Inositol 1,4,5-trisphosphate receptor/Ca2+ channel modulatory role of chromogranin A, a Ca2+ storage protein of secretory granules.

The secretory granules of neuroendocrine cells, which contain large amounts of Ca(2+) and chromogranins, have been demonstrated to release Ca(2+) in response to inositol 1,4,5-trisphosphate (IP(3)), indicating the IP(3)-sensitive intracellular Ca(2+) store role of secretory granules. In our previous study, chromogranin A (CGA) was shown to interact with several secretory granule membrane proteins, including the IP(3) receptor (IP(3)R), 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 IP(3)-mediated Ca(2+) release property of the IP(3)R reconstituted into liposomes in the presence and absence of CGA. Presence of CGA in the IP(3)R-reconstituted liposome significantly enhanced the IP(3)-mediated Ca(2+) release from the liposomes. Moreover, the number of IP(3) bound to the reconstituted IP(3)R increased. The fluorescence energy transfer and IP(3)R Trp fluorescence quenching studies indicated that the structure of reconstituted IP(3)R becomes more ordered and exposed in the presence of CGA, suggesting that the coupled CGA in the liposome caused structural changes of the IP(3)R, changing it to a structure that is better suited to IP(3) binding and subsequent Ca(2+) release. These results appear to underscore the physiological significance of IP(3)R-CGA coupling in the secretory granules.

The secretory granules of adrenal medullary chromaffin cells have been shown to release Ca 2ϩ in response to IP 3 1 (1), and this observation has also been extended to the secretory granules of zymogen-secreting pancreatic acinar cells (2), further demonstrating the IP 3 -sensitive intracellular Ca 2ϩ store role of secretory granules. Recently, direct participation of secretory granule calcium in the control of cytoplasmic Ca 2ϩ concentration has also been shown in the secretory granules of goblet cells (3); uptake of Ca 2ϩ by secretory granules was temporally and spatially matched by simultaneous reduction of Ca 2ϩ concentration in the surrounding cytoplasm, whereas IP 3 -medi-ated release of Ca 2ϩ by the secretory granules resulted in the simultaneous increase of cytoplasmic Ca 2ϩ concentration in the immediate vicinity of the secretory granules, clearly indicating the participation of secretory granule calcium in the control of cytoplasmic Ca 2ϩ concentration. Moreover, the IP 3 -sensitive Ca 2ϩ store role of secretory granules of bovine adrenal medullary chromaffin cells was attributed to the presence of high capacity, low affinity Ca 2ϩ storage protein CGA, which binds 30 -50 mol of Ca 2ϩ /mol, inside the secretory granule (1,4). IP 3 mediates release of Ca 2ϩ from intracellular Ca 2ϩ stores by binding to the IP 3 R, which can also function as a Ca 2ϩ channel (5). The IP 3 R, which has been found in the endoplasmic reticulum, nuclei, and plasma membrane (6 -8), is known to exist in at least three types, i.e. type I, II, and III, and to form homo-or heterotetrameric structures (9 -13). 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 IP 3 R (14). 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). It also undergoes pH-and Ca 2ϩ -dependent conformational changes (16) and forms a homodimer at pH 7.5 and a homotetramer at pH 5.5 (17,18). Furthermore, a tetrameric CGA has been shown to bind four molecules of an intraluminal loop peptide of the IP 3 R (19), suggesting the interaction of tetrameric CGA with tetrameric IP 3 R in the cell.
In our recent study, it was shown that purified IP 3 Rs interact directly with CGA at the intravesicular pH 5.5 and dissociate from it at a near physiological pH 7.5 (20). Further, cotransfection of IP 3 R and CGA into COS-7 cells followed by coimmunoprecipitation also demonstrated coimmunoprecipitation of these two proteins (20), indicating that IP 3 R and CGA exist in a complexed state in vivo. These results strongly suggested that coupling of Ca 2ϩ storage protein CGA to the IP 3 R/ Ca 2ϩ channel might serve important physiological roles in the secretory vesicles not only during secretory vesicle biogenesis (14) but also in controlling IP 3 -mediated Ca 2ϩ mobilization in the cell.
