Characterization of a cytosolic and a luminal Ca2+ binding site in the type I inositol 1,4,5-trisphosphate receptor

To study the Ca2+ regulation of the inositol 1,4,5-trisphosphate receptor (InsP(3)R) at the molecular level, we expressed various cytosolic and luminal regions of the mouse type I InsP(3)R as glutathione S-transferase fusion proteins. Ca-45(2+) and ruthenium red overlay studies and Stains-all spectra and staining revealed both a cytosolic and a luminal Ca2+ binding site, The luminal Ca2+ binding site was mapped to the nonconserved acidic subregion of the luminal loop between amino acids 2463 and 2528, A K-0.5 of 0.3 mu M and a Hill coefficient of 1.1 were determined by Ca-45(2+) overlay by quantification of Ca-45(2+) binding on blots, The cytosolic Ca2+ binding site was localized in a region just preceding the transmembrane domain M1. The Ca2+ binding was mapped to a 23-amino acid stretch between amino acids 2124 and 2146, This cytosolic region showed a single high affinity site for Ca2+, with a K-0.5, of 0.8 mu M and a Hill coefficient of 1.0, Neither of the identified Ca2+ binding regions contained an EF-hand motif, We conclude that the type I InsP(3)R has at least two quite distinct types of Ca2+ binding sites, which are localized in different structural regions of the protein.