Characterization of a Cytosolic and a Luminal Ca2+ Binding Site in the Type I Inositol 1,4,5-Trisphosphate Receptor

To study the Ca 2+ 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. 45 Ca 2+ and ruthenium red overlay studies and Stains-all spectra and staining revealed both a cytosolic and a luminal Ca 2+ binding site. The luminal Ca 2+ 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 μ M and a Hill coefficient of 1.1 were determined by 45 Ca 2+ overlay by quantification of 45 Ca 2+ binding on blots. The cytosolic Ca 2+ binding site was localized in a region just preceding the transmembrane domain M1. The Ca 2+ 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 Ca 2+ , with a K 0.5 of 0.8 μ M and a Hill coefficient of 1.0. Neither of the identified Ca 2+ binding regions contained an EF-hand motif. We conclude that the type I InsP 3 R has at least two quite distinct types of Ca 2+ binding sites, which are localized in different structural regions of the protein.