Therefore, we have investigated in this report the physiological significance of CGA coupling to the IP 3 R using IP 3 R-reconstituted liposomes in the presence and absence of CGA, and found that CGA coupling to the IP 3 R in the proteoliposomes causes structural changes of IP 3 R so as to facilitate not only the IP 3 binding but also the Ca 2ϩ release activity of the channel.

EXPERIMENTAL PROCEDURES
Materials-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 Ϫ20°C. Cholesterol and IP 3 were obtained from Sigma. All radioactive reagents were from NEN Life Science Products. Other chemicals were of the highest grade commercially available.
Purification of CGA and IP 3 R Antibody-Chromogranin A from bovine adrenal medulla was prepared from the secretory vesicle lysates of chromaffin cells as described previously (Fig. 1) (16). An IP 3 R peptide (DEEEVWLFWRDSNKEI, in single-letter code) corresponding to residues 692-707 of the type I IP 3 R (22, 23) was synthesized with a carboxyl-terminal cysteine. A polyclonal antibody was raised in rabbits against the peptide coupled to keyhole limpet hemocyanin and affinitypurified on the immobilized peptide following the procedure described (14).
Purification of IP 3 R from Cerebellum-Bovine cerebella were mixed with 3 volumes of buffer I (50 mM Tris-HCl, pH 7.4, 0.32 M sucrose, 1 mM EDTA, 1 mM ␤-mercaptoethanol, 0.1 mM PMSF, 10 M leupeptin, and 10 M 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 ϫ g for 10 min at 4°C, and the supernatants were recentrifuged at 105,000 ϫ 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 ␤-mercaptoethanol, 0.1 mM PMSF, 10 M leupeptin, and 10 M 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 ϫ 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 ␤-mercaptoethanol, 0.1 mM PMSF, 10 M leupeptin, and 10 M pepstatin) and applied to an IP 3 R antibody-coupled immunoaffinity column (0.35 ϫ 1 cm) equilibrated with buffer C (see below). The IP 3 R antibody-coupled column was prepared by coupling 0.6 mg of affinity purified anti-peptide IP 3 R antibody (14) 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), and stored in 20 mM Tris-HCl, pH 7.5, containing 0.02% sodium azide until use. The proteinloaded column was washed with 20 bed volumes of buffer C to remove unbound proteins, and the IP 3 R 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 ␤-mercaptoethanol, 0.1 mM PMSF, 10 M leupeptin, and 10 M 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 ␤-mercaptoethanol), and then applied to a benzamidine-Sepharose column equilibrated with buffer D to remove any residual proteases from the IP 3 R sample. The IP 3 R containing flow-through was collected and stored at Ϫ70°C until use (Fig. 1). The purified bovine cerebellum IP 3 R bound approximately 320 pmol of IP 3 /mg of protein, which is comparable to other purified IP 3 Rs (9, 10), as determined according to the published method (10).
Reconstitution of IP 3 R and Encapsulation of CGA into Liposomes-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 l. 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 CaCl 2 , and 1% CHAPS; B: 20 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM CaCl 2 , and 1% CHAPS) containing 17 g/ml IP 3 R and 60 g/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 ϫ 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 mM NaCl) and then were dialyzed against buffer E for 48 h at 4°C. The resulting proteoliposomes were passed through Chelex 100 to remove Ca 2ϩ . 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 Ϯ 50 nm when assayed by light scattering (24). The proteoliposomes produced were estimated to contain ϳ300 M Ca 2ϩ and were stable for at least 3 days as determined by Ͻ10% 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 l 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 l of 500 mM Tris-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 ϫ 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 IP 3 R inserted into the liposomes (data not shown).
Removal of Ca 2ϩ Contamination-Removal of Ca 2ϩ contamination was conducted according to the method described previously (25). Ca 2ϩ contamination during all experiments was checked using the fluorescence of Ca 2ϩ indicator, indo-1, before measurements. IP 3 -induced Ca 2ϩ Release Measurement-Ca 2ϩ 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 IP 3 -induced Ca 2ϩ release, the data were acquired every 20 ms after addition of indicated concentration of IP 3 to 1.7 ml of the proteoliposome solution. The fluorescent intensities of indo-1 were calibrated to free Ca 2ϩ concentrations using Ca 2ϩ -EGTA buffering system (26).
