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

Abstract

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. 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 secretory (14.Yoo S.H. J. Biol. Chem. 1994; 269: 12001-12006Abstract Full Text PDF PubMed Google Scholar) but also in IP3-mediated Ca2+ in the cell. we have in this the physiological significance of CGA coupling to the IP3R IP3R-reconstituted liposomes in the presence and absence of CGA, and found that CGA coupling to the IP3R in the structural changes of IP3R as to not the IP3 binding but also the Ca2+ release of the from and further from of in at and IP3 from from of the Chromogranin A from bovine adrenal was from the secretory of chromaffin cells as (16.Yoo S.H. Albanesi J.P. J. Biol. Chem. 1990; 265: 14414-14421Abstract Full Text PDF PubMed Google Scholar). IP3R peptide in to of the type IP3R T. S. Miyawaki A. K. N. Mikoshiba K. Nature. 1989; 342: PubMed Scopus Google Scholar, G.A. III, Südhof T.C. J. Biol. Chem. 1990; 265: Full Text PDF PubMed Google Scholar) was with a A antibody was in the peptide coupled to and the peptide the (14.Yoo S.H. J. Biol. Chem. 1994; 269: 12001-12006Abstract Full Text PDF PubMed Google Scholar). with of pH and and into in a followed by in a The at at and the at to the membrane The was in pH and to the membrane protein concentration of The membrane was and at at The was with an of pH and and to an IP3R with The IP3R was by coupling of affinity purified IP3R antibody (14.Yoo S.H. J. Biol. Chem. 1994; 269: 12001-12006Abstract Full Text PDF PubMed Google Scholar) to of the protein A from the protein A or to of to the 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 in pH The was with of to proteins, and the IP3R was by of pH and The was by pH and with an of pH and and to a with to from the IP3R The IP3R was and at The purified bovine IP3R bound of of which is to 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 N. Niinobe M. Mikoshiba K. EMBO J. 1990; 9: 61-67Crossref PubMed Scopus (232) Google Scholar), as to the N. Niinobe M. Mikoshiba K. EMBO J. 1990; 9: 61-67Crossref PubMed Scopus (232) Google Scholar). bovine bovine and in to a of and The concentration was in a of The was a of and the was by The in A or pH and pH and IP3R and CGA The of A or at The by at at and with pH and to The with pH and at The to The of was and by of was as a function of time the at measured with the The of the liposomes was by S. J. Chem. 1993; PubMed Scopus Google Scholar). The to contain and at least as by in To the of CGA into we the fluorescence of which is and to of was with of at in the presence or absence of and of pH was to the to the To the was to and was at the with the the at was measured at the of In the of CGA was also by the and with and followed by of the These not the of CGA in the but also indicated that the presence of CGA not the the of IP3R into the liposomes not of Ca2+ was to the T. T. Biochemistry. 1990; PubMed Scopus Google Scholar). Ca2+ was the fluorescence of Ca2+ from the was by the fluorescence changes of at by a with a The fluorescence was measured at the of of with of and of the of Ca2+ the of indicated concentration of IP3 to of the The of to Ca2+ T. 1989; PubMed Scopus Google Scholar). In to the of IP3R antibody the Ca2+ release from the IP3R-reconstituted the IP3R was with the at the of of or of IP3R the at the time of Ca2+ release was with the IP3 release of also measured by the fluorescence of of IP3 and with the fluorescence of of To the of Ca2+ of Ca2+ was in these which was a high concentration to the with of IP3 as of at of was a from with of at The of was by a and with the control the fluorescence quenching of Trp in IP3R by a of to was to the the concentration of and the fluorescence at the of was measured with the at at To the IP3R with and fluorescein or from was to of IP3R, and the was to at of the an of and was to the or IP3R molecules by and The receptor molecules reconstituted into the liposomes with Ca2+ in the presence and absence of CGA, and the fluorescence energy transfer was at by the of in the of at the of the of The in the presence