretion, contraction of smooth muscle, and neural signals(
      • Berridge M.J.
      ). For these multiple cell signaling, the mechanisms of transducing the IP3 signal into a Ca2+ signal, i.e. IP3-induced Ca2+ 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 IP3R(
      • Champeil P.
      • Combettes L.
      • Berthon B.
      • Doucet E.
      • Orlowski S.
      • Claret M.
      ,
      • Meyer T.
      • Wensel T.
      • Stryer L.
      ,
      • Finch E.A.
      • Turner T.J.
      • Goldin S.M.
      ,
      • Somlyo A.V.
      • Horiuti K.
      • Trentham D.R.
      • Kitazawa T.
      • Somlyo A.P.
      ,
      • Kindman L.A.
      • Meyer T.
      ), the regulation of IICR by modulators such as protein kinase A(
      • Supattapone S.
      • Danoff S.K.
      • Theibert A.
      • Joseph S.K.
      • Steiner J.
      • Snyder S.H.
      ,
      • Hajnoczky G.
      • Gao E.
      • Nomura T.
      • Hoek J.B.
      • Thomas A.P.
      ,
      • Nakade S.
      • Rhee S.K.
      • Hamanaka H.
      • Mikoshiba K.
      ), ATP (
      • Ferris C.D.
      • Huganir R.L.
      • Snyder S.H.
      ,
      • Maeda N.
      • Kawasaki T.
      • Nakade S.
      • Yokota N.
      • Taguchi T.
      • Kasai M.
      • Mikoshiba K.
      ), GTP(
      • Ghosh T.K.
      • Mullaney J.M.
      • Tarazi F.I.
      • Gill D.L.
      ,
      • Hajnoczky G.
      • Lin C.
      • Thomas A.P.
      ), and Ca2+(
      • Finch E.A.
      • Turner T.J.
      • Goldin S.M.
      ,
      • Iino M.
      ,
      • Watras J.
      • Bezprozvanny I.
      • Ehrlich B.E.
      ,
      • Iino M.
      • Endo M.
      ,
      • Missiaen L.
      • De Smedt H.
      • Droogmans G.
      • Casteels R.
      ,
      • Combettes L.
      • Champeil P.
      ,
      • Missiaen L.
      • De Smedt H.
      • Parys J.B.
      • Casteels R.
      ,
      • Missiaen L.
      • Parys J.B.
      • De Smedt H.
      • Himpens B.
      • Casteels R.
      ), and the phenomenon of “quantal Ca2+ release” originally described by Muallem et al.(
      • Muallem S.
      • Pandol S.J.
      • Beeker T.G.
      ), where submaximal concentrations of IP3 cause the partial release of Ca2+ 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 IP3R and Ca2+ pools assayed (e.g. different constitution of phospholipase C, IP3-metabolizing enzymes, Ca2+-binding proteins, Ca2+ pumps, and modulators in each cell type). For example, the degree of the cooperativity of Ca2+ release, which is an important issue for understanding the channel opening mechanism, differed among the reports. Some reports show no cooperativity of IICR(
      • Finch E.A.
      • Turner T.J.
      • Goldin S.M.
      ,
      • Watras J.
      • Bezprozvanny I.
      • Ehrlich B.E.
      ), others show positive cooperativity (nH = 2)(
      • Champeil P.
      • Combettes L.
      • Berthon B.
      • Doucet E.
      • Orlowski S.
      • Claret M.
      ,
      • Somlyo A.V.
      • Horiuti K.
      • Trentham D.R.
      • Kitazawa T.
      • Somlyo A.P.
      ) (nH = 4)(
      • Meyer T.
      • Wensel T.
      • Stryer L.
      ). In addition, recent molecular cloning studies have revealed that there are at least three types of the IP3R from distinct genes(
      • Furuichi T.
      • Yoshikawa S.
      • Miyawaki A.
      • Wada K.
      • Maeda N.
      • Mikoshiba K.
      ,
      • Sudhof T.C.
      • Newton C.L.
      • Archer III, B.T.
      • Ushkaryov Y.A.
      • Mignery G.A.
      ,
      • Blondel O.
      • Takeda J.
      • Janssen H.
      • Seino S.
      • Bell G.I.
      ). One of the major arguments on IICR derives from the fact that multiple IP3R types can coexist within a single cell type(
      • Sugiyama T.
      • Yamamoto-Hino M.
      • Miyawaki A.
      • Furuichi T.
      • Mikoshiba K.
      • Hasegawa M.
      ,
      • Sugiyama T.
      • Furuya A.
      • Monkawa T.
      • Yamamoto-Hino M.
      • Satoh S.
      • Ohmori K.
      • Miyawaki A.
      • Hanai N.
      • Mikoshiba K.
      • Hasegawa M.
      ). To further characterize the channel opening mechanism, the kinetic study of IICR should be examined using a purified single type of the IP3R.
      The cerebellum is known to be the richest source of IP3R type 1 (IP3R1) among rodent tissues tested. A recent immunohistochemical study indicated that rat cerebellum contains three IP3R types whose expressing cell types are quite distinct; IP3R1 is well known to be enriched in Purkinje cells, IP3R type 3 (IP3R3) is present in Bergmann glia and astrocytes, and IP3R type 2 (IP3R2) is also present, but not in neurons and astrocytes(
      • Yamamoto-Hino M.
      • Miyawaki A.
      • Kawano H.
      • Sugiyama T.
      • Furuichi T.
      • Hasegawa M.
      • Mikoshiba K.
      ). The differential localization of each IP3R type in cerebellar cell types indicates that most IP3R•channel complexes in the cerebellum are homotetramers within single cells. In the present study, we have purified the cerebellar IP3R using an immunoaffinity column coupled with an anti-IP3R1 antibody as described previously(
      • Nakade S.
      • Rhee S.K.
      • Hamanaka H.
      • Mikoshiba K.
      ). The population of the purified IP3R has been found to be almost homogeneous, containing little IP3R2 and IP3R3. Therefore the results derived from the analysis of this purified IP3R reflect the properties of IP3R1. In this study, we have investigated the kinetics of IICR mediated by the purified and reconstituted IP3R using the fluorescent Ca2+ indicator fluo-3 and have defined the cooperativity, quantal Ca2+ release, and biphasic nature of IICR.