In experiments designed to determine the effect of IP 3 R antibody (Ab) on the Ca 2ϩ release from the IP 3 R-reconstituted liposomes, the IP 3 R Ab was mixed with the reaction solution at the ratio of 1 g of IP 3 R/10 or 50 g of IP 3 R Ab. After preincubating the sample at 35°C for 30 min, the time course of IP 3 -induced Ca 2ϩ release was monitored with the same method described above. IP 3 dose-dependent release of Ca 2ϩ was also measured by the fluorescence intensity of indo-1 after addition of IP 3 and compared with the fluorescence intensity after addition of Triton X-100 instead of IP 3 . To

FIG. 1. SDS-PAGE of purified bovine IP 3 R and chromogranin
A. Coomassie Blue-stained purified bovine IP 3 R (0.5 g) and CGA (1.2 g) on a 7.5% SDS gel. exclude the possibility of Ca 2ϩ regulation of IP 3 -induced Ca 2ϩ release, 10 M of indo-1 was used in these experiments, which was a high enough concentration to buffer the released Ca 2ϩ .
IP 3 Binding to IP 3 R-Reconstituted proteoliposomes were incubated with various concentrations of IP 3 containing 1/1000 as much [ 3 H]IP 3 . 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 ϫ g. The radioactivity of each filtrate was determined by a liquid scintillation counter (Beckman LS6000LL) and compared with the control values without proteoliposomes.
Fluorescence Quenching-For the collisional fluorescence quenching of Trp residues in IP 3 R 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.
Fluorescence Resonance Energy Transfer-To label the IP 3 R with 7-diethylamino-3-(4Ј-maleimidylphenyl)-4-methylcoumarin (CPM) and fluorescein 5-maleimide (F-mal), respectively, CPM or F-mal, both from Molecular Probes, was added dropwise to 2 g of IP 3 R, 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 IP 3 R molecules were separated by Sephadex G-25 and dialyzed extensively against buffer E. The labeled receptor molecules were reconstituted into the liposomes along with Ca 2ϩ 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.
Effect of Ca 2ϩ on the IP 3 -mediated Ca 2ϩ Release from the Proteoliposomes-The proteoliposomes were produced in the presence of 45 Ca 2ϩ to include ϳ50,000 cpm of 45 Ca 2ϩ in buffer E (20 mM HEPES, pH 7.5, 100 mM NaCl) according to the procedure described. To remove residual Ca 2ϩ bound to the vesicle surface, the sample was applied to Sephadex G25 column (10 ϫ 00 mm) equilibrated with buffer E and, after collection of the vesicle fractions, the liposomes were pelleted by centrifugation (100,000 ϫ 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 CaCl 2 and incubated for 10 min at 35°C. After further incubation of the sample for 10 min in the presence of 0.2 M of IP 3 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 ϫ 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.
Other Procedures-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). The concentrations of fluorescent probes were determined spectrophotometrically using the following values as the molar extinction coefficients: 5700 cm Ϫ1 M Ϫ1 at 336 nm for 1,5-IAEDANS, 77,000 cm Ϫ1 M Ϫ1 at 494 nm and 83,000 cm Ϫ1 M Ϫ1 at 492 nm for F-mal, and 33,000 cm Ϫ1 M Ϫ1 at 384 nm for CPM.

Maintenance of pH Gradient in the Liposomes-To mimic an
in 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.
