Inositol 1,4,5-Trisphosphate Receptor/Ca2+Channel Modulatory Role of Chromogranin A, a Ca2+Storage Protein of Secretory Granules
2000; Elsevier BV; Volume: 275; Issue: 20 Linguagem: Inglês
10.1074/jbc.m909391199
ISSN1083-351X
AutoresSeung Hyun Yoo, Choon Ju Jeon,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoThe secretory granules of neuroendocrine cells, which contain large amounts of Ca2+ and chromogranins, have been demonstrated to release Ca2+ in response to inositol 1,4,5-trisphosphate (IP3), indicating the IP3-sensitive intracellular Ca2+ store role of secretory granules. In our previous study, chromogranin A (CGA) was shown to interact with several secretory granule membrane proteins, including the IP3 receptor (IP3R), at the intravesicular pH 5.5 (Yoo, S. H. (1994) J. Biol. Chem. 269, 12001–12006). To examine the functional aspect of this coupling, we measured the IP3-mediated Ca2+ release property of the IP3R reconstituted into liposomes in the presence and absence of CGA. Presence of CGA in the IP3R-reconstituted liposome significantly enhanced the IP3-mediated Ca2+ release from the liposomes. Moreover, the number of IP3 bound to the reconstituted IP3R increased. The fluorescence energy transfer and IP3R Trp fluorescence quenching studies indicated that the structure of reconstituted IP3R becomes more ordered and exposed in the presence of CGA, suggesting that the coupled CGA in the liposome caused structural changes of the IP3R, changing it to a structure that is better suited to IP3 binding and subsequent Ca2+ release. These results appear to underscore the physiological significance of IP3R-CGA coupling in the secretory granules. The secretory granules of neuroendocrine cells, which contain large amounts of Ca2+ and chromogranins, have been demonstrated to release Ca2+ in response to inositol 1,4,5-trisphosphate (IP3), indicating the IP3-sensitive intracellular Ca2+ store role of secretory granules. In our previous study, chromogranin A (CGA) was shown to interact with several secretory granule membrane proteins, including the IP3 receptor (IP3R), at the intravesicular pH 5.5 (Yoo, S. H. (1994) J. Biol. Chem. 269, 12001–12006). To examine the functional aspect of this coupling, we measured the IP3-mediated Ca2+ release property of the IP3R reconstituted into liposomes in the presence and absence of CGA. Presence of CGA in the IP3R-reconstituted liposome significantly enhanced the IP3-mediated Ca2+ release from the liposomes. Moreover, the number of IP3 bound to the reconstituted IP3R increased. The fluorescence energy transfer and IP3R Trp fluorescence quenching studies indicated that the structure of reconstituted IP3R becomes more ordered and exposed in the presence of CGA, suggesting that the coupled CGA in the liposome caused structural changes of the IP3R, changing it to a structure that is better suited to IP3 binding and subsequent Ca2+ release. These results appear to underscore the physiological significance of IP3R-CGA coupling in the secretory granules. d-myo-inositol 1,4,5-triphosphate 5-IAEDANS, (5-(((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid 4,5-triphosphate receptor fluorescein 5-isothiocyanate chromogranin A fluorescein 5- maleimide 7-diethylamino-3-(4′-maleimidylphenyl)-4-methyl-coumarin 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid antibody phenylmethylsulfonyl fluoride The secretory granules of adrenal medullary chromaffin cells have been shown to release Ca2+ in response to IP31 (1.Yoo S.H. Albanesi J.P. J. Biol. Chem. 1990; 265: 13446-13448Abstract Full Text PDF PubMed Google Scholar), and this observation has also been extended to the secretory granules of zymogen-secreting pancreatic acinar cells (2.Gerasimenko O.V. Gerasimenko J.V. Belan P.V. Petersen O.H. Cell. 1996; 84: 473-480Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar), further demonstrating the IP3-sensitive intracellular Ca2+ store role of secretory granules. Recently, direct participation of secretory granule calcium in the control of cytoplasmic Ca2+concentration has also been shown in the secretory granules of goblet cells (3.Nguyen T. Chin W.