Inositol 1,4,5-Trisphosphate Receptor in Chromaffin Secretory Granules and Its Relation to Chromogranins

Abstract

The inositol 1,4,5-trisphosphate (IP₃)-mediated intracellular Ca²⁺ releases in secretory cells play vital roles in controlling not only the intracellular Ca²⁺ concentrations but also the Ca²⁺-dependent exocytotic processes. Of intracellular organelles that release Ca²⁺ in response to IP₃, secretory granules stand out as the most prominent organelle and are responsible for the majority of IP₃-dependent Ca²⁺ releases in the cytoplasm of chromaffin cells. Bovine chromaffin granules were the first granules that demonstrated the IP₃-mediated Ca²⁺ release as well as the presence of the IP₃ receptor (IP₃R) in granule membranes. Secretory granules contain all three (type 1, 2, and 3) IP₃R isoforms, and 58–69% of total cellular IP₃R isoforms are expressed in bovine chromaffin granules. Moreover, secretory granules contain large amounts (2–4 mM) of chromogranins and secretogranins; chromogranins A and B, and secretogranin II being the major species. Chromogranins A and B, and secretogranin II are high-capacity, low-affinity Ca²⁺ binding proteins, binding 30–93 mol of Ca²⁺/mol of protein with dissociation constants of 1.5–4.0 mM. Due to this high Ca²⁺ storage properties of chromogranins secretory granules contain ~40 mM Ca²⁺. Furthermore, chromogranins A and B directly interact with the IP₃Rs and modulate the IP₃R/Ca²⁺ channels, i.e., increasing the open probability and the mean open time of the channels 8- to 16-fold and 9- to 42-fold, respectively. Coupled chromogranins change the IP₃R/Ca²⁺ channels to a more ordered, release-ready state, whereby making the IP₃R/Ca²⁺ channels significantly more sensitive to IP₃.

Introduction

Secretory cells are distinguished from other cells in that their main physiological function in an organism is to produce, store, and secrete a variety of small and large molecules of physiological importance. At the center of physiological functions of secretory cells lie the secretory granules, which have traditionally been thought to merely store and carry the secretory products until they are released in Ca²⁺-dependent regulatory secretory processes. The first signal in the regulated exocytosis is the sudden increase in the cytoplasmic Ca²⁺ concentrations, which can result from either release from intracellular Ca²⁺ stores or influx from outside.

Despite the presence of high concentrations of Ca²⁺, reaching 15–40 mM (Haigh et al. 1989; Petersen and Tepikin 2008; Winkler and Westhead 1980), in secretory granules of secretory cells, the identity of intracellular Ca²⁺ source that leads to initiation of exocytotic processes has not been attributed to secretory granules until recently (reviewed in Yoo 2010). The first demonstration of IP₃-induced Ca²⁺ release from secretory granules was done with bovine chromaffin granules (Yoo and Albanesi 1990), and the first evidence for the existence of the IP₃R on secretory granule membrane has also been obtained from bovine chromaffin granules (Yoo 1994). Since then the IP₃-sensitive Ca²⁺ store role of secretory granules has been shown with many different secretory cells such as pancreatic β-cells (Xie et al. 2006; Srivastava et al. 1999), acinar cells (Gerasimenko et al. 1996, 2006), airway goblet cells, mast cells (Nguyen et al. 1998; Quesada et al. 2001, 2003), in addition to chromaffin cells (Santodomingo et al. 2008).

In dual measurements of the intragranular Ca²⁺ as well as the cytoplasmic Ca²⁺ concentrations in the immediate vicinity of secretory granules using optical sectioning method, it was demonstrated that the IP₃-mediated Ca²⁺ release from secretory granules of airway goblet cells directly control the cytoplasmic Ca²⁺ concentrations (Nguyen et al. 1998). The IP₃-mediated intragranular Ca²⁺ releases and sequestrations were in exactly 180° out-of-phase with the oscillatory fluctuations of cytoplasmic Ca²⁺ concentrations in the immediate vicinity of the granules (Nguyen et al. 1998); the cytoplasmic Ca²⁺ concentrations rose when the intragranular Ca²⁺ concentrations decreased, and the cytoplasmic Ca²⁺ concentrations decreased when the intragranular Ca²⁺ concentrations rose, clearly demonstrating the control of cytoplasmic Ca²⁺ concentrations by secretory granules.

