A Functional Interaction between Chromogranin B and the Inositol 1,4,5-Trisphosphate Receptor/Ca2+ Channel*

Chromogranins A and B (CGA and CGB) are high capacity, low affinity calcium (Ca2+) storage proteins found in many cell types most often associated with secretory granules of secretory cells but also with the endoplasmic reticulum (ER) lumen of these cells. Both CGA and CGB associate with inositol 1,4,5-trisphosphate receptor (InsP3R) in a pH-dependent manner. At an intraluminal pH of 5.5, as found in secretory vesicles, both CGA and CGB bind to the InsP3R. When the intraluminal pH is 7.5, as found in the ER, CGA totally dissociates from InsP3R, whereas CGB only partially dissociates. To investigate the functional consequences of the interaction between the InsP3R and CGB monomers or CGA/CGB heteromers, purified mouse InsP3R type I were fused to planar lipid bilayers and activated by 2 μm InsP3. In the presence of luminal CGB monomers or CGA/CGB heteromers the InsP3R/Ca2+ channel open probability and mean open time increased significantly. The channel activity remained elevated when the pH was changed to 7.5, a reflection of CGB binding to the InsP3R even at pH 7.5. These results suggest that CGB may play an important modulatory role in the control of Ca2+ release from the ER. Furthermore, the difference in the ability of CGA and CGB to regulate the InsP3R/Ca2+ channel and the variability of CGA/CGB ratios could influence the pattern of InsP3-mediated Ca2+ release.

(1). Levels of CGB expression in cells can be used as markers for a number of physiological and medically important pathophysiological conditions (2)(3)(4). In normal brain tissue CGB expression is enhanced after neuronal activation, providing a marker for stimulated neurons (3). In addition to the tissuespecific distribution, a regionally specific distribution of CGB has been found intracellularly in neuronally differentiated pheochromocytoma (PC12) cells (5). In these cells CGB levels are elevated in the neurites rather than in the soma, which correlates with the initiation site for intracellular calcium (Ca 2ϩ ) signals. The levels of CGB and chromogranin-derived peptides can be diagnostic markers for pathophysiological conditions. For example, levels of CGB are greatly reduced in the cerebrospinal fluid of chronic schizophrenia subjects (6,7). Moreover, the levels of CGB and chromogranin-derived peptides are diagnostically significant as neuronal markers for synaptic degeneration in Alzheimer's disease (4).
At the cellular level CGB is believed to have many intra-and extracellular functions. CGB functions as a heparin binding extracellular matrix protein, mediating adhesion of cells and supporting neurite outgrowth (8). CGB is a prohormone with numerous di-and tribasic amino acid cleavage sites that act as targets for proteolytic enzymes such as the prohormone convertase (9,10). Furthermore, chromogranin B is known to bind Ͼ90 mol of Ca 2ϩ /mol with a dissociation constant (K d ) of 1.5 mM (11), distinguishing itself as a very efficient Ca 2ϩ storage protein. Intracellularly, CGB has also been suggested to participate in packaging and sorting other proteins into the secretory vesicles of neuroendocrine cells, thus playing key roles in secretory granule biogenesis (12)(13)(14). Indeed, CGB has recently been shown to induce secretory granule biogenesis (15). CGB also localizes to the nucleus and controls transcription of many genes, including those for transcription factors (16).
In secretory granules both CGA and CGB have been shown to interact with the InsP 3 R at the intravesicular pH of 5.5 (11,17). Purified InsP 3 R interacts directly with CGA and CGB at pH 5.5. CGA dissociates from the InsP 3 R at pH 7.5, whereas CGB remains partially associated (18). Both chromogranin proteins form a complex with the InsP 3 R in vivo (11). The functional aspect of this coupling has been investigated for CGA alone using single channel experiments and Ca 2ϩ flux studies (19). In the presence of CGA the open probability and mean open time of the InsP 3 R channel increases 10-fold.