to in pH to the To Ca2+ bound to the the was to with of the the liposomes by The was and of at The with indicated concentration of and at further of the in the presence of of IP3 in the the was with The liposomes by at at followed by in The was with and the of and was by acid to the The of by J. PubMed Scopus Google Scholar). The of the as the at at and at and at To physiological pH the inside pH of the liposomes was at 5.5 that of was at To the inside pH of the is at pH was in the and fluorescence increase to pH was measured shown in of the to the pH 5.5 of the by not the fluorescence of of the to pH or 7.5 of the liposome the fluorescence of indicating the of pH 5.5 inside the This indicated that the inside pH of the was suggesting the of these subsequent The fluorescence was measured at with the of in the presence of A the time of Ca2+ from the IP3R-reconstituted as by the fluorescence of at is a Ca2+ which is to membrane and has a Ca2+ the fluorescence at that at the IP3 Ca2+ was from the with the and the fluorescence changes a of IP3 further increase of the fluorescence was by more IP3 not The of Ca2+ by IP3 was to be of the Ca2+concentration the fluorescence the liposomes by also the of Ca2+ release the IP3R the IP3R of IP3R at the of of of IP3R of the Ca2+ release. of more IP3R at the of the Ca2+ release more A and CGA was inside the the Ca2+ release was with the and the a of the presence of CGA whereas the of IP3 a in the absence of CGA These results that the of IP3R becomes more as a of CGA the pH of inside the liposome was at 7.5 the of Ca2+ release was to that in the absence of CGA our previous 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, S.H. Lewis M.S. Biochemistry. 1995; 34: 632-638Crossref PubMed Scopus (42) Google Scholar). a control IP3R was with or of the in the presence or absence of CGA, and the fluorescence was measured to the of CGA the of reconstituted In we not in the fluorescence of the reconstituted indicating that the of IP3R reconstituted was not by the presence of CGA not it has also been shown that from the IP3R can be by Ca2+ M. M. Nature. 1992; PubMed Scopus Google Scholar), was a that the Ca2+ release might not IP3-mediated Ca2+ release. To this we the of Ca2+ of indicated concentration of the presence of of which is a high concentration to the Ca2+ shown in presence of CGA at pH 5.5 significantly the amounts of with in the absence of CGA, the in the amounts of Ca2+ in as IP3 increased. the presence of CGA to this the pH of the liposomes was To the release of Ca2+ in the presence of CGA is to binding of IP3 to the the of IP3 bound to the IP3R was as a function of IP3 shown in the presence of CGA enhanced the that in the absence of CGA. the IP3R was a of mol of IP3 to bind mol of CGA was also inside the liposomes at the intravesicular pH of the IP3 binding to the shown with the IP3R the pH of the liposomes was CGA the IP3 binding to the the was the of the presence of CGA, indicating that the affinity of IP3R IP3 has not To conformational changes of the IP3R by interaction with CGA, we quenching of the IP3R Trp fluorescence by shown in the IP3R Trp fluorescence was by of the presence of CGA. The fluorescence at was and the results to the PubMed Scopus Google is the in the absence of is the in the presence of is the quenching and is the concentration of from the was the reconstituted IP3R in the absence of CGA. This to CGA was this it is that at least Trp of the IP3R exposed to the CGA is and that CGA conformational changes of In to further examine the of CGA the property of IP3R in the reconstituted we the fluorescence energy transfer the and IP3Rs shown in the reconstituted IP3Rs fluorescence energy transfer as by the increase of fluorescence the This indicated that the IP3R molecules have a to exist as in the CGA was in the the by suggesting that the of IP3Rs was enhanced by interaction with CGA. In of the of the Ca2+ release of IP3R T. A. T. K. T. M. EMBO J. PubMed Scopus Google Scholar), we the of the in response to a of IP3 in the presence of of Ca2+ shown in IP3 Ca2+ in the presence of CGA in the absence in the Ca2+ concentration of in the Ca2+ concentration the in the of Ca2+ indicating the of Ca2+ the Ca2+ release property of These results clearly that IP3 amounts of Ca2+ from the in the presence of CGA is of Ca2+ in the liposome a control we measured Ca2+ release release of Ca2+ be in the presence of to Ca2+ not the that demonstrated the Ca2+ release from chromaffin granules (1.Yoo S.H. Albanesi J.P. J. Biol. Chem. 1990; 265: 13446-13448Abstract Full Text PDF PubMed Google Scholar), Petersen and (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) have shown the IP3-mediated Ca2+ release from the granules, the of secretory granules of and neuroendocrine cells as the IP3-sensitive intracellular Ca2+ store (1.Yoo S.H. Albanesi J.P. J. Biol. Chem. 1990; 265: 13446-13448Abstract Full Text PDF PubMed Google Scholar, S. 1995; Full Text PDF PubMed Scopus Google Scholar). Recently, indicating direct participation of secretory granule calcium in the control of cytoplasmic Ca2+ has been 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); the 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 secretory granules resulted in the simultaneous increase of cytoplasmic Ca2+ concentration in the immediate vicinity of the secretory granules, clearly demonstrating the participation of secretory granules in the control of cytoplasmic Ca2+concentration. in cells, and neuroendocrine cells, and known to be proteins of neuroendocrine cells H. 1992; PubMed Scopus Google Scholar, 1995; PubMed Scopus Google Scholar, J. 1989; PubMed Scopus Google Scholar, W.B. P. Sci. 1991; Full Text PDF PubMed Scopus Google Scholar). In the secretory granules of bovine adrenal chromaffin cells, in J. 1989; PubMed Scopus Google Scholar, H. PubMed Scopus Google Scholar), and bind S.H. Albanesi J.P. 1990; PubMed Scopus Google Scholar), J.S. M.S. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar), in to Ca2+ M. Biochemistry. PubMed Scopus Google Scholar, M. PubMed Scopus Google Scholar). Chromogranin A binds mol of with of at pH and mol of with of at pH 5.5 S.H. Albanesi J.P. J. Biol. Chem. 1991; 266: 7740-7745Abstract Full Text PDF PubMed Google Scholar). to the high Ca2+ binding of chromogranins, of intravesicular calcium bound to chromogranins, in the Ca2+ concentration of M. Biochemistry. PubMed Scopus Google Scholar). In of the that the secretory granules of bovine chromaffin H. PubMed Scopus Google Scholar), the calcium storage of the secretory granules be roles in the control of calcium in the In this the high capacity, low affinity Ca2+ binding property of to be the secretory granules to store an large of It that the secretory granules roles in the intracellular Ca2+ the that the secretory granule of adrenal medullary chromaffin cells is a major IP3-sensitive intracellular Ca2+ store (1.Yoo S.H. Albanesi J.P. J. Biol. Chem. 1990; 265: 13446-13448Abstract Full Text PDF PubMed Google Scholar) the presence of IP3R the secretory granule the presence of IP3R the secretory granule membrane was not the intravesicular matrix protein CGA was shown to interact with several integral membrane proteins of secretory granules including the IP3R (14.Yoo S.H. J. Biol. Chem. 1994; 269: 12001-12006Abstract Full Text PDF PubMed Google Scholar), which was the first demonstrating a coupling an ion channel and a cognate ion storage protein. it was not known the coupled CGA the IP3R/Ca2+ The results in the the purified IP3R and CGA to the important roles these molecules in the cytoplasm, which the secretory granules to control the intracellular Ca2+ in neuroendocrine The Ca2+ in and that the of Ca2+ in response to of IP3 was significantly enhanced as a of coupled CGA. These results not a direct coupling but also a functional coupling the IP3R/Ca2+ channel and CGA at the intravesicular pH of at a near physiological pH of CGA to the IP3-mediated and the of the intravesicular pH in the coupling of CGA to the IP3R can bind not exist in a state in the four molecules of or of IP3R interact with to form a or a heterotetrameric IP3R (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), a CGA is also known to form a at the near physiological pH 7.5 and a at the intravesicular pH 5.5 (17.Yoo S.H. Lewis M.S. J. Biol. Chem. 1992; 267: 11236-11241Abstract Full Text PDF PubMed Google Scholar). Further, a tetrameric CGA has also been shown to bind four molecules of intraluminal loop peptide of IP3R (19.Yoo S.H. Lewis M.S. Biochemistry. 