In addition to storing large amounts of Ca²⁺ inside, the sheer number of secretory granules in secretory cells, often numbering tens of thousands per cell (Burgoyne 1991; Huh et al. 2005a; Nordmann 1984; Plattner et al. 1997; Vitale et al. 1995), overwhelms other subcellular organelles in number as well as in total cell volume. Hence, secretory granules contain by far the highest amounts of cellular calcium, and in the case of bovine chromaffin cells secretory granules occupy ~20% of total cell volume (Burgoyne 1991; Huh et al. 2005a; Plattner et al. 1997) and contain >60% of total cell calcium (Haigh et al. 1989). Such a high Ca²⁺ storing capacity of secretory granules is due to the presence of large amounts (2–4 mM) of high-capacity Ca²⁺-storage proteins chromogranins A and B, along with some secretogranins of which secretogranin II being the major one (Helle 2000; Montero-Hadjadje et al. 2008; Taupenot et al. 2003; Winkler and Fischer-Colbrie 1992). Moreover, the IP₃R/Ca²⁺ channels are richly expressed in the membranes of secretory granules: all three isoforms of IP₃R are expressed in secretory granules (Srivastava et al. 1999; Yoo et al. 2001) and 58–69% of total cellular IP₃R isoforms are represented in secretory granules of bovine chromaffin cells (Huh et al. 2005c). The overall role of secretory granules in IP₃-dependent Ca²⁺ signaling in the cytoplasm of neuroendocrine cells has recently been reviewed (Yoo 2010). Hence, in the present review the specific functions of and the relationship between chromogranins and the IP₃Rs in secretory granules will be discussed.

IP₃Rs in Secretory Granules

Soon after the discovery of the IP₃R in the membranes of bovine chromaffin granules (Yoo 1994), the presence of the IP₃R in insulin-containing secretory granules of pancreatic β-cells has also been shown (Blondel et al. 1994). Although the presence of the IP₃R in secretory granules of pancreatic β-cells has initially been questioned due to potential cross-reactivity of the IP₃R antibody used in the study (Ravazzola et al. 1996) and some studies were not able to detect the presence of the IP₃R (Mitchell et al. 2001), it is clear that the IP₃Rs are heavily expressed in secretory granules of β-cells (Srivastava et al. 1999; Xie et al. 2006) and other cells. The bulk of the remaining cellular IP₃Rs is distributed between the ER and nucleus, and the nuclear IP₃Rs are primarily localized in numerous small IP₃-sensitive nucleoplasmic Ca²⁺ store vesicles (Huh et al. 2006; Yoo et al. 2005).

Utilizing the information on the cellular volumes of each subcellular organelle in bovine chromaffin cells (Huh et al. 2005a), it is possible to estimate the relative concentrations and total amounts of each IP₃R isoform in subcellular organelles of these cells (Huh et al. 2005c). By taking the respective IP₃R concentration in the nucleus as 1, the relative concentrations of the IP₃R1 in the endoplasmic reticulum (ER) and secretory granules are shown to be 0.92–1.20 and 3.68–3.77, respectively (Huh et al. 2005c; Huh and Yoo 2003). Likewise, the relative concentrations of the IP₃R2 in the ER and secretory granules are shown to be 0.87–1.55 and 3.05–3.48, respectively, while those of the IP₃R3 in the ER and secretory granules are estimated to be 0.74–0.94 and 1.88–3.32, respectively (Huh et al. 2005c; Huh and Yoo 2003). These results indicate that the concentrations of the IP₃Rs in secretory granules are 2–3-fold higher than those of the ER.