-C. Verdugo P. Nature. 1998; 395: 908-912Crossref PubMed Scopus (160) Google Scholar); uptake of Ca2+ by secretory granules was temporally and spatially matched by simultaneous reduction of Ca2+ concentration in the surrounding cytoplasm, whereas IP3-mediated release of Ca2+ by the secretory granules resulted in the simultaneous increase of cytoplasmic Ca2+ concentration in the immediate vicinity of the secretory granules, clearly indicating the participation of secretory granule calcium in the control of cytoplasmic Ca2+concentration. Moreover, the IP3-sensitive Ca2+store role of secretory granules of bovine adrenal medullary chromaffin cells was attributed to the presence of high capacity, low affinity Ca2+ storage protein CGA, which binds 30–50 mol of Ca2+/mol, inside the secretory granule (1.Yoo S.H. Albanesi J.P. J. Biol. Chem. 1990; 265: 13446-13448Abstract Full Text PDF PubMed Google Scholar, 4.Yoo S.H. Albanesi J.P. J. Biol. Chem. 1991; 266: 7740-7745Abstract Full Text PDF PubMed Google Scholar). IP3 mediates release of Ca2+ from intracellular Ca2+ stores by binding to the IP3R, which can also function as a Ca2+ channel (5.Ferris C.D. Huganir R.L. Supattapone S. Snyder S.H. Nature. 1989; 42: 87-89Crossref Scopus (366) Google Scholar). The IP3R, which has been found in the endoplasmic reticulum, nuclei, and plasma membrane (6.Mignery G.A. Südhof T.C. Takei K. DeCamilli P. Nature. 1989; 342: 192-195Crossref PubMed Scopus (394) Google Scholar, 7.Matter N. Ritz M.-F. Freyermuth S. Rogue P. Malviya A. J. Biol. Chem. 1993; 268: 732-736Abstract Full Text PDF PubMed Google Scholar, 8.Khan A.A. Steiner J.P. Klein M.G. Schneider M.F. Snyder S.H. Science. 1992; 257: 815-818Crossref PubMed Scopus (164) Google Scholar), is known to exist in at least three types,i.e. type I, II, and III, and to form homo- or heterotetrameric structures (9.Supattapone S. Worley P.F. Baraban J.M. Snyder S.H. J. Biol. Chem. 1988; 263: 1530-1534Abstract Full Text PDF PubMed Google Scholar, 10.Maeda N. Niinobe M. Mikoshiba K. EMBO J. 1990; 9: 61-67Crossref PubMed Scopus (232) Google Scholar, 11.Chadwick C.C. Saito A. Fleischer S. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2132-2136Crossref PubMed Scopus (204) Google Scholar, 12.Monkawa T. Miyawaki A. Sugiyama T. Yoneshima H. Yamamoto-Hino M. Furuich T. Saruta T. Hasegawa M. Mikoshiba K. J. Biol. Chem. 1995; 270: 14700-14704Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 13.Joseph S.K. Lin C. Pierson S. Thomas A.P. Maranto A.R. J. Biol. Chem. 1995; 270: 23310-23316Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). In our previous study, chromogranin A was shown to interact with several integral membrane proteins of secretory granules of bovine adrenal medullary chromaffin cells, including the IP3R (14.Yoo S.H. J. Biol. Chem. 1994; 269: 12001-12006Abstract Full Text PDF PubMed Google Scholar). This was the first time an ion channel protein was shown to be physically linked to a cognate ion storage protein. Chromogranin A, which is the major secretory granule matrix protein of bovine adrenal chromaffin cells, interacts with the secretory granule membrane at the intravesicular pH of 5.5 but dissociates from it at the near physiological pH of 7.5 (15.Yoo S.H. Biochemistry. 