Time Course of IP 3 -induced Ca 2ϩ Release- Fig. 3A shows the time course of IP 3 -induced Ca 2ϩ efflux from the IP 3 R-reconstituted liposomes, as assayed by the fluorescence change of indo-1 (1 M) at 393 nm. Indo-1 is a fluorescent Ca 2ϩ indicator, which is non-permeable to membrane and has a dual emission character on Ca 2ϩ binding; the fluorescence at 390 nm increases, while that at 465 nm decreases. When increasing the IP 3 concentrations successively, Ca 2ϩ was released from the proteoliposomes with only the fast rate, and the fluorescence changes virtually reached a plateau after addition of 4 M IP 3 (Fig. 3A). No further increase of the emission fluorescence was observed by adding more IP 3 (data not shown). The total amount of Ca 2ϩ released by IP 3 was estimated to be approximately 50% of the total encapsulated Ca 2ϩ concentration considering the maximal fluorescence signal obtained when the liposomes were lysed by 1% Triton X-100 (Fig. 3A).
We also tested the specificity of Ca 2ϩ release through the IP 3 R using the IP 3 R Ab (Fig. 3, A and B). Addition of IP 3 R Ab at the ratio of 1 g of IP 3 R/10 g of IP 3 R Ab inhibited about 70% of the IP 3 -induced Ca 2ϩ release. Addition of more IP 3 R Ab, at the IP 3 R/IP 3 R Ab ratio of 1:50 (w/w), inhibited the Ca 2ϩ release only slightly more (Fig. 3, A and B). When CGA was present inside the vesicle, the IP 3 -induced Ca 2ϩ release was biexponential with the fast and the slow rate (Fig. 3C). Interestingly, 1 M IP 3 released almost a maximumal amount of Ca 2ϩ in the presence of CGA (Fig. 3C), whereas the same amount of IP 3 released only a half-maximum in the absence of CGA (Fig. 3A). These results indicate that the Ca 2ϩ channel activity of IP 3 R 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. 3C), the kinetic profile of IP 3 -induced Ca 2ϩ release was very similar to that obtained in the absence of CGA (Fig. 3A), confirming our previous result, which indicated CGA interaction with the IP 3 R at pH 5.5 but not at pH 7.5 (14,19).
As a control experiment, IP 3 R 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 IP 3 R. In both cases, we could not find any difference in the fluorescence intensities of the reconstituted samples, indicating that the amount of IP 3 R reconstituted was not affected by the presence of CGA (data not shown).
IP 3 Dose-dependent Ca 2ϩ Release-Since it has also been shown that Ca 2ϩ release from the IP 3 R can be regulated by cytosolic Ca 2ϩ concentrations (29), there was a possibility that the IP 3 -induced Ca 2ϩ release might not represent net IP 3mediated Ca 2ϩ release. To address this possibility, we determined the amount of Ca 2ϩ released after one-time dose of each indicated concentration of IP 3 in the presence of 10 M of indo-1, which is a high enough concentration to buffer the released Ca 2ϩ (Fig. 4). As shown in Fig. 4, presence of CGA at pH 5.5 significantly increased the amounts of Ca 2ϩ released, compared with those in the absence of CGA, although the difference in the amounts of Ca 2ϩ released in both cases diminished as IP 3 concentrations increased. However, even the presence of CGA failed to exert this effect when the pH of the liposomes was 7.5.
CGA Effect on IP 3 Binding to IP 3 R-To determine whether the increased release of Ca 2ϩ in the presence of CGA is due to increased binding of IP 3 to the proteoliposome, the amount of IP 3 bound to the IP 3 R was determined as a function of increasing IP 3 concentrations (Fig. 5). As shown in Fig. 5, the presence of CGA enhanced the IP 3 binding 2-fold over that in the absence of CGA. When the IP 3 R alone was reconstituted, a maximum of 0.4 mol of IP 3 appeared to bind 1 mol of IP 3 R. However, when CGA was also present inside the liposomes at the containing 1 M of indo-I at 35°C. The inside pH of the proteoliposome was 5.5 unless stated otherwise. A, IP 3 -induced Ca 2ϩ release from the proteoliposomes and its inhibition by IP 3 R Ab. Two different concentrations of IP 3 R Ab, at a ratio of 1 g of IP 3 R/10 or 50 g of IP 3 R Ab, were added. TX-100, Triton X-100. B, percent changes of released Ca 2ϩ from the liposomes described in A. Addition of preimmune serum at the IP 3 R:preimmune ratio of 1:50 (w/w) did not affect the Ca 2ϩ release. Each indicated concentration of IP 3 represents the amount of cumulative IP 3 added. C, IP 3 -induced Ca 2ϩ release in the presence of encapsulated CGA at the inside pH of 5.5 and 7.5. intravesicular pH of 5.5, the IP 3 binding increased to about double the value shown with the IP 3 R alone. But when the pH of the liposomes was 7.5, CGA showed no effect on the IP 3 binding to the IP 3 R. Nevertheless, the half-maximal value was almost the same for both cases regardless of the presence of CGA, indicating that the affinity of IP 3 R for IP 3 has not changed.