Furthermore, the relative amounts of each IP₃R isoform present in each organelle were also estimated. Secretory granules of bovine chromaffin cells are shown to contain ~69% of total cellular IP₃R1 while the ER contains ~15% (Huh et al. 2005c). Moreover, secretory granules contain ~64 and ~58% of total cellular IP₃R2 and IP₃R3, respectively (Huh et al. 2005c), while the ER contains ~20 and ~16% of total cellular IP₃R2 and IP₃R3, respectively (Huh et al. 2005c). These estimates show that the majority (58–69%) of cellular IP₃Rs of bovine chromaffin cells is expressed in secretory granules. Though it is likely that the percentages of cellular IP₃Rs expressed in secretory granules of other secretory cells will be lower than those of chromaffin cells given that the number of secretory granules in many secretory cells is lower than that of chromaffin cells, it nevertheless points out that the major portion of cellular IP₃Rs is expressed in secretory granules. Considering that the relative concentrations of IP₃Rs in secretory granules are 2–3-fold higher than those of the ER, it appears highly likely that secretory granules of secretory cells contain the most IP₃Rs in most, if not all, types of secretory cells.

Interestingly, the relative concentrations of each IP₃R isoform present in secretory granules that had been induced in otherwise nonsecretory NIH3T3 cells have also been shown to be 3–5-fold higher than those in the nucleus and ER (Huh et al. 2005b). Along with the expression patterns of the IP₃Rs in the subcellular organelles of chromaffin cells, these results suggest that chromogranin-expressing secretory granules have intrinsic tendency to accumulate the IP₃R/Ca²⁺ channels in the granule membranes, presumably due to the IP₃R-binding properties of chromogranins A and B (Yoo et al. 2000) (see below). Though it is not possible to accurately estimate the relative amounts of each IP₃R isoform in the subcellular organelles of the nonsecretory cells in which the formation of secretory granules is induced, owing to lack of information on the cell volume of each organelle, it appeared nonetheless apparent that secretory granules contain more of all three isoforms of IP₃R than either the ER or the nucleus (Huh et al. 2005b).

Chromogranins in Secretory Granules

Besides the usual hormones, chromogranins A and B are the major constituents of most secretory granules, followed by significantly less abundant secretogranin II (Helle 2000; Montero-Hadjadje et al. 2008; Taupenot et al. 2003; Winkler and Fischer-Colbrie 1992). Hence these proteins are considered marker proteins of secretory granules, the signature organelle of a variety of secretory cells. Though the granin proteins are expressed in secretory granules of secretory cells, not all secretory granules express the same combinations of the granin proteins (Helle 2000; Montero-Hadjadje et al. 2008; Taupenot et al. 2003; Winkler and Fischer-Colbrie 1992). Bovine chromaffin cells express predominantly chromogranin A, followed by chromogranin B and secretogranin II, whereas humans and rats express more chromogranin B than other granins.

The CGA concentration in bovine chromaffin granules is known to be >1800 μM (Simon and Aunis 1989; Winkler and Westhead 1980) while that in the ER is estimated to be >140 μM, whereas the CGB concentrations in the bovine chromaffin granules and the ER are estimated to be >200 and >120 μM, respectively (Yoo 2010). Unlike the more abundant chromogranins A and B, the third member of the granin protein family secretogranin II is expressed significantly less than chromogranins, and the amounts of SgII present in secretory granules and the ER of bovine chromaffin cells are estimated to be >30 and >10 μM, respectively (Yoo 2010). Taken together, chromaffin granules appear to contain ~6-fold more granin proteins than the ER, containing >2 mM compared to >0.3 mM of the ER. Interestingly, chromogranin B and secretogranin II, but not chromogranin A, are also expressed in the nucleus (Yoo et al. 2002, 2007), localizing in the numerous small IP₃-sensitive nucleoplasmic Ca²⁺ store vesicles (Huh et al. 2006; Yoo et al. 2005).