      EXPERIMENTAL PROCEDURES

      Materials

      The following reagents were purchased: IP3, 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 Ca2+Contamination

      Removal of Ca2+ contamination is necessary to measure the calcium release and to improve the sensitivity of the fluorometric measurements. We removed the Ca2+ contamination according to the method of Meyer et al.(
      • Meyer T.
      • Wensel T.
      • Stryer L.
      ). 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. Ca2+ contamination in all solutions, cuvettes, and stir bars was checked using the Ca2+ indicator fluo-3 before the measurements. IP3 stock solution was also passed over the polymetal sponge to remove Ca2+. Passing IP3 stock solution over the polymetal sponge did not cause any changes in IP3 concentrations, which was checked using IP33H radio receptor assay kit (DuPont NEN).

      Purification of IP3R

      IP3R was purified from mouse cerebellar microsomal fraction by using an immunoaffinity column conjugated with a polyclonal antibody against IP3R1 by the method reported previously(
      • Nakade S.
      • Rhee S.K.
      • Hamanaka H.
      • Mikoshiba K.
      ).

      Monoclonal Antibody

      Monoclonal antibodies, 18A10, KM1083, and KM1082 against IP3R type 1, IP3R type 2, and IP3R type 3, respectively, were prepared as described elsewhere(
      • Sugiyama T.
      • Furuya A.
      • Monkawa T.
      • Yamamoto-Hino M.
      • Satoh S.
      • Ohmori K.
      • Miyawaki A.
      • Hanai N.
      • Mikoshiba K.
      • Hasegawa M.
      ,
      • Maeda N.
      • Niinobe M.
      • Nakahira K.
      • Mikoshiba K.
      ,
      • Maeda N.
      • Niinobe M.
      • Mikoshiba K.
      ).

      Reconstitution of the Immunoaffinity-purified IP3R 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 CaCl2) containing 1% CHAPS. The immunoaffinity-purified IP3R was concentrated by using Centriprep 100 (Amicon) to give a protein concentration of 100 μg/ml. The concentrated IP3R solution was mixed with buffer A containing lipids and detergent to give final IP3R, 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 IP3R/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 (IP3R in lipid vesicles) were pelleted by centrifugation at 100,000 × g for 30 min at 2°C, washed with buffer B (buffer A without Ca2++ 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 Ca2+ and were used for IICR assay.