Structural and Motional Changes of IP 3 R by Interaction with CGA-To investigate possible conformational changes of the IP 3 R by its interaction with CGA, we utilized collisional quenching of the IP 3 R Trp fluorescence by iodide (Fig. 6). As shown in Fig. 6, the IP 3 R 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).
F o 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 Ϫ ] is the molar concentration of iodide. The K sv value estimated from the slope was 5.20 M Ϫ1 for the reconstituted IP 3 R in the absence of CGA. This value decreased to 3.76 M Ϫ1 when CGA was present. From this experiment, it is clear that at least some Trp residues of the IP 3 R are less exposed to the solvent when CGA is present and that CGA induced conformational changes of IP 3 R. In order to further examine the effect of CGA on the molecular property of IP 3 R in the reconstituted vesicles, we carried out the fluorescence resonance energy transfer study using the CPM-and F-mal-labeled IP 3 Rs (Fig. 7). As shown in Fig. 7, the reconstituted IP 3 Rs 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 IP 3 R 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 IP 3 Rs was enhanced by their interaction with CGA.

Effect of Ca 2ϩ on IP 3 -induced Ca 2ϩ
Release from the Proteoliposomes-In view of the potential inhibitory effect of increasing Ca 2ϩ concentrations on the IP 3 -induced Ca 2ϩ release activity of IP 3 R (31), we examined the Ca 2ϩ release activity of the proteoliposome in response to a fixed amount of IP 3 in the presence of increasing concentrations of Ca 2ϩ (Fig. 8). As shown in Fig. 8, 0.2 M IP 3 induced far greater Ca 2ϩ releases in the presence of CGA than in the absence in the Ca 2ϩ concentration range of 0.1-1.0 M. However, in the higher Ca 2ϩ concentration range, the differences in the amount of Ca 2ϩ released disappeared, indicating the inhibitory effect of Ca 2ϩ on the Ca 2ϩ release property of IP 3 R. These results clearly show that IP 3 releases far greater amounts of Ca 2ϩ from the proteoliposome in the presence of CGA when there is no inhibitory amount of Ca 2ϩ in the liposome solution. As a control experiment, we measured Ca 2ϩ -induced Ca 2ϩ release without IP 3 . However, no appreciable release of stored Ca 2ϩ could be observed in the presence of up to 2 M free Ca 2ϩ (data not shown).

DISCUSSION
Since the original study that demonstrated the IP 3 -induced Ca 2ϩ release from chromaffin granules (1), Petersen and colleagues (2) have shown the IP 3 -mediated Ca 2ϩ release from the zymogen granules, thus supporting the concept of secretory granules of endocrine and neuroendocrine cells serving as the IP 3 -sensitive intracellular Ca 2ϩ store (1,32). Recently, additional evidence indicating direct participation of secretory granule calcium in the control of cytoplasmic Ca 2ϩ has been obtained in the secretory granules of goblet cells (3); the uptake of Ca 2ϩ by secretory granules was temporally and spatially matched by simultaneous reduction of Ca 2ϩ concentration in the surrounding cytoplasm, whereas IP 3 -mediated release of Ca 2ϩ by secretory granules resulted in the simultaneous increase of cytoplasmic Ca 2ϩ concentration in the immediate vicinity of the secretory granules, clearly demonstrating the active participation of secretory granules in the control of cytoplasmic Ca 2ϩ concentration.