Chromogranin A binds 32 mol of Ca²⁺/mol of protein with a dissociation constant (Kd) of 4.0 mM at a near physiological pH 7.5, but this Ca²⁺-binding capacity increases to 55 mol of Ca²⁺/mol with a Kd of 2.7 mM at the intragranular pH 5.5 (Yoo and Albanesi 1991). This pattern of more Ca²⁺ binding at pH 5.5 is also observed in chromogranin B that it binds 50 mol of Ca²⁺/mol with a Kd of 3.1 mM at pH 7.5, but this increases to 93 mol of Ca²⁺/mol with a Kd of 1.5 mM at pH 5.5 (Yoo et al. 2001, 2007). Similar pattern is also shown with secretogranin II that it binds 30 mol Ca²⁺/mol with a Kd of 3.0 mM at pH 7.5 but binds 61 mol Ca²⁺/mol with a Kd of 2.2 mM at pH 5.5 (Yoo et al. 2007). These results indicate that the granin proteins can bind significantly more Ca²⁺ at the intragranular pH 5.5 than at a near physiological pH, and this difference in the Ca²⁺-binding properties of the granin proteins at different pH milieus will apparently affect the Ca²⁺ storage capacity of these proteins in secretory granules and the ER. Hence, combined with the markedly higher concentrations of chromogranins and secretogranin II in secretory granules, the acidic intragranular pH will greatly facilitate more Ca²⁺ binding by the granin proteins, thereby resulting in 40 mM Ca²⁺ in secretory granules (Haigh et al. 1989; Winkler and Westhead 1980) compared to 3 mM Ca²⁺ in the ER (Meldolesi and Pozzan 1998; Garcia et al. 2006) that maintains a physiological pH.

Moreover, in accordance with the higher Ca²⁺ storage capacity at the acidic intragranular pH, even a slight increase in the intragranular pH by an alkalinization agent changes the Ca²⁺ storage property in secretory granules and induces Ca²⁺ releases from secretory granules (Camacho et al. 2006, 2008; Haynes et al. 2006; Mundorf et al. 1999, 2000), which has been shown to be sufficient to initiate secretory processes in bovine chromaffin cells (Camacho et al. 2008; Mundorf et al. 2000). Under normal conditions, most (>99.9%) of the intragranular Ca²⁺ stays bound to chromogranins and only a very small amount of granular Ca²⁺ stays in free state, ranging in 20–100 μM (Bulenda and Gratzl 1985; Gerasimenko et al. 1996; Mitchell et al. 2001; Nguyen et al. 1998; Quesada et al. 2003; Santodomingo et al. 2008). However, an increase in intragranular pH will reduce the degree of calcium binding by the granular chromogranins and increase the concentrations of free Ca²⁺, a condition conducive for release of more Ca²⁺ into the cytoplasm.

Interaction of the IP₃R by Chromogranins and IP₃R/Ca²⁺ Channel Modulation

As though to complement the presence of other parties in the same organelle, the IP₃R/Ca²⁺ channels and chromogranins work side by side to coordinate the IP₃-dependent intracellular Ca²⁺ storage and control roles of secretory granules. In addition to the high-capacity Ca²⁺ storage roles of chromogranins, both chromogranins A and B directly interact with the IP₃Rs and modulate the IP₃R/Ca²⁺ channels (Thrower et al. 2002, 2003; Yoo et al. 2000; Yoo and Jeon 2000). Yet chromogranins A and B differ significantly in both their IP₃R binding strength and the IP₃R/Ca²⁺ channel modulating activity (Figs. 1, 2).