1993; 32: 8213-8219Crossref PubMed Scopus (51) Google Scholar). It also undergoes pH- and Ca2+-dependent conformational changes (16.Yoo S.H. Albanesi J.P. J. Biol. Chem. 1990; 265: 14414-14421Abstract Full Text PDF PubMed Google Scholar) and forms a homodimer at pH 7.5 and a homotetramer at pH 5.5 (17.Yoo S.H. Lewis M.S. J. Biol. Chem. 1992; 267: 11236-11241Abstract Full Text PDF PubMed Google Scholar, 18.Thiele C. Huttner W.B. J. Biol. Chem. 1998; 273: 1223-1231Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Furthermore, a tetrameric CGA has been shown to bind four molecules of an intraluminal loop peptide of the IP3R (19.Yoo S.H. Lewis M.S. Biochemistry. 1995; 34: 632-638Crossref PubMed Scopus (42) Google Scholar), suggesting the interaction of tetrameric CGA with tetrameric IP3R in the cell. In our recent study, it was shown that purified IP3Rs interact directly with CGA at the intravesicular pH 5.5 and dissociate from it at a near physiological pH 7.5 (20.Yoo S.H. So S.H. Kwon H.S. Lee J.S. Kang M.K. Jeon C.J. J. Biol. Chem. 2000; 275: 12553-12559Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Further, cotransfection of IP3R and CGA into COS-7 cells followed by coimmunoprecipitation also demonstrated coimmunoprecipitation of these two proteins (20.Yoo S.H. So S.H. Kwon H.S. Lee J.S. Kang M.K. Jeon C.J. J. Biol. Chem. 2000; 275: 12553-12559Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), indicating that IP3R and CGA exist in a complexed state in vivo. These results strongly suggested that coupling of Ca2+ storage protein CGA to the IP3R/Ca2+ channel might serve important physiological roles in the secretory vesicles not only during secretory vesicle biogenesis (14.Yoo S.H. J. Biol. Chem. 1994; 269: 12001-12006Abstract Full Text PDF PubMed Google Scholar) but also in controlling IP3-mediated Ca2+ mobilization in the cell. Therefore, we have investigated in this report the physiological significance of CGA coupling to the IP3R using IP3R-reconstituted liposomes in the presence and absence of CGA, and found that CGA coupling to the IP3R in the proteoliposomes causes structural changes of IP3R so as to facilitate not only the IP3 binding but also the Ca2+ release activity of the channel. Phospholipids were purchased from Avanti Polar Lipids (Albaster, AL) and were used without further purification. Fluorescence probes were from Molecular Probes (Eugene, OR). Chloroform solutions of lipids were stored in sealed ampules under argon gas at −20 °C. Cholesterol and IP3 were obtained from Sigma. All radioactive reagents were from NEN Life Science Products. Other chemicals were of the highest grade commercially available. Chromogranin A from bovine adrenal medulla was prepared from the secretory vesicle lysates of chromaffin cells as described previously (Fig. 1) (16.Yoo S.H. Albanesi J.P. J. Biol. Chem. 1990; 265: 14414-14421Abstract Full Text PDF PubMed Google Scholar). An IP3R peptide (DEEEVWLFWRDSNKEI, in single-letter code) corresponding to residues 692–707 of the type I IP3R (22.Furuichi T. Yoshikawa S. Miyawaki A. Wada K. Maeda N. Mikoshiba K. Nature. 1989; 342: 32-38Crossref PubMed Scopus (824) Google Scholar, 23.Mignery G.A. Newton C.L. Archer III, B.T. Südhof T.C. J. Biol. Chem. 1990; 265: 12679-12685Abstract Full Text PDF PubMed Google Scholar) was synthesized with a carboxyl-terminal cysteine. A polyclonal antibody was raised in rabbits against the peptide coupled to keyhole limpet hemocyanin and affinity-purified on the immobilized peptide following the procedure described (14.Yoo S.H. J. Biol. Chem. 1994; 269: 12001-12006Abstract Full Text PDF PubMed Google Scholar). Bovine cerebella were mixed with 3 volumes of buffer I (50 mmTris-HCl, pH 7.4, 0.32 m sucrose, 1 mm EDTA, 1 mm β-mercaptoethanol, 0.1 mm PMSF, 10 μm leupeptin, and 10 μm pepstatin) and were broken into small pieces in a blender, followed by homogenization in a glass-Teflon