Chromogranins are widely distributed in neurons, endocrine cells, and neuroendocrine cells, and they are known to be marker proteins of neuroendocrine cells (33)(34)(35)(36). In the secretory granules of bovine adrenal chromaffin cells, chromogranins are present in 1-2 mM range (35,37), and bind ATP (38), catecholamine (39), in addition to Ca 2ϩ (40,41). Chromogranin A binds 32 mol of Ca 2ϩ /mol with dissociation constant of 2.7 mM at pH 7.5, and 55 mol of Ca 2ϩ /mol with dissociation constant of 4 mM at pH 5.5 (4). Due to the high capacity Ca 2ϩ binding of chromogranins, most (Ͼ99.9%) of 40 mM intravesicular calcium stay bound to chromogranins, resulting in the free Ca 2ϩ concentration of only 24 M (40). In view of the fact that the secretory granules occupy approximately 10% of bovine chromaffin cell volume (37), the calcium storage capacity of the secretory granules must be playing key roles in the overall control of calcium in the cells. In this regard, the high capacity, low affinity Ca 2ϩ binding property of chromogranins proves to be essential, enabling the secretory granules to store an exceptionally large amount of Ca 2ϩ . It appears therefore very natural that the secretory granules play pivotal roles in controlling the intracellular Ca 2ϩ concentrations.
Although the discovery that the secretory granule of adrenal medullary chromaffin cells is a major IP 3 -sensitive intracellular Ca 2ϩ store (1) implied the presence of IP 3 R on the secretory granule membrane, the presence of IP 3 R on the secretory granule membrane was not addressed until the intravesicular matrix protein CGA was shown to interact with several integral membrane proteins of secretory granules including the IP 3 R (14), which was the first example demonstrating a physical coupling between an ion channel and a cognate ion storage protein. Nevertheless, it was not known what effect the coupled CGA exert on the IP 3 R/Ca 2ϩ channel.
The present results obtained in the reconstitution experiments using the purified IP 3 R and CGA are considered to reflect the important roles these molecules play in the cytoplasm, which enable the secretory granules to control the intracellular Ca 2ϩ levels in neuroendocrine cells. The Ca 2ϩ flux experiments in Figs. 3C and 4 show that the amount of Ca 2ϩ released in response to submaximal levels of IP 3 was significantly enhanced as a result of coupled CGA. These results suggest not only a direct physical coupling but also a functional coupling between the IP 3 R/Ca 2ϩ channel and CGA at the intravesicular pH of 5.5. Nevertheless, at a near physiological pH of 7.5, CGA failed to exert any effect on the IP 3 -mediated Ca 2ϩ release (Figs. 3C and 4), underscoring the importance of the intravesicular acidic pH environment in the coupling of CGA to the IP 3 R. Although IP 3 R monomers can bind IP 3 , they do not exist in a monomeric state in the cell; rather, four molecules of same or different types of IP 3 R interact with each other to form either a homotetrameric or a heterotetrameric IP 3 R (9 -13), forming a Ca 2ϩ channel. Interestingly, CGA is also known to form a dimer at the near physiological pH 7.5 and a tetramer at the intravesicular pH 5.5 (17). Further, a tetrameric CGA has also been shown to bind four molecules of intraluminal loop peptide of IP 3 R (19), thus suggesting the potential interaction between a tetrameric CGA and a tetrameric IP 3 R. Therefore, the results showing the direct interaction between the purified IP 3 R and CGA (20), combined with the co-immunoprecipitation of transfected IP 3 R and CGA (20), strongly suggest the importance of this coupling in fine-tuning the intracellular Ca 2ϩ control mechanisms in the cell. In view of the fact that CGA and IP 3 R interact with each other at pH 5.5 and dissociate at pH 7.5 (14,20), the lack of any effect of CGA on the IP 3mediated Ca 2ϩ release at pH 7.5 is probably due to the failure of CGA to couple with the IP 3 R. This is also reflected in the fact that the number of IP 3 bound to the IP 3 R remained virtually the same when the liposome pH was maintained at 7.5, regardless of the presence of CGA (Fig. 5).