Despite the role of secretory granules of secretory cells and ER as major InsP 3 -sensitive intracellular Ca 2ϩ stores (13,20,21) and the abundance of CGB in these (10) and a variety of other cell types (1), the functional interaction of InsP 3 R and CGB is less well characterized. Given also the tendency of CGA/CGB mixture to spontaneously form a CGA/CGB heterodimer at a near physiological pH 7.5 and a CGA 2 CGB 2 heterotetramer at the intragranular pH 5.5 (22), it is important to understand the effects of CGB and CGA/CGB heteromers. In this study, we examined the effect of CGB and CGA/CGB heteromers on the channel gating properties of the InsP 3 R type I. CGB increased the open probability and mean open time of the channel by almost 20-fold. However, in contrast to CGA, this functional effect was less sensitive to changes in pH when compared with the effect of CGA alone. These results show the functional interaction of InsP 3 R with CGB monomers and CGA/CGB heteromers and suggest that regulation of these interactions plays a physiologically important role in determining the pattern of InsP 3 -mediated Ca 2ϩ release.

MATERIALS AND METHODS
Antibody-An InsP 3 R peptide specific to terminal 10 -13 amino acids of type 1 (HPPHMNVNPQQPA) was synthesized with a carboxyl-ter-FIG. 1. pH-dependent interaction of InsP 3 R type I with CGA, CGB, and CGA/CGB. Purified InsP 3 R type I (0.5-0.7 g) was reacted with the GST fusion proteins of CGA, CGB, and an equimolar mixture of GST-CGA and GST-CGB at both pH 5.5 and 7.5. The bound InsP 3 R type I was separated on 7.5% SDS-gels and analyzed by immunoblot using a type 1-specific InsP 3 R antibody specific for the type I isoforms (11).

FIG. 2. Effects of CGB and CGA/CGB on InsP 3 -induced Ca 2؉ release from InsP 3 R-reconstituted liposomes.
InsP 3 -induced Ca 2ϩ efflux through the proteoliposomes (300 M Ca 2ϩ inside) was determined by the fluorescence change of 10 M indo-I at 393 nm after a series of incremental additions of InsP 3 (2.0 M final) to the proteoliposome solution. A, the InsP 3 -induced Ca 2ϩ release was measured both in the presence of encapsulated CGB at intraliposomal pH of 5.5 (q) and 7.5 (E) and in the absence at pH 5.5 (f) and 7.5 (Ⅺ). The figure shows the amount of released Ca 2ϩ expressed as percentage of maximum releasable Ca 2ϩ . InsP 3 -induced fluorescent changes were compared with that obtained by the addition of 1% Triton X-100 (this value was set at 100%). B, the InsP 3 -induced Ca 2ϩ release was measured in the presence of an equimolar mixture of CGA and CGB at an intraliposomal pH of 5.5 (q) and 7.5 (E) and in the absence at pH 5.5 (OE) and 7.5 (‚). The figure shows the amount of released Ca 2ϩ expressed as percentage of maximum releasable Ca 2ϩ . InsP 3 -induced fluorescent changes were compared with that obtained by the addition of 1% Triton X-100 (this value was set at 100%). minal cysteine, and anti-rabbit polyclonal antibody was raised. The polyclonal anti-rabbit antibody was affinity-purified on the immobilized peptide following the procedure described (23), and the specificity of the antibody was been confirmed (11).
InsP 3 R Interaction with Glutathione S-Transferase (GST)-CGA/ CGB-To construct GST fusion proteins, bovine CGA cDNA (24) and CGB cDNA (25) were amplified by PCR and subcloned into vector pGEX-5T (Amersham Biosciences). GST-CGA and GST-CGB fusion proteins were expressed in Escherichia coli BL21 cells and purified on glutathione-agarose beads. The binding reactions were carried out either in a pH 5.5 buffer (20 mM sodium acetate, pH 5.5, 0.1 M KCl, 2 mM CaCl 2 , and 0.1% Triton X-100) or in a pH 7.5 buffer (20 mM Tris-HCl, pH 7.5, 0.1 M KCl, 2 mM CaCl 2 , and 0.1% Triton X-100), and the interaction was determined by incubating an excess amount of purified GST-CGA or -CGB fusion protein (20 g) with the purified InsP 3 R (0.5-0.7 g) in 0.5 ml of buffer supplemented with 1ϫ protease inhibitor mixture (Roche Applied Science) for 1 h at 4°C. After incubation the reaction mixture was rinsed with 0.5 ml of buffer to remove unbound proteins. Elution of the bound InsP 3 R was carried out by using three bed volumes of the pH 7.5 buffer but with 1 M KCl. The eluted InsP 3 R was resolved on a 7.5% SDS-polyacrylamide gel and identified by chemiluminescence-based immunoblot analysis using the affinity-purified InsP 3 R antibody.