Fig. 1.

pH-dependent interaction of IP₃R with chromogranin A and single channel activity of the IP₃R/Ca²⁺ channel. a Purified IP₃R (0.5–0.7 μg) was reacted with glutathione S-transferase (GST)–CGA fusion protein in the presence (2 mM) and absence of Ca²⁺ at pH 5.5 and 7.5, respectively. The bound IP₃R was separated on a 7.5% SDS gel and immunoblotted using the IP₃R antibody. Notice that CGA dissociated completely at pH 7.5. b Single channel activity of the IP₃R incorporated into planar lipid bilayers in the absence and presence of CGA. Trace i, IP₃R single channel activated by adding 2 μM IP₃ to the cis compartment. Openings are defined as downward deflections from the base line. Trace ii, conditions are same as for trace i, except that CGA (1 μg) was added to the trans compartment at pH 5.5. Trace iii, the pH of the trans compartment was changed to 7.5. The open probability (P ₀) and the mean open time of the channel are shown. a and b are modified from (Yoo et al. 2000) and (Thrower et al. 2002), respectively

Fig. 2.

pH-dependent interaction of IP₃R with chromogranin B and single channel activity of the IP₃R/Ca²⁺ channel. a Purified IP₃R (0.5–0.7 μg) was reacted with glutathione S-transferase (GST)–CGB fusion protein in the presence (2 mM) and absence of Ca²⁺ at pH 5.5 and 7.5, respectively. The bound IP₃R was separated on a 7.5% SDS gel and immunoblotted using the IP₃R antibody. Notice that some CGB still remained bound at pH 7.5. b Single channel activity of the IP₃R incorporated into planar lipid bilayers in the absence and presence of CGB. Trace i, IP₃R single channel activated by adding 2 μM IP₃ to the cis compartment. Openings are defined as downward deflections from the base line. Trace ii, conditions are same as for trace i, except that CGB (1 μg) was added to the trans compartment at pH 5.5. Trace iii, the pH of the trans compartment was changed to 7.5. The open probability (P ₀) and the mean open time of the channel are shown. a and b are modified from (Yoo et al. 2000) and (Thrower et al. 2003), respectively

Chromogranin A interacts with the IP₃R only at pH 5.5, but dissociates completely from the IP₃R at pH 7.5 (Fig. 1a), whereas chromogranin B interacts with the IP₃R at both pH 5.5 and 7.5 (Fig. 2a), although the interaction at pH 7.5 is significantly weaker than at pH 5.5. This means that only CGB will bind the IP₃Rs in the ER while both CGA and CGB bind the IP₃Rs in secretory granules. Underscoring the physiological significance of the CGB interaction with the IP₃Rs at pH 7.5, the CGB–IP₃R coupling in the ER has indeed been shown to play essential roles in controlling the cytoplasmic Ca²⁺ concentrations (Choe et al. 2004). Since the interaction of chromogranins to the IP₃R changes the conformation of the IP₃R/Ca²⁺ channel that it becomes a more ordered structure better suited for Ca²⁺ release in response to IP₃ (Thrower et al. 2002, 2003; Yoo et al. 2000; Yoo and Jeon 2000), coupling of chromogranins to the IP₃R/Ca²⁺ channel greatly increases the channel open probability and the mean open time.

The modulatory role of chromogranins on the IP₃R/Ca²⁺ channels was first demonstrated in Ca²⁺ flux studies using CGA-encapsulated liposomes (Yoo and Jeon 2000). The IP₃R/Ca²⁺ channels that are incorporated in the liposome opened and released intraliposomal Ca²⁺ in response to IP₃, and the amount of Ca²⁺ released through the IP₃R/Ca²⁺ channel was shown to be far greater in the presence of coupled chromogranins than in the absence (Thrower et al. 2003; Yoo and Jeon 2000). Further, in planar bilayer single channel recording studies the IP₃R/Ca²⁺ channel was shown to have an open probability of 4% and a mean open time of 8.6 ms (Thrower et al. 2002) (Fig. 1b). But when chromogranin A was coupled with the IP₃R/Ca²⁺ channel at pH 5.5, the open probability increased to 33%, a 8.2-fold increase, and the mean open time of the channel increased to 71.7 ms, a 8.3-fold increase (Thrower et al. 2002). However, when the pH was changed to 7.5, the IP₃R/Ca²⁺ channel-activating effect of CGA virtually disappeared and the channel open probability and the mean open time returned to 3% and 10.0 ms, respectively (Fig. 1b), due to complete dissociation of CGA from the IP₃R (Fig. 1a).

