Structural and functional analyses were used to investigate the regulation of the inositol 1,4,5-trisphosphate (InsP 3 ) receptor (InsP 3 R) by Ca 2؉ . To define the structural determinants for Ca 2؉ binding, cDNAs encoding GST fusion proteins that covered the complete linear cytosolic sequence of the InsP 3 R-1 were expressed in bacteria. The fusion proteins were screened for Ca 2؉ and ruthenium red binding through the use of 45 The inositol 1,4,5-trisphosphate receptor (InsP 3 R) 1 is an intracellular Ca 2ϩ release channel that is modulated by various physiological ligands such as inositol 1,4,5-trisphosphate (InsP 3 ), Ca 2ϩ , nucleotides, calmodulin (CaM), FK506BP, phosphatases, and kinases (reviewed in Refs. 1 and 2). The InsP 3 R can be divided into three functionally different domains as follows: an N-terminal ligand-binding domain, a modulatory domain, and a channel domain near the C terminus (3, 4). Among the various modulators, Ca 2ϩ itself plays a pivotal role and may be considered as a co-agonist exerting both positive and negative effects on InsP 3 -induced Ca 2ϩ release. Cytosolic Ca 2ϩ has a bell-shaped effect on the InsP 3 R, with low concentrations stimulating the release and high concentrations inhibiting the release (5-8). Both phases are time-dependent, but the inhibition develops more slowly than the stimulation (6, 9 -11). Although these kinetically distinct effects are believed to be crucial for the generation of Ca 2ϩ oscillations and waves (12), little is known about how Ca 2ϩ interacts with the InsP 3 R. Regulation might be exerted by direct binding of Ca 2ϩ to the InsP 3 R and/or via Ca 2ϩ -sensitive protein(s), e.g. via Ca 2ϩ /CaMdependent protein kinase II and protein phosphatase 2B (13) or via a Ca 2ϩ -sensitizing factor (14). Studies in which effects of Sr 2ϩ were compared with those of Ca 2ϩ revealed that at least two interaction sites must exist: a stimulatory site that is modestly sensitive to