For bilayer experiments the InsP 3 R type I was solubilized in 1% CHAPS and purified from mouse cerebellum using heparin affinity and Con-A-Sepharose column chromatography, as described previously (27). The purified InsP 3 R was then incorporated into liposomes by the addition of 15 g of purified protein to 1 ml of liposome solution (consisting of phosphatidylcholine in bilayer buffer), mixing, and then incubating on ice for 10 min.
Flux Studies-InsP 3 R proteoliposomes were formed as described previously (26). Flux experiments were carried out using three types of proteoliposomes at both pH 5.5 and 7.5. Proteoliposomes either had 1) CGB encapsulated in them, 2) CGA and CGB encapsulated in them at the molar ratio of 1:1, or 3) no added chromogranin to be used for control experiments. Ca 2ϩ efflux from the proteoliposomes was measured by observing changes in indo-1 fluorescence. Fluorometric measurements were carried out at 35°C by using a Shimadzu RF-5301 PC spectrofluorometer equipped with a temperature-controlled cuvette holder. Fluorescence intensity was measured at the emission wavelength of 393 nm (excitation of 355 nm) with 10 nm of excitation band slit width and 10 nm of emission band slit width. For the kinetic analysis of InsP 3induced Ca 2ϩ release, the data were acquired every 20 ms after each addition of the indicated InsP 3 concentration to 0.5 ml of the proteoliposome solution. The fluorescent intensities of indo-1 were calibrated to free Ca 2ϩ concentrations using a Ca 2ϩ -EGTA buffering system (28). Fluorescence intensity after the addition of Triton X-100 was used to determine total intravesicular Ca 2ϩ .
Potassium Iodide Quench Analysis-For the collisional fluorescence quenching of Trp residues in InsP 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.
Bilayer Experiments-Planar lipid bilayers were formed by painting a solution of phosphatidylethanolamine/phosphatidylserine (3:1; 30 mg/ml in decane) across a 100-m aperture in a Teflon sheet bisecting a Lucite chamber. The hole was pre-painted with phosphatidylcholine/ phosphatidylserine (3:1) before membrane formation. The two compartments are defined cis (corresponding to the cytosol) and trans (corresponding to the lumen of the ER). The cis (cytosolic) compartment consisted of 250 mM HEPES-Tris, pH 7.35, 0.5 mM EGTA, 300 nM [Ca 2ϩ ] free , 0.5 mM ATP, 2 M ruthenium red. The trans (luminal) compartment consisted of 250 mM HEPES adjusted to pH 5.5 (because purified InsP 3 R was used in these experiments the pH could be changed using 70 mM HCl) and 53 mM BaOH 2 . Single channel currents were amplified using a bilayer clamp amplifier (Warner Instruments) and recorded on digital tape. Data were filtered with an eight-pole Bessel filter to 500 Hz, digitized to 2 KHz, transferred to a personal computer, and analyzed using pClamp 9.0 (Axon Instruments) software package.
InsP 3 R proteoliposomes were added to the cis compartment and mixed followed by the addition of 2 M InsP 3 to the same compartment. Upon InsP 3 R activation, single channel activity was recorded. Either CGB (1 g) or a mixture of CGA/CGB (1 g) was added to the trans compartment and mixed. InsP 3 R single channel activity was recorded. The pH inside the trans compartment was changed by the addition of Tris (final concentration 110 mM) to pH 7.5, and InsP 3 R single channel activity was recorded.