homogenizer. The homogenates were then centrifuged at 2,000 × g for 10 min at 4 °C, and the supernatants were recentrifuged at 105,000 × g for 1 h to precipitate the membrane pellet. The pellet was resuspended in buffer II (50 mm Tris-HCl, pH 8.0, 1 mm EDTA, 1 mm β-mercaptoethanol, 0.1 mm PMSF, 10 μm leupeptin, and 10 μm pepstatin) containing 1% Triton X-100 to give the membrane protein concentration of approximately 2 mg/ml. The membrane solution was stirred for 1 h and then centrifuged at 32,000 × g for 1 h at 4 °C. The supernatant obtained was mixed with an equal volume of buffer III (20 mm Tris-HCl, pH 7.5, 0.1 m NaCl, 1% Triton X-100, 1 mm β-mercaptoethanol, 0.1 mm PMSF, 10 μm leupeptin, and 10 μm pepstatin) and applied to an IP3R antibody-coupled immunoaffinity column (0.35 × 1 cm) equilibrated with buffer C (see below). The IP3R antibody-coupled column was prepared by coupling 0.6 mg of affinity purified anti-peptide IP3R antibody (14.Yoo S.H. J. Biol. Chem. 1994; 269: 12001-12006Abstract Full Text PDF PubMed Google Scholar) either to 1.2 ml of the immobilized protein A resin from the ImmunoPure protein A IgG orientation kit (Pierce) or to 0.2 g of CNBr-activated Sepharose 4B according to the method described previously (8.Khan A.A. Steiner J.P. Klein M.G. Schneider M.F. Snyder S.H. Science. 1992; 257: 815-818Crossref PubMed Scopus (164) Google Scholar), and stored in 20 mmTris-HCl, pH 7.5, containing 0.02% sodium azide until use. The protein-loaded column was washed with 20 bed volumes of buffer C to remove unbound proteins, and the IP3R was eluted by 10 ml of elution buffer (0.1 m glycine, pH 2.8, 0.2% Triton X-100, 0.5 m NaCl, 1 mm β-mercaptoethanol, 0.1 mm PMSF, 10 μm leupeptin, and 10 μm pepstatin). The eluate was immediately neutralized by adding 1 m Tris-HCl, pH 9.5, and mixed with an equal volume of buffer IV (50 mm Tris-HCl, pH 8.0, 0.2% Triton X-100, 0.5 m NaCl, and 1 mm β-mercaptoethanol), and then applied to a benzamidine-Sepharose column equilibrated with buffer D to remove any residual proteases from the IP3R sample. The IP3R containing flow-through was collected and stored at −70 °C until use (Fig. 1). The purified bovine cerebellum IP3R bound approximately 320 pmol of IP3/mg of protein, which is comparable to other purified IP3Rs (9.Supattapone S. Worley P.F. Baraban J.M. Snyder S.H. J. Biol. Chem. 1988; 263: 1530-1534Abstract Full Text PDF PubMed Google Scholar,10.Maeda N. Niinobe M. Mikoshiba K. EMBO J. 1990; 9: 61-67Crossref PubMed Scopus (232) Google Scholar), as determined according to the published method (10.Maeda N. Niinobe M. Mikoshiba K. EMBO J. 1990; 9: 61-67Crossref PubMed Scopus (232) Google Scholar). Phosphatidylcholine (from bovine brain), phosphatidylserine (from bovine brain), and cholesterol dissolved in chloroform were mixed to give a molar ratio of 60%, 20%, and 20%, respectively. The final lipid concentration was 5 mm in a total volume of 500 μl. The solvent was evaporated under a stream of argon gas, and the residual chloroform was removed by speed vacuuming. The dry lipids were hydrated in buffer A or B solution (A: 20 mm sodium acetate, pH 5.5, 100 mm NaCl, 1 mm CaCl2, and 1% CHAPS; B: 20 mmHEPES, pH 7.5, 100 mm NaCl, 1 mmCaCl2, and 1% CHAPS) containing 17 μg/ml IP3R and 60 μg/ml CGA (Fig.1). The mixtures were dialyzed for 72 h against excess volume of buffer C (buffer A or B without CHAPS) at 4 °C. The resulting proteoliposomes were pelleted by centrifugation at 100,000 × g for 30 min at 4 °C and washed with buffer D (20 mm HEPES, pH 7.5, 100 mm NaCl, 1 m KCl, and 3 m urea) twice to remove unreconstituted proteins. The pellets were resuspended with buffer E (20 mm HEPES, pH 7.5, 100 mmNaCl) and then were dialyzed against buffer E for 48 h at 4 °C. The resulting