      IP3-induced Ca2+Release Measurements

      Ca2+ 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 IP3 to give the desired IP3 concentration. The data were acquired every 200 ms. The fluorescent intensities of fluo-3 were calibrated to free Ca2+ concentrations using Ca2+-EGTA buffering system(
      • Tsien R.
      • Pozzan T.
      ). The calibration curve gave the dissociation constant of fluo-3 for Ca2+ of 170 nM, which was used to estimate the free and total Ca2+ concentrations. To exclude the possibility of Ca2+ regulation of IICR, we used 10 μM fluo-3, whose concentration was high enough to buffer the released Ca2+ and to keep deviations of extravesicular free Ca2+ concentration within 10-30 nM. We also examined IICR using 1 μM fluo-3, where the deviations of free Ca2+ concentration were 150-300 nM, to compare the effects of changes in free Ca2+ concentration on IICR.

      RESULTS

      Immunoaffinity Purification of IP3R

      We purified the IP3R by the immunoaffinity method using the anti-IP3R1 antibody as described previously(
      • Nakade S.
      • Rhee S.K.
      • Hamanaka H.
      • Mikoshiba K.
      ). To investigate the homogeneity of the IP3R1, existence of IP3R type 2 (IP3R2) and type 3 (IP3R3) in the purified IP3R was analyzed by immunoblotting with monoclonal antibodies to each type of IP3R. The same amount of [3H]IP3 binding activity of cerebellar microsomal fraction and the purified IP3R (1.5 pmol of IP3R/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 IP3R1 and little with mAbs KM1083 and KM1082 against IP3R2 and IP3R3, respectively. The purified IP3R also showed strong immunoreactivity with mAb 18A10 and little with mAbs KM1083 and KM1082, and the contents of IP3R2 and IP3R3 in the purified receptors which might form heterotetramer with IP3R1 (
      • Monkawa T.
      • Miyawaki A.
      • Sugiyama T.
      • Yoneshima H.
      • Yamamoto-Hino M.
      • Furuichi T.
      • Saruta T.
      • Hasegawa M.
      • Mikoshiba K.
      ) were very small and decreased after the immunoaffinity purification in comparison with the cerebellar microsomal fraction. These results showed that the purified IP3R was chiefly composed of homotetramers of IP3R1. A recent immunohistochemical study also indicated that rat cerebellum contains three IP3R types whose expressing cell types are quite distinct; IP3R1 is well known to be enriched in Purkinje cells, IP3R type 3 (IP3R3) is in Bergmann glia and astrocytes, and IP3R type 2 (IP3R2) is also present, but not in neurons and astrocytes(
      • Yamamoto-Hino M.
      • Miyawaki A.
      • Kawano H.
      • Sugiyama T.
      • Furuichi T.
      • Hasegawa M.
      • Mikoshiba K.
      ). The differential localization of each IP3R type in cerebellar cell types indicate that most IP3R•channel complexes in the cerebellum are homotetramers within single cells.
      Figure thumbnail gr1
      Figure 1:Immunoblots of the immunoaffinity-purified IP3R. The purified IP3R was analyzed by Western blotting to investigate its homogeneity. The same amounts of [3H]IP3 binding activity of cerebellar microsomal fraction and the purified IP3R (1.5 pmol of IP3R/lane) were applied to the gel, followed by immunoblotting with monoclonal antibodies 18A10, KM1083, and KM1082 against IP3R1, IP3R2, and IP3R3, respectively. Lanes 1, 3, and 5, the solubilized cerebellar membrane fraction with 1% of CHAPS. Lanes 2, 4, and 6, the immunopurified IP3R. The arrow indicates the position of IP3R.

      Reconstitution of the Immunoaffinity-purified IP3R

      The immunoaffinity-purified IP3R was reconstituted into lipid vesicles by the dialysis method described previously(
      • Nakade S.
      • Rhee S.K.
      • Hamanaka H.
      • Mikoshiba K.
      ). 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). IP3-induced Ca2+ efflux from the proteoliposomes was monitored as fluorescence changes of fluo-3, whose values were used to calculate total Ca2+ concentrations outside the proteoliposomes. The profiles of IICR were highly reproducible. Free Ca2+ concentrations prior to addition of IP3 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 IP3, 10 μM of Ca2+ ionophore Br-A23187 was added to estimate the fraction of liposomes with the purified IP3R. About 6% of the total released Ca2+ by Br-A23187 responded to IP3, indicating 6% of the liposome were reconstituted with the purified IP3R.

