Inositol 1,4,5-Trisphosphate Receptor/Ca2+Channel Modulatory Role of Chromogranin A, a Ca2+Storage Protein of Secretory Granules
Journal of Biological Chemistry
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.
... 69, 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.