proteoliposomes were passed through Chelex 100 to remove Ca2+. The formation of proteoliposomes was monitored using light scattering during dialysis against buffer C and by electron microscopy; some portion of sample was collected as a function of time and, the emission values at 450 nm were measured with the same excitation wavelength (excitation band slit width: 1.5/emission band slit width: 5 nm). The average diameter of the liposomes was 280 ± 50 nm when assayed by light scattering (24.Kolchens S. Ramaswami V. Birgenheier J. Nett L. O'Brien D.F. Chem. Phys. Lipids. 1993; 65: 1-10Crossref PubMed Scopus (63) Google Scholar). The proteoliposomes produced were estimated to contain ∼300 μmCa2+ and were stable for at least 3 days as determined by <10% deviation in light scattering values. To test the encapsulation of CGA into liposomes, we used the fluorescence of FITC which is amine-reactive and non-permeable to membranes. 50 μl of proteoliposome was incubated with 5 mm of FITC for 2 h at room temperature in the presence or absence of 2% Triton X-100, and then 10 μl of 500 mmTris-HCl, pH 8.0 was added to the mixture to stop the reaction. To remove free FITC, the sample was applied to Sephadex G-25 column (10 × 200 mm) and then was extensively dialyzed against buffer E at 4 °C. After labeling the proteoliposomes with the probe, the emission intensity at 519 nm was measured at the excitation wavelength of 494 nm. In addition, the encapsulation of CGA was also confirmed by lysing the CGA-containing and non-CGA-containing proteoliposomes with Triton X-100 and separating on SDS-polyacrylamide gels, followed by quantitation of the proteins. These procedures not only confirmed the encapsulation of CGA in the proteoliposomes but also indicated that the presence of CGA did not affect the the amount of IP3R inserted into the liposomes (data not shown). Removal of Ca2+ contamination was conducted according to the method described previously (25.Meyer T. Wensel T. Stryer L. Biochemistry. 1990; 29: 32-37Crossref PubMed Scopus (141) Google Scholar). Ca2+ contamination during all experiments was checked using the fluorescence of Ca2+indicator, indo-1, before measurements. Ca2+ efflux from the proteoliposomes was observed by measuring the fluorescence changes of indo-1. Fluorometric measurements were performed at 35 °C by using a Shimadzu RF-5301 PC spectrofluorometer equipped with a temperature-controlled cuvette holder. The fluorescence intensity was measured at the emission wavelength of 393 nm (excitation of 355 nm) with 1.5 nm of excitation band slit width and 10 nm of emission band slit width. For the kinetic analysis of IP3-induced Ca2+ release, the data were acquired every 20 ms after addition of indicated concentration of IP3 to 1.7 ml of the proteoliposome solution. The fluorescent intensities of indo-1 were calibrated to free Ca2+ concentrations using Ca2+-EGTA buffering system (26.Tsien R. Pozzan T. Methods Enzymol. 1989; 172: 230-262Crossref PubMed Scopus (395) Google Scholar). In experiments designed to determine the effect of IP3R antibody (Ab) on the Ca2+ release from the IP3R-reconstituted liposomes, the IP3R Ab was mixed with the reaction solution at the ratio of 1 μg of IP3R/10 or 50 μg of IP3R Ab. After preincubating the sample at 35 °C for 30 min, the time course of IP3-induced Ca2+ release was monitored with the same method described above. IP3 dose-dependent release of Ca2+was also measured by the fluorescence intensity of indo-1 after addition of IP3 and compared with the fluorescence intensity after addition of Triton X-100 instead of IP3. To exclude the possibility of Ca2+ regulation of IP3-induced Ca2+ release, 10 μmof indo-1 was used in these experiments, which was a high enough concentration to buffer the