The IP₃R/Ca²⁺ channel-activating effect of CGB is more pronounced than that of CGA: when CGB was coupled to the IP₃R/Ca²⁺ channel at pH 5.5, the channel open probability increased from 5 to 80%, a 16-fold increase, and the mean open time increased from 5.2 to 217.4 ms, a 42-fold increase (Thrower et al. 2003) (Fig. 2b). But in contrast to CGA that dissociates completely from the IP₃Rs at pH 7.5, CGB remains coupled at pH 7.5, albeit to a reduced degree (Yoo et al. 2000) (Fig. 2a). As though to reflect the weakened interaction at pH 7.5, the channel-activating effect of CGB at pH 7.5 was approximately half of that shown at pH 5.5, increasing the channel open probability 8-fold and the mean open time 24-fold (Thrower et al. 2003) (Fig. 2b). Yet the channel-activating effect of CGB at pH 7.5 was still greater than that shown with CGA at pH 5.5 (Thrower et al. 2002, 2003). The prominent role of CGB and the IP₃R/Ca²⁺ channels is also manifested in neurons of hippocampus (Jacob et al. 2005; Nicolay et al. 2007) and in the neurites of neuronally differentiated PC12 cells (Johenning et al. 2002) that CGB and the IP₃Rs are shown to play key roles in controlling the IP₃-dependent Ca²⁺ concentrations in these cells.

Conclusion

The presence of chromogranins appears to be critical in the IP₃-dependent intracellular Ca²⁺ control in secretory cells. Considering that the main function of secretory cells is to store and release a variety of bioactive molecules in response to subtle changes in cytoplasmic Ca²⁺ concentrations, under no other molecular arrangements in secretory granules is the coupling between the major intracellular Ca²⁺ channels in the form of the IP₃R/Ca²⁺ channel and the proteins that double as the Ca²⁺ storage and the Ca²⁺ channel modulating proteins expected to control more effectively the IP₃-dependent intracellular Ca²⁺ concentrations. Hence, consistent with the substantially higher concentrations of chromogranins in secretory granules than in the ER and the greater IP₃R/Ca²⁺ channel-activating effects of chromogranins at pH 5.5 than at the pH of the ER, the secretory granule IP₃R/Ca²⁺ channels are at least 6–7-fold more sensitive to IP₃ than those of the ER (Huh et al. 2007; Yoo 2010).

The crucial roles of chromogranins and the IP₃R/Ca²⁺ channels uncovered in chromaffin cells are also presumed to apply to other secretory cells. Secretory granules of pancreatic β-cells contain not only chromogranins (Giordano et al. 2008; Hutton 1989; Winkler and Fischer-Colbrie 1992) but also all the IP₃R isoforms, and the IP₃-induced Ca²⁺ release from the granules has been demonstrated to initiate insulin secretion (Srivastava et al. 1999; Xie et al. 2006). In addition, chromogranin B has recently been shown to be expressed in cardiomyocytes that are not considered traditional secretory cells, and was identified as a key regulator of the IP₃-dependent Ca²⁺ release in cardiomyocytes (Heidrich et al. 2008), which has a strong implication in cardiac hypertrophy and heart failure. The significance of chromogranin–IP₃R coupling is further confirmed in induced secretory granules, which result from transfected chromogranins in nonsecretory cells (Beuret et al. 2004; Huh et al. 2003; Kim et al. 2001), that the IP₃Rs are cotargeted with chromogranins to the secretory granule membranes (Huh et al. 2005b). Taken together, these results suggest that the physiological partnership between chromogranins and the IP₃R/Ca²⁺ channels in the Ca²⁺ homeostasis goes beyond neuroendocrine cells to include other non-traditional secretory cells, such as cardiomyocytes (Heidrich et al. 2008) and astrocytes (Hur et al. 2010).