      Time Course of IICR by the Immunoaffinity-purified IP3R

      Fig. 2 shows a typical profile of IICR by the immunoaffinity-purified IP3R reconstituted into lipid vesicles. Five-hundred nanomolar IP3-induced Ca2+ release from the liposomes followed a constant leakage of Ca2+ (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 Ca2+ 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 (Equation 1) (Fig. 2C, the solid line) with the fast and slow rate constants (kfast = 0.51 ± 0.01 s−1 (71 ± 1%), kslow = 0.042 ± 0.001 s−1 (29 ± 1%)), indicating that the purified IP3R has two states for IICR.
      Δ[Ca2+]total=T(1AfastekfasttAslowekslowt)
      (Eq. 1)


      Figure thumbnail gr2
      Figure 2:Typical profile of IP3-induced Ca2+ release from proteoliposomes reconstituted with the purified IP3R. Changes of fluorescence of the Ca2+ indicator fluo-3 ([fluo-3] = 10 μM) were recorded after injection of IP3 (500 nM). The total Ca2+ concentration was estimated from the fluorescent intensity as described in the text. A, IP3-induced Ca2+ release from the liposomes was followed by a constant leakage of Ca2+ (the solid line). B, the net IICR was obtained by extrapolating and subtracting the constant Ca2+ 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 Ca2+, A is the amplitude of the fast and slow components (percent) (Afast+ Aslow = 100%), k is the rate constant (s−1), and t is time (s).

      Kinetic Analysis of IICR

      Different concentrations of IP3 were added to obtain dose-response curves. Fig. 3 shows typical time courses of IICR observed using the same batch of proteoliposomes. Submaximal concentrations of IP3 caused partial Ca2+ releases, and rates of Ca2+ release were dependent on the IP3 concentration. Each profile of IICR consisted of the sum of two single exponentials as described in Fig. 2C and Fig. 6.
      Figure thumbnail gr3
      Figure 3:Time course of IP3-induced Ca2+ release following the injection of different IP3 concentrations. IP3-induced Ca2+ release at different concentrations of IP3 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 IP3.
      Figure thumbnail gr6
      Figure 6:Biexponential analysis of IP3-induced Ca2+ release: IP3 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 and in the text (Equation 1). 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 IP3. A and C were measured at 10 μM fluo-3 (values are mean ± S.D., n = 3-4; initial free Ca2+ concentration = 100 nM; deviations of free Ca2+ concentration by the released Ca2+ = 10-30 nM) and B and D at 1 μM fluo-3 (values are mean ± S.D., n = 2-5; initial free Ca2+ concentration = 200 nM; deviations of free Ca2+ concentration by the released Ca2+ = 150-300 nM).
      Relative amounts of released Ca2+ at various concentration of IP3 are shown in Fig. 4, A (n = 3-4) and B (n = 2-5). The amount of released Ca2+ increased as a function of IP3 concentration, indicating that the single type of IP3R is capable of producing the quantal response of Ca2+ release.
      Figure thumbnail gr4
      Figure 4:The amounts of released Ca2+ plotted as a function of IP3 concentration. The amounts of released Ca2+ were plotted as a function of IP3 concentration. The data were normalized to the amplitude for 5.0 μM IP3. A, 10 μM fluo-3 (values are mean ± S.D., n = 3-4, initial free Ca2+ concentration = 100 nM, deviations of free Ca2+ concentration by the released Ca2+ = 10-30 nM). B, 1 μM fluo-3 (values are mean ± S.D., n = 2-5, initial free Ca2+ concentration = 200 nM, deviations of free Ca2+ concentration by the released Ca2+ = 150-300 nM).
      The initial rates of Ca2+ release varied with IP3 concentrations and saturated above 1 μM IP3 at both fluo-3 concentrations of 10 μM (Fig. 5A, n = 3-4; deviations of [Ca2+]free = 10-30 nM) and 1 μM (Fig. 5B, n = 2-5; deviations of [Ca2+]free = 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 IP3 (20-200 nM) were calculated to be 1.8 ± 0.1 (Fig. 5, C and D), indicating that the IICR of the purified IP3R exhibited positive cooperativity. As the EC50 value and the Hill coefficient of IICR at both concentrations of fluo-3 were calculated to be the same, the changes of free Ca2+ concentration by the released Ca2+ had no significant effect on the sensitivity for IP3 and the cooperativity of IICR.
      Figure thumbnail gr5
      Figure 5:Analysis of IP3-induced Ca2+ release. Initial rates were measured from the initial and fast slope of IICR. A and B, normalized initial rates of Ca2+ release were plotted as a function of the concentration of IP3. 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 Ca2+ concentration = 100 nM; deviations of free Ca2+ concentration by the released Ca2+ = 10-30 nM), B and D at 1 μM fluo-3 (values are mean ± S.D., n = 2-5, initial free Ca2+ concentration = 200 nM; deviations of free Ca2+ concentration by the released Ca2+ = 150-300 nM).