These experiments were repeated in the presence of increasing concentrations of InsP 3 (over the range 0.2-2 M) to the cis compartment and carried out in the presence and absence of 1 g of CGB or the CGA/CGB mixture in the trans compartment at pH 5.5. InsP 3 R single channel activity was recorded. Because CGB does not completely dissociate from the InsP 3 R at pH 7.5, the InsP 3 concentration-response was repeated in the presence of luminal CGB or the CGA/CGB mixture at this pH. All data are presented as the mean Ϯ S.E. of at least four similar experiments.

RESULTS
pH-dependent Interaction of InsP 3 R with CGA, CGB, and CGA/CGB-To determine whether there was a direct interaction between the purified InsP 3 R and CGA or CGB, GST fusion forms of CGA and CGB were expressed in E. coli BL21 and purified. The interaction between the InsP 3 R and GST-CGA and -CGB fusion proteins was examined at pH 5.5 and 7.5 ( Fig.  1). As shown in Fig. 1 (left side of the figure), CGA, CGB, and a mixture of CGA and CGB (CGA/CGB) all interacted with the InsP 3 R at pH 5.5. When tested at pH 7.5 CGA failed to interact with the InsP 3 R (Fig. 1, right side), but CGB still interacted with the InsP 3 R, albeit at a reduced level (Fig. 1, right side), reflecting a stronger affinity of CGB for the InsP 3 R.
Effects of CGB and CGA/CGB on InsP 3 -mediated Ca 2ϩ Release-Ca 2ϩ release studies were employed to investigate the effects of CGB on the InsP 3 concentration-response for InsP 3 R type I. As previously described for CGA (19), InsP 3 -induced Ca 2ϩ release from InsP 3 R-reconstituted liposomes was monitored at two different pH values both in the presence and absence of CGB ( Fig. 2A). InsP 3 -induced Ca 2ϩ efflux was determined using proteoliposomes containing 300 M Ca 2ϩ . The total amount of InsP 3 -releasable Ca 2ϩ was estimated to be 62-70%. The InsP 3 -induced Ca 2ϩ release obtained in the absence of CGB gave a K app value for InsP 3 of 0.68 M. When CGB was present inside the vesicle at pH 5.5, the pH value at which CGA and CGB coupled with the InsP 3 R (18), InsP 3 -induced Ca 2ϩ release was markedly enhanced (see Fig. 2A), yielding a K app value for InsP 3 of 0.16 M. Interestingly, when the pH was maintained at 7.5, the fluxes measured at each InsP 3 concen-

TABLE II The effect of the chromogranins on the open probability of the InsP 3 R
Open probabilities for the InsP 3 R single channel currents in the absence and presence of CGA, CGB, and CGA/CGB at pH 5.5 and 7.5. Values are generated from at least 4 experiments using each condition. tration were similar to those seen at pH 5.5 in the presence of CGB (K app value for InsP 3 of 0.17 M). This result is distinct from that obtained with CGA where pH had a profound effect, and the response at pH 7.5 was similar to that measured in the absence of CGA (19). Ca 2ϩ release from the InsP 3 R-reconstituted liposomes was also monitored in the presence of an equimolar mixture of CGA/CGB (Fig. 2B). The K app value for InsP 3 was approximately the same in the presence of the mixtures as that measured in the presence of CGB alone (K app value for InsP 3 was 0.15 M at pH 5.5 and 0.18 M at pH 7.5).