released Ca2+. Reconstituted proteoliposomes were incubated with various concentrations of IP3 containing 1/1000 as much [3H]IP3. After 10 min of incubation at 35 °C, 0.1 volume of each sample was filtered through a spun concentrator (Microcon from Amicon) with molecular weight cutoff of 100,000 at 5000 × g. The radioactivity of each filtrate was determined by a liquid scintillation counter (Beckman LS6000LL) and compared with the control values without proteoliposomes. For the collisional fluorescence quenching of Trp residues in IP3R by iodide, a varying amount of KI, up to 0.16 m final, was added to the reaction mixtures while maintaining the total concentration of KI plus KCl constant, and the fluorescence intensity at the emission wavelength of 340 nm was measured with the excitation at 295 nm at 35 °C. To label the IP3R with 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin (CPM) and fluorescein 5-maleimide (F-mal), respectively, CPM or F-mal, both from Molecular Probes, was added dropwise to 2 μg of IP3R, and the labeling was allowed to proceed overnight at 4 °C. Upon completion of the labeling, an excess of glutathione and mercaptoethanol was added to stop the reaction. CPM- or F-mal- labeled IP3R molecules were separated by Sephadex G-25 and dialyzed extensively against buffer E. The labeled receptor molecules were reconstituted into the liposomes along with Ca2+ in the presence and absence of CGA, and the fluorescence resonance energy transfer was determined at 35 °C by measuring the emission spectra of F-mal in the range of 500–580 nm at the excitation wavelength of 384 nm, the excitation wavelength of CPM. The proteoliposomes were produced in the presence of45Ca2+ to include ∼50,000 cpm of45Ca2+ in buffer E (20 mm HEPES, pH 7.5, 100 mm NaCl) according to the procedure described. To remove residual Ca2+ bound to the vesicle surface, the sample was applied to Sephadex G25 column (10 × 00 mm) equilibrated with buffer E and, after collection of the vesicle fractions, the liposomes were pelleted by centrifugation (100,000 × g, 30 min, 4 °C). The pellet was then redissolved and dialyzed against excess volume of buffer E for 12 h at 4 °C. The proteoliposomes were mixed with each indicated concentration of CaCl2 and incubated for 10 min at 35 °C. After further incubation of the sample for 10 min in the presence of 0.2 μm of IP3 in the reaction mixtures, the sample was diluted with buffer F (buffer E plus 1.5 m KCl). The liposomes were pelleted by centrifugation at 100,000 ×g for 30 min at 35 °C followed by washing in buffer D twice. The pellet was then dissolved with 1% Triton X-100, and the radioactivity of each fraction (pellet and supernatant) was determined by scintillation counting. Protein concentrations were determined using bicinchoninic acid according to the manufacturer's instruction (Sigma). The concentrations of non-fluorescent phospholipids were determined by phosphorus assay (28.Vaskovsky V.E. Kostetsky E.Y. Vasendin I.M. J. Chromatogr. 1975; 114: 129-141Crossref PubMed Scopus (599) Google Scholar). The concentrations of fluorescent probes were determined spectrophotometrically using the following values as the molar extinction coefficients: 5700 cm−1m−1 at 336 nm for 1,5-IAEDANS, 77,000 cm−1m−1 at 494 nm and 83,000 cm−1m−1 at 492 nm for F-mal, and 33,000 cm−1m−1 at 384 nm for CPM. To mimic anin vivo physiological pH environment, the inside pH of the liposomes was maintained at 5.5 while that of outside was kept at 7.5. To determine whether the inside pH of the proteoliposome is stably maintained at pH 5.5, oxonol V (Molecular Probes) was encapsulated in the proteoliposome and its fluorescence increase upon exposure to higher pH environment was measured (Fig.2). As shown in Fig. 2, exposure of the