Furthermore, the result in Fig. 4 shows that 1 M IP 3 releases almost the maximal amount of Ca 2ϩ that can be released. Given that approximately 2 molecules of IP 3 bound/ tetrameric IP 3 R at 1 M IP 3 (Fig. 5), the result in Fig. 4 suggests that almost a maximal amount of Ca 2ϩ can be released when 2 molecules of IP 3 are bound per tetrameric IP 3 R, although a maximum of 4 molecules of IP 3 can be bound per tetrameric IP 3 R at 4 M IP 3 (Fig. 5). This result is in general agreement with previous results, which indicated that one molecule of IP 3 opens the IP 3 R/Ca 2ϩ channel, which exhibits four conductance states (42). Although it has also been sug- gested that three or more IP 3 molecules are needed to open the IP 3 R/Ca 2ϩ channel (43), the general consensus is that only one IP 3 molecule is necessary to open the IP 3 R/Ca 2ϩ channel (42,44,45).
Since the pH of intravesicular milieu of secretory vesicles of neuroendocrine cells is 5.5 (46,47) and that of the extravesicular or extracellular environment is 7.4, the coupling of IP 3 R, an integral membrane protein, and CGA, an intravesicular matrix protein, at pH 5.5 and dissociation at pH 7.5 are in line with the physiological demands of the environments these molecules experience as they travel from the trans-Golgi network where the secretory vesicles are formed to the bloodstream. During exocytosis, the secretory vesicle contents will be exposed to a new pH environment of the extracellular space, which maintains a pH of 7.4. This change of pH from the intravesicular pH of 5.5 to 7.4 will automatically cause dissociation of the vesicular contents from the vesicle membrane, thus allowing the secretory cargo to move into the extracellular space and then on to the bloodstream.
The fluorescence resonance energy transfer (Fig. 7) measurements suggested that the enhanced rate of Ca 2ϩ release is due to the structural stabilization of the IP 3 R by coupled CGA. This result suggested that the presence of CGA in the liposome changes the conformation of the IP 3 R, probably changing it to a structure that is better suited to IP 3 binding (Fig. 5) and subsequent Ca 2ϩ release. Furthermore, quenching experiments designed to measure the Trp fluorescence of the IP 3 R also indicated that at least some Trp residues of the IP 3 R are less exposed when CGA is present (Fig. 6). Therefore, it appears that CGA play pivotal roles in controlling the Ca 2ϩ release property of the IP 3 R in neuroendocrine cells, not only by binding and freeing of the intravesicular Ca 2ϩ but also by modulating the channel activity of the IP 3 Rs. IP 3 binding to the IP 3 R has been known to cause conformational changes of the IP 3 R (48). Further, it has been known that CGA assumes different conformations in different pH environment (16) and that CGA in different conformations exhibits different Ca 2ϩ -binding capacity and affinity (4). In light of these observations, it is natural to think that the conformational changes of the IP 3 R that occur as a result of IP 3 binding will be transmitted instantly to the coupled CGA in the secretory granules, causing their conformational changes. It may then be assumed that the resulting conformational changes in CGA will concomitantly result in lowering of CGA's affinity for Ca 2ϩ , thus freeing some Ca 2ϩ from the protein and making them available for release through the IP 3 R channel to the cytoplasm.
In view of the fact that the secretory granules contain up to 40 mM Ca 2ϩ and most (Ͼ99.9%) of it remains bound to chromogranins in the secretory granules (40), the coupling of calcium storage proteins to the IP 3 R/Ca 2ϩ channel appears to reflect the efficient and intricate structural organization of an intracellular Ca 2ϩ store whose Ca 2ϩ storage/release function should be strictly and subtly controlled for the Ca 2ϩ store to respond appropriately to the changing cytoplasmic Ca 2ϩ concentrations. Moreover, the insight into the molecular organization of Ca 2ϩ control mechanism of secretory granules is certain to shed further light on elucidation of Ca 2ϩ storage/release mechanisms of other intracellular Ca 2ϩ stores. Given the phys-iological needs of cells to tightly control the cellular Ca 2ϩ , the physical coupling of Ca 2ϩ storage proteins to Ca 2ϩ channels seems to be a natural consequence.