Changes in InsP 3 R Conformation-To investigate possible conformational changes of the InsP 3 R by its interaction with monomeric CGB or a heteromeric CGA/CGB, we utilized collisional quenching of the intrinsic tryptophan (Trp) fluorescence of the InsP 3 R by iodide (Fig. 3). By determining the extent of quenching it was possible to determine whether the Trp environment of the InsP 3 R is changed as a result of a change in the InsP 3 R conformation. As shown in Fig. 3, the InsP 3 R Trp fluorescence was quenched by iodide in the presence of both CGB or the CGA/CGB mixture. The emission fluorescence at 340 nm was measured, and the results were plotted according to the Stern-Volmer equation (29), where 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 quench-ing constant, and [I Ϫ ] is the molar concentration of iodide. The K sv value estimated from the slope was 2.94 M Ϫ1 for the reconstituted InsP 3 R in the absence of CGB. When CGB was present, this value decreased to 2.51 and 1.55 M Ϫ1 at the intraliposomal pH 7.5 and 5.5, respectively (Fig. 3A). From this experiment it is clear that at least some Trp residues of the InsP 3 R are less exposed to the solvent when CGB is present, demonstrating the CGB-induced conformational changes of the InsP 3 R. Furthermore, the K sv values suggest that the Trp residues are less exposed when the intraliposomal pH was maintained at 5.5 than at 7.5.
There also were conformational changes of the InsP 3 R in the presence of a CGA/CGB mixture in the liposome (Fig. 3B). The K sv value of 3.11 in the absence of CGA/CGB was decreased to 2.25 and 2.06 M Ϫ1 at the intraliposomal pH 7.5 and 5.5, respectively, in the presence of the CGA/CGB mixture, indicating the conformational changes of the InsP 3 R in the presence of CGA/CGB.
Effects of CGB and CGA/CGB on InsP 3 R Channel Activity-The effects of CGB and CGA/CGB on InsP 3 R function were investigated at the single channel level using InsP 3 R incorporated into planar lipid bilayers. In the absence of luminal CGB and in the presence of cytosolic-free Ca 2ϩ (300 nM) and InsP 3 (2 M) InsP 3 R single channel activity was observed (see Fig. 4A, trace iii). Single channel currents of ϳ2 pA were seen, and a  Table I). The open probability (Po) was 5 Ϯ 1% (n ϭ 4; Table II).
After the addition of 1 g of CGB to the luminal compartment a dramatic increase in channel activity was observed (Fig. 4A, trace ii). Although the size of the single channel current remained unaltered, significant differences in mean open times and Po were seen. Two populations of mean open time were apparent, but the values were noticeably increased over control values (Fig. 4B, middle panel) Table I). Furthermore, the Po increased from 5% in control conditions to 80 Ϯ 9% (n ϭ 4; Table II) in the presence of CGB.
After changing the pH of the luminal compartment to pH 6.5, a condition causing partial dissociation of CGB from the InsP 3 R, the Po was reduced to 63 Ϯ 2%. Although this is a decrease in channel activity, the Po was still elevated when compared with control levels (Fig. 4A, trace iv). A further change in luminal pH to 7.5 (Fig. 4A, trace v) caused the Po to fall even further, but the value of 40 Ϯ 2% still exceeded control levels, indicating that CGB remained coupled to the InsP 3 R. The mean open times were reduced to 4.8 Ϯ 0.6 and 122 Ϯ 0.7 ms (n ϭ 4; Fig. 4B, bottom panel and Table I). Addition of heparin, an InsP 3 R-specific antagonist, to the cytoplasmic compartment inhibited channel activity completely (Fig. 4A,  trace vi).
In the next series of experiments a mixture of CGA/CGB was tested. In the absence of CGA/CGB the amplitude of the single channel currents was 2 pA (see Fig. 5A, trace ii), the Po was 3.0 Ϯ 1.0% (n ϭ 4), and the mean open time was similar to that obtained in the previous control experiments (7.2 Ϯ 0.2 ms). After the addition of 1 g of CGA/CGB to the luminal compartment (pH 5.5) a large increase in channel activity was observed  (Fig. 5A, trace iii). The Po was 77% Ϯ 3% (n ϭ 4), and two populations of mean open time were evident (11.1 Ϯ 0.4 and 84.4 Ϯ 0.3 ms; Table I). Although the values for the open times were increased over control values (Fig. 5B, top panel) the longer population of mean open times was less than that seen with CGB alone and more closely resembled that seen with CGA alone.