internalized oxonol V to the pH 5.5 environment upon lysis of the proteoliposome by Triton X-100 treatment did not change the fluorescence of oxonol V. However, exposure of the internalized oxonol V to pH 6.5 or 7.5 environment upon lysis of the liposome increased the fluorescence of oxonol V, indicating the maintenance of pH 5.5 inside the proteoliposome. This result indicated that the inside pH of the proteoliposome was well maintained, suggesting the suitability of these proteoliposomes for subsequent experiments. The emission fluorescence was measured at 630 nm with the excitation wavelength of 610 nm in the presence of 1% Triton X-100. Fig. 3 A shows the time course of IP3-induced Ca2+ efflux from the IP3R-reconstituted liposomes, as assayed by the fluorescence change of indo-1 (1 μm) at 393 nm. Indo-1 is a fluorescent Ca2+ indicator, which is non-permeable to membrane and has a dual emission character on Ca2+ binding; the fluorescence at 390 nm increases, while that at 465 nm decreases. When increasing the IP3 concentrations successively, Ca2+ was released from the proteoliposomes with only the fast rate, and the fluorescence changes virtually reached a plateau after addition of 4 μm IP3 (Fig.3 A). No further increase of the emission fluorescence was observed by adding more IP3 (data not shown). The total amount of Ca2+ released by IP3 was estimated to be approximately 50% of the total encapsulated Ca2+concentration considering the maximal fluorescence signal obtained when the liposomes were lysed by 1% Triton X-100 (Fig. 3 A). We also tested the specificity of Ca2+ release through the IP3R using the IP3R Ab (Fig. 3, Aand B). Addition of IP3R Ab at the ratio of 1 μg of IP3R/10 μg of IP3R Ab inhibited about 70% of the IP3-induced Ca2+ release. Addition of more IP3R Ab, at the IP3R/IP3R Ab ratio of 1:50 (w/w), inhibited the Ca2+ release only slightly more (Fig. 3, A and B). When CGA was present inside the vesicle, the IP3-induced Ca2+ release was biexponential with the fast and the slow rate (Fig. 3 C). Interestingly, 1 μmIP3 released almost a maximumal amount of Ca2+in the presence of CGA (Fig. 3 C), whereas the same amount of IP3 released only a half-maximum in the absence of CGA (Fig. 3 A). These results indicate that the Ca2+channel activity of IP3R becomes far more efficient as a result of CGA encapsulation. However, when the pH value of inside the liposome was kept at 7.5 (Fig. 3 C), the kinetic profile of IP3-induced Ca2+ release was very similar to that obtained in the absence of CGA (Fig. 3 A), confirming our previous result, which indicated CGA interaction with the IP3R at pH 5.5 but not at pH 7.5 (14.Yoo S.H. J. Biol. Chem. 1994; 269: 12001-12006Abstract Full Text PDF PubMed Google Scholar, 19.Yoo S.H. Lewis M.S. Biochemistry. 1995; 34: 632-638Crossref PubMed Scopus (42) Google Scholar). As a control experiment, IP3R was labeled with FITC or 1,5-IAEDANS after completion of the reconstitution in the presence or absence of CGA, and the fluorescence intensity was measured to determine the effect of CGA on the amount of reconstituted IP3R. In both cases, we could not find any difference in the fluorescence intensities of the reconstituted samples, indicating that the amount of IP3R reconstituted was not affected by the presence of CGA (data not shown). Since it has also been shown that Ca2+release from the IP3R can be regulated by cytosolic Ca2+ concentrations (29.Iino M. Endo M. Nature. 