      Analysis of Biphasic Nature of IICR and Quantal Ca2+Release

      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 (Equation 1) 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 IP3. The amplitudes of both states (Afast and Aslow) were plotted as a function of the concentrations of IP3 (Fig. 6, C (n = 3-4) and D (n = 2-5)). Afast increased as the concentration of IP3 increased, whereas Aslow decreased. Considering these amplitudes with the amount of total released Ca2+ (Fig. 4), the amounts of released Ca2+ by the fast and slow phases were then calculated. The amounts of released Ca2+ by the fast and slow components relative to the total released Ca2+ at 5 μM IP3 were plotted as a function of the concentrations of IP3 (Fig. 7, A (n = 3-4) and B (n = 2-5)). The amount of released Ca2+ by the fast component increased as a function of the concentration of IP3, whereas the amount by the slow component remained almost constant over the range of 10-5000 nM IP3 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−1, was mainly responsible for the quantal Ca2+ release.
      Figure thumbnail gr7
      Figure 7:The amounts of released Ca2+ by the fast and slow components of IP3-induced Ca2+ release. The amounts of total released Ca2+ in and the amplitude of the two components of IICR allowed us to calculate the amounts of released Ca2+ by the fast (squares) and slow (circles) components. A, 10 μM fluo-3 (values are mean ± S.D., n = 3-4; initial free Ca2+ concentration = 100 nM; deviations of free Ca2+ concentration by the released Ca2+ = 10-30 nM). B, 1 μM fluo-3 (values are mean ± S.D., n = 2-5; initial free Ca2+ concentration = 200 nM; deviations of free Ca2+ concentration by the released Ca2+ = 150-300 nM).

