Localization of three types of the inositol 1,4,5-trisphosphate receptor/Ca2+ channel in the secretory granules and coupling with the Ca2+ storage proteins chromogranins A and B

Although the role of secretory granules as the inositol 1,4,5-trisphosphate (IP3)-sensitive intracellular Ca2+ store and the presence of the IP3 recep tor (IP3R)/Ca2+ channel on the secretory granule membrane have been establi shed, the identity of the IP3R types present in the secretory granules is n ot known. We have therefore investigated the presence of different types of IP3R in the secretory granules of bovine adrenal medullary chromaffin cell s using immunogold electron microscopy and found the existence of all three types of IP3R in the secretory granules. To determine whether these IP(3)R s interact with CGA and CGB, each IP3R isoform was cotransfected with CGA o r CGB into NIH3T3 or COS-7 cells, and the expressed IP3R isoform. and CGA o r CGB were co-immunoprecipitated. From these studies it was shown that all three types of IP3R form complexes with CGA and CGB in the cells. To furthe r confirm whether the IP3R isoforms and CGA and CGB form a complex in the s ecretory granules the potential interaction between all three isoforms of I P3R and CGA and CGB was tested by co-immunoprecipitation experiements of th e mixture of secretory granule lysates and the granule membrane proteins. T he three isoforms of IP3R were shown to form complexes with CGA and CGB, in dicating the complex formation between the three isoforms of IP3R and CGA a nd CGB in the secretory granules. Moreover, the pH-dependent Ca2+ binding p roperty of CGB was also studied using purified recombinant CGB, and it was shown that CGB bound 93 mol of Ca2+/mol with a dissociation constant (K-d) of 1.5 mm at pH 5.5 but virtually no Ca2+ at pH 7.5. The high capacity, low affinity Ca2+-binding property of CGB at pH 5.5 is comparable with that of CGA and is in line with its role as a Ca2+ storage protein in the secretor y granules.