A subsequent change in the luminal pH to 7.5 (Fig. 5A, trace iv) caused Po to fall, but the value of 48 Ϯ 2% again exceeded control levels. The elevated Po indicates a continued coupling of CGA/CGB with the InsP 3 R, potentially via the CGB component of the heteromer. The mean open times were 7.3 Ϯ 0.3 and 120.9 Ϯ 0.3 ms, values closer to CGB alone at pH 7.5 (n ϭ 4; Fig. 5B, bottom panel; Table I). The addition of heparin to the cytoplasmic compartment inhibited channel activity completely (Fig. 5A, trace v).
As with previous studies demonstrating the effect of CGA on the activity of the InsP 3 R (19), several control experiments were done. 1) The addition of CGB or CGA/CGB in the absence of cytosolic InsP 3 did not potentiate any InsP 3 R channel activity; 2) the addition of CGB or CGA/CGB to the luminal compartment in the absence of InsP 3 R had no effect upon the bilayer currents; 3) the addition of CGB or CGA/CGB to the cytoplasmic compartment in the presence of InsP 3 R and InsP 3 did not affect channel activity.
Effect of CGB or CGA/CGB on the InsP 3 Concentration-Response for InsP 3 R-Single channel activity was observed over a range of InsP 3 concentrations both in the presence of CGB at pH 5.5 and 7.5 and in its absence (Fig. 6A). Starting at an InsP 3 concentration of 0.2 M, the Po was greater in the presence of CGB (luminal pH 5.5) when compared with control levels, with an ϳ18-fold increase in Po at 2 M InsP 3 (Fig. 6A, middle  panel). At pH 7.5 (Fig. 6A, right panel) the channel again has a higher Po at the lower InsP 3 concentration range, although at 2 M InsP 3 the Po is less than that observed at pH 5.5.
Similar experiments to examine the InsP 3 concentration dependence were done in the presence of the CGA/CGB heteromer (Fig. 6B). Again, in the presence of CGA/CGB, over a range of InsP 3 concentrations starting at 0.2 M, the open probability was greater in the presence of CGA/CGB (luminal pH 5.5) when compared with control levels (Fig. 6B, middle panel). Repetition of the experiment at pH 7.5 (Fig. 6B, right panel) indicated that the Po at the lower InsP 3 concentrations were similar to those seen for CGA/CGB at pH 5.5, but at 2 M InsP 3 the Po was less at pH 7.5 than that seen at pH 5.5.
The InsP 3 concentration dependence of channel activity after the addition of either CGB or the mixture of CGA/CGB was compared at pH 5.5 and 7.5 (Fig. 7). Regardless of the pH used the effect of CGB alone or CGA/CGB was similar (Fig. 7), suggesting that the properties of CGB, especially the ability to bind to the InsP 3 R at pH 7.5, predominates.

DISCUSSION
In the present study we examined the functional interaction between the InsP 3 R type I and CGB monomers or CGA/CGB heteromers and found that both CGB and CGA/CGB heteromers enhanced activation of the InsP 3 R/Ca 2ϩ channel in the presence of InsP 3 . A direct physical interaction between the InsP 3 R and CGA or CGB was demonstrated (17), and the molecules co-localized in intact cells (5). Furthermore, a mixture of CGA/CGB interacted with the purified InsP 3 R at both pH 5.5 and 7.5, although the interaction at pH 7.5 was markedly reduced compared with that at pH 5.5 (Fig. 1). The stronger interaction between the InsP 3 R and the chromogranins at acidic pH is consistent with the physiological roles of secretory granules because the intragranular pH of secretory granules decreases and the Ca 2ϩ content increases as the granules move from the trans-Golgi network to the plasma membrane.