      DISCUSSIONS

      Measurements of IP3-induced Ca2+Release by the Immunoaffinity-purified IP3R

      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 IP3Rs: the presence of multiple IP3R types in single cells may affect the kinetics of IICR. (ii) Metabolism of IP3: IP3 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 IP3Rs, quantal release by submaximal doses, inactivation by IP3 itself) are dependent on IP3 doses. (iii) Ca2+ pump: the activity of the ATP-driven Ca2+ pump affects IICR by refilling Ca2+ stores following Ca2+ release. This prevents us from evaluating the cooperativity of IICR by reducing the net IICR to a great extent at low concentrations of IP3 than at high concentrations(
      • Champeil P.
      • Combettes L.
      • Berthon B.
      • Doucet E.
      • Orlowski S.
      • Claret M.
      ). (iv) Molecules sensing changes in Ca2+ concentration: dynamic changes in cytosolic and luminal Ca2+ concentrations have been argued to be involved in functional regulation of IICR properties by modifying the function of the IP3R itself and by activating IP3R modulator proteins (e.g. protein kinases (e.g. Ca2+-calmodulin-dependent protein kinase and protein kinase C) and phosphatases (e.g. calcineurin) and Ca2+-binding proteins (e.g. calmodulin) and IP3-metabolizing enzymes (e.g. IP3 kinase)). (v) Heterogeneity in IICR-Ca2+ pools: there is a subcellular heterogeneity in IP3 sensitive Ca2+ stores, e.g. subsurface cisternae, calciosomes, nuclear membranes, etc., which may have different IICR properties. Artificial effects on IP3-sensitive Ca2+ stores by experimental conditions must be considered, e.g. fusion of cisternae membranes by excess treatment with saponin (
      • Renard-Rooney D.C.
      • Hajnoczky G.
      • Seitz M.B.
      • Schneider T.G.
      • Thomas A.P.
      ) and induction of formation of cisternal stacks mediated by IP3Rs by nonphysiological treatment(
      • Takei K.
      • Mignery G.A.
      • Mugnaini E.
      • Sudhof T.C.
      • De Camilli P.
      ).
      In this study, we have investigated the kinetics of IICR of the immunoaffinity-purified and reconstituted IP3R, excluding the possibility of modulation of IICR by factors other than Ca2+ and IP3 itself. Furthermore, as the immunoaffinity-purified IP3R showed very strong immunoreactivity with the monoclonal antibody against IP3R type 1 and little with the monoclonal antibodies against IP3R types 2 and 3, the population of the immunoaffinity-purified IP3R was almost homogeneous of IP3R1 but contained very small amounts of IP3R2 and IP3R3. Therefore the results derived from the analysis of this purified IP3R reflect mainly the properties of IP3R1. Due to the absence of IP3 metabolizing enzymes, in our system, applied IP3 doses should be constant throughout each experiment. However, we must consider the regulation of the IP3R by changes in free Ca2+ concentration. Feedback regulations of IICR by the released Ca2+ have been observed in permeabilized cells (
      • Iino M.
      ,
      • Iino M.
      • Endo M.
      ) and microsomal systems(
      • Finch E.A.
      • Turner T.J.
      • Goldin S.M.
      ,
      • Watras J.
      • Bezprozvanny I.
      • Ehrlich B.E.
      ). On the other hand, the high concentrations of Ca2+ chelators and Ca2+ indicators caused artificial effects on IICR in those experiments(
      • Combettes L.
      • Champeil P.
      ,
      • Richardson A.
      • Taylor C.W.
      ). In this study, to avoid problems concerning the regulation of IICR by the released Ca2+, we used high enough concentration of fluo-3 to keep extravesicular free Ca2+ concentration within 100-130 nM. We also used 1 μM fluo-3, 200-500 nM free Ca2+ concentration, to compare the effect of changes of extravesicular free Ca2+ by IICR on the kinetics of Ca2+ release. At both fluo-3 concentrations, where the extravesicular free Ca2+ 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 Ca2+ release was essentially the same, indicating little feedback regulation by the released Ca2+ in our system. We also observed similar kinetics of Ca2+ release at the initial extravesicular free Ca2+ concentration of 300 nM (data not shown), where Ca2+ release using cerebellar microsomes was shown to be inhibited(
      • Bezprozvanny I.
      • Watras J.
      • Ehrlich B.E.
      ). Feedback regulation by the released Ca2+ or the regulation by Ca2+ outside of pools may be mediated by the action(s) of other molecules which can sense changes of Ca2+ concentration.

      Fundamental Properties of the Immunoaffinity-purified IP3R

      The extent of cooperativity of Ca2+ 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 (
      • Finch E.A.
      • Turner T.J.
      • Goldin S.M.
      ,
      • Watras J.
      • Bezprozvanny I.
      • Ehrlich B.E.
      ) or positive cooperativity (nH = 2) (
      • Champeil P.
      • Combettes L.
      • Berthon B.
      • Doucet E.
      • Orlowski S.
      • Claret M.
      ,
      • Somlyo A.V.
      • Horiuti K.
      • Trentham D.R.
      • Kitazawa T.
      • Somlyo A.P.
      ) (nH = 4) (
      • Meyer T.
      • Wensel T.
      • Stryer L.
      ) has been demonstrated. The Hill plots of the initial rates of Ca2+ release by the purified IP3R (Fig. 5, C and D) showed a positive cooperativity (nH = 1.8 ± 0.1) at submaximal concentrations of IP3. This cooperativity was observed in the presence of both 10 μM and 1 M fluo-3, indicating that at least two molecules of IP3 is needed for channel opening and that the positive cooperativity could not be due to sensitization of IICR by the rise in free Ca2+ concentration, i.e. by the released Ca2+. This result shows that the positive cooperativity of IICR could be mediated by a single type of IP3R.
      The dose-response curves of IICR mediated by the purified IP3R showed that the amount of released Ca2+ increased as a function of IP3 concentration (Fig. 4) and provided evidence to suggest that the purified IP3R, which was chiefly composed of IP3R1, can exhibit the quantal response of Ca2+ release. Ferris et al.(
      • Ferris C.D.
      • Cameron A.M.
      • Huganir R.L.
      • Snyder S.H.
      ) have also reported that conventionally purified and reconstituted cerebellar IP3Rs showed the quantal response and suggested that heterogeneity of IP3R types was a possible mechanism underlying quantal Ca2+ release. However, it is likely that the quantal Ca2+ release is an intrinsic property of IP3R1.