1992; 360: 76-78Crossref PubMed Scopus (243) Google Scholar), there was a possibility that the IP3-induced Ca2+ release might not represent net IP3-mediated Ca2+ release. To address this possibility, we determined the amount of Ca2+ released after one-time dose of each indicated concentration of IP3in the presence of 10 μm of indo-1, which is a high enough concentration to buffer the released Ca2+ (Fig.4). As shown in Fig. 4, presence of CGA at pH 5.5 significantly increased the amounts of Ca2+released, compared with those in the absence of CGA, although the difference in the amounts of Ca2+ released in both cases diminished as IP3 concentrations increased. However, even the presence of CGA failed to exert this effect when the pH of the liposomes was 7.5. To determine whether the increased release of Ca2+ in the presence of CGA is due to increased binding of IP3 to the proteoliposome, the amount of IP3 bound to the IP3R was determined as a function of increasing IP3 concentrations (Fig. 5). As shown in Fig. 5, the presence of CGA enhanced the IP3binding 2-fold over that in the absence of CGA. When the IP3R alone was reconstituted, a maximum of 0.4 mol of IP3 appeared to bind 1 mol of IP3R. However, when CGA was also present inside the liposomes at the intravesicular pH of 5.5, the IP3 binding increased to about double the value shown with the IP3R alone. But when the pH of the liposomes was 7.5, CGA showed no effect on the IP3 binding to the IP3R. Nevertheless, the half-maximal value was almost the same for both cases regardless of the presence of CGA, indicating that the affinity of IP3R for IP3 has not changed. To investigate possible conformational changes of the IP3R by its interaction with CGA, we utilized collisional quenching of the IP3R Trp fluorescence by iodide (Fig.6). As shown in Fig. 6, the IP3R Trp fluorescence was quenched by iodide regardless of the presence of CGA. The emission fluorescence at 340 nm was measured, and the results were plotted according to the Stern-Volmer equation (30.Eftink M.R. Ghiron C.A. Anal. Biochem. 1981; 114: 199-227Crossref PubMed Scopus (1618) Google Scholar).Fo/F=Ksv[I−]+1Equation 1 Fo is the emission intensity in the absence of iodide, F is the intensity in the presence of iodide,K sv is the Stern-Volmer quenching constant, and [I−] is the molar concentration of iodide. TheK sv value estimated from the slope was 5.20m−1 for the reconstituted IP3R in the absence of CGA. This value decreased to 3.76m−1 when CGA was present. From this experiment, it is clear that at least some Trp residues of the IP3R are less exposed to the solvent when CGA is present and that CGA induced conformational changes of IP3R. In order to further examine the effect of CGA on the molecular property of IP3R in the reconstituted vesicles, we carried out the fluorescence resonance energy transfer study using the CPM- and F-mal-labeled IP3Rs (Fig. 7). As shown in Fig. 7, the reconstituted IP3Rs exhibited efficient fluorescence resonance energy transfer as evidenced by the increase of F-mal emission fluorescence under the excitation wavelength for CPM. This result indicated that the IP3R molecules have a strong tendency to exist as oligomers (tetramers) in the membranes. When CGA was present in the liposomes, the emission intensity increased by approximately 30%, suggesting that the organizational order of IP3Rs was enhanced by their interaction with CGA. In view of the potential inhibitory effect of increasing Ca2+concentrations on the IP3-induced Ca2+ release activity of IP3R (31.Miyakawa T. Maeda A. Yamazawa T. Hirose K. Kurosaki T. Iino M. EMBO J. 1999; 18: 1303-1308Crossref PubMed Scopus (338) Google Scholar), we examined the Ca2+release activity of the proteoliposome in response to a fixed amount of IP3 in the presence of increasing concentrations of Ca2+ (Fig. 8). As shown in Fig. 8, 0.2 μm IP3 induced far greater Ca2+ releases in the presence of CGA than in the absence in the Ca2+ concentration range of 0.1–1.0 μm. However, in the higher Ca2+ concentration r
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