1995; 34: 632-638Crossref PubMed Scopus (42) Google Scholar), suggesting the interaction a tetrameric CGA and a tetrameric the results the direct interaction the purified IP3R and CGA (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), with the of IP3R and CGA (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), strongly the of this coupling in the intracellular Ca2+ control in the cell. In of the that CGA and IP3R interact with at pH 5.5 and dissociate at pH 7.5 (14.Yoo S.H. J. Biol. Chem. 1994; 269: 12001-12006Abstract Full Text PDF PubMed Google Scholar, 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), the of of CGA the IP3-mediated Ca2+ release at pH 7.5 is to the of CGA to with the This is also in the that the number of to the IP3R the the liposome pH was at of the presence of CGA Furthermore, the in that the of can be that molecules of IP3 IP3R at the in that a of Ca2+ can be molecules of IP3 bound tetrameric IP3R, a of molecules of IP3 can be bound tetrameric IP3R at IP3 This is in with previous which indicated that of IP3 the IP3R/Ca2+ which four J. J. 1991; PubMed Google Scholar). it has also been suggested that three or more IP3 molecules to the IP3R/Ca2+ channel T. Science. 1988; PubMed Scopus Google Scholar), the is that IP3 is to the IP3R/Ca2+ channel J. J. 1991; PubMed Google Scholar, P. G.A. J. 1990; PubMed Google Scholar, P.F. Baraban J.M. Supattapone S. Snyder S.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the pH of intravesicular of secretory vesicles of neuroendocrine cells is 5.5 J. Biol. Chem. Full Text PDF PubMed Google Scholar, Biochemistry. PubMed Scopus Google Scholar) and that of the or is the coupling of IP3R, an integral membrane and CGA, an intravesicular matrix at pH 5.5 and at pH 7.5 in with the physiological of the these molecules as from the the secretory vesicles to the the secretory be exposed to a pH of the which a pH of This of pH from the intravesicular pH of 5.5 to of the from the the secretory to into the and to the The fluorescence energy transfer suggested that the enhanced of Ca2+ release is to the structural of the IP3R by coupled CGA. This suggested that the presence of CGA in the liposome changes the of the IP3R, changing it to a structure that is better suited to IP3 binding and subsequent Ca2+ release. Furthermore, quenching to the Trp fluorescence of the IP3R also indicated that at least Trp of the IP3R exposed CGA is it that CGA roles in the Ca2+ release property of the IP3R in neuroendocrine cells, not by binding and of the intravesicular Ca2+ but also by the channel of the IP3 binding to the IP3R has been known to conformational changes of the IP3R G.A. Südhof T.C. EMBO J. 1990; 9: PubMed Scopus Google Scholar). Further, it has been known that CGA in pH (16.Yoo S.H. Albanesi J.P. J. Biol. Chem. 1990; 265: 14414-14421Abstract Full Text PDF PubMed Google Scholar) and that CGA in and affinity S.H. Albanesi J.P. J. Biol. Chem. 1991; 266: 7740-7745Abstract Full Text PDF PubMed Google Scholar). In of these it is to that the conformational changes of the IP3R that as a of IP3 binding be to the coupled CGA in the secretory granules, conformational It be that the conformational changes in CGA in of affinity Ca2+ from the protein and release the IP3R channel to the In of the that the secretory granules contain to Ca2+ and of it bound to in the secretory granules M. Biochemistry. PubMed Scopus Google Scholar), the coupling of calcium storage proteins to the to the and structural of an intracellular Ca2+ store Ca2+ function be and the Ca2+ store to to the changing cytoplasmic Ca2+ Moreover, the into the of Ca2+ control of secretory granules is to further of Ca2+ of intracellular Ca2+ the physiological of cells to control the the coupling of Ca2+ storage proteins to to be a T. H. with the and S. H. So in the IP3R and