      Detailed Kinetic Analysis of Ca2+Release

      The profiles of IICR mediated by the purified IP3R 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−1, respectively. We also analyzed the contribution of the fast and slow components to the total amounts of released Ca2+, which were estimated as described in. The amounts of released Ca2+ by the fast component increased as a function of the concentration of IP3, whereas those by the slow component were constant. These results suggest that the fast component is kinetically the state of low affinity for IP3 and high permeability of Ca2+, and the slow component is of high affinity and low permeability. Consistent with this view, the studies of IP3 binding in permeabilized hepatocytes and a liver plasma membrane-enriched fraction displayed the existence of two states with high and low affinity for IP3(
      • Pietri F.
      • Hilly M.
      • Mauger J.P.
      ,
      • Pietri F.
      • Hilly M.
      • Claret M.
      • Mauger J.P.
      ). Since our data show that the fast phase of Ca2+ release increases with increasing IP3 concentrations and the slow phase remains constant, it appears that the fast phase is the determinant of the amount of Ca2+ release and is responsible for the quantal Ca2+ release.
      Recently, heterogeneity of IP3R densities in pools, which had equal sensitivity to IP3, was reported to be responsible for biphasic Ca2+ release(
      • Hirose K.
      • Iino M.
      ). 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 IP3 concentrations, and the ratio of the amounts of released Ca2+ by the fast and slow components must be constant. Because in such an assumption, the amplitudes and the ratio of the amounts of released Ca2+ should reflect the distribution of such heterogeneity, i.e. proportion of IP3-sensitive Ca2+ pools with high and low density of IP3R reflect the amplitudes and the ratio of amounts of the released Ca2+ 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 Ca2+ were dependent on IP3 concentrations, indicating that the biphasic nature of Ca2+ release was not due to such heterogeneity of receptor density. A possibility of heterogeneity in the size of individual Ca2+ 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 IP3R has two states with different affinity for IP3, i.e. a low affinity and a high affinity state. This could arise from alternative splicing leading to the production of variants of IP3R1(
      • Nakagawa T.
      • Okano H.
      • Furuichi T.
      • Aruga J.
      • Mikoshiba K.
      ). Alternatively, there may be two different states of a single IP3R due to an IP3-dependent inactivation or a Ca2+-dependent interconversion.
      In the absence and presence of changes in extravesicular free Ca2+ concentrations, we observed the biphasic nature of IICR, indicating that the changes in the free Ca2+ concentration may not be responsible for the biphasic kinetics. However, we cannot rule out the possibility that the released Ca2+ causes an instantaneous and local rise in Ca2+ 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 Ca2+ instantaneously rises near the channel pore, in cooperation with IP3, the inactivation of the IP3R reported by Hajnoczky et al.(
      • Hajnoczky G.
      • Thomas A.P.
      ) may be caused by the interconversion of IP3R. This hypothesis could be supported by the observations in Fig. 3 of (
      • Hajnoczky G.
      • Thomas A.P.
      ), where the degree of inactivation (interconversion) varied with the cytosolic free Ca2+ concentrations during the preincubation with IP3 and IP3R, whereas no significant change in IICR at various cytosolic free Ca2+ concentrations were observed without preincubation.
      We demonstrated here the positive cooperativity of IICR and the quantal Ca2+ release phenomenon of IICR by the purified IP3R, which was mainly composed of a single type of IP3R (IP3R1), and the biphasic nature of IICR, which had kinetically two states to release Ca2+. The purification and reconstitution of other types of IP3Rs 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 IP3R. Also, this will allow us to observe the effects of modulators, such as protein kinase A, ATP, Ca2+, etc. on type-specific IICR.

      Acknowledgments

      We thank Drs. Michio Niinobe and Shinji Nakade for their help in purification of IP3R 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.

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