The interaction of the InsP 3 R with CGB monomers and with CGA/CGB heterotetramers at pH 5.5 elicited an open probability for the InsP 3 -gated Ca 2ϩ channel of 80 and 77%, respectively, which is the highest activity of the InsP 3 R observed to date in a bilayer. This functional interaction at pH 5.5 is likely related to the association of these proteins in secretory granules (18). An elevation of intracellular Ca 2ϩ is necessary for the process of fusion and exocytosis of secretory vesicles, and it is likely that the InsP 3 R/CGB interaction assists in this process. As the vesicles mature, the intraluminal pH becomes more acidic, and the Ca 2ϩ is elevated (30). The mature vesicles accumulate near the plasma membrane, and exocytosis is more likely to occur for more acidic secretory granules (30). The increased association between CGB and the InsP 3 R as the vesicles mature will enhance the probability of exocytosis because the protein complex shows an increased sensitivity of the InsP 3 R to activation by InsP 3 . This enhanced channel activity will quickly release Ca 2ϩ into the microregion between the vesicle and the plasma membrane where the fusion apparatus awaits the Ca 2ϩ required for exocytosis.
The functional interaction between the InsP 3 R and CGB remains even when the pH is at a near physiological level, suggesting that this protein association has roles in numerous locations in a cell. The addition of CGB or CGA/CGB heteromers to the InsP 3 R at pH 7.5 elicited an open probability of 40 and 48%, respectively. Again, the level of activation at this pH was elevated from those reported previously when using isolated InsP 3 R (31,32). The association between the InsP 3 R and CGB at pH 7.5 can have important regulatory roles in the ER, where CGB is found intraluminally and the InsP 3 R is associated with the ER membrane. Although the distribution of the InsP 3 R is assumed to be relatively homogeneous, CGB has been shown to be distributed in a regionally specific manner, as seen in neuronally differentiated PC12 cells (5). In these cells CGB is preferentially localized to the neurites, which is the region where intracellular Ca 2ϩ signals initiate (5). It is likely that this scenario will be found in other cell types as additional signaling microdomains are identified, and the association of CGB with the InsP 3 R may become an important modulator and amplifier of intracellular Ca 2ϩ signaling.
Interestingly, the effect of adding a mixture of CGA/CGB is the same as adding CGB alone. When mixed in vitro, CGA and CGB form a CGA-CGB heterodimer at pH 7.5 and a CGA 2 CGB 2 heterotetramer at pH 5.5 (22). In the experiments presented here the response of the InsP 3 -gated channel was similar after the addition of CGB alone or a mixture of CGA/CGB at a ratio of 1:1 or 10:1. Unlike CGA, which dissociated completely from the InsP 3 R at pH 7.5 (18) and was without any activating effect on the InsP 3 R/Ca 2ϩ channel (19), the InsP 3 R/Ca 2ϩ channelactivating effect of CGB at pH 7.5 implies an important physiological role. Because the ratios of CGA to CGB vary among cells (10) and the intraluminal pH levels of the organelles in which CGA and CGB are found also differ, the InsP 3 R/Ca 2ϩ channel-activating roles of CGA and CGB will also differ. We suggest, therefore, that the different ratios of CGA to CGB found in cells could be very important in modulating the time needed to achieve maturity in different Ca 2ϩ storage organelles because the pH dependence of the enhanced Ca 2ϩ release will be diminished in CGB-containing vesicles. Similarly, this difference in the ability of CGA and CGB to regulate the InsP 3 R could influence the pattern of InsP 3 -mediated Ca 2ϩ release as CGA/CGB ratios vary in different regions of the cell. In particular, the ability of CGB to activate the InsP 3 R/Ca 2ϩ channel at pH 7.5 is likely to have a big impact on InsP 3 -mediated Ca 2ϩ release from the ER.
In conclusion, we have identified a functional interaction between the intracellular Ca 2ϩ release channel, the InsP 3 R, with the Ca 2ϩ storage protein, chromogranin. Interactions oc-cur with CGB, CGA, and with CGA/CGB heteromers. In all of these cases the interactions elicit the highest increase in the purified InsP 3 R activity observed. Because these interactions can be modulated, the chromogranins have the potential to serve as robust amplifiers of Ca 2ϩ release in a variety of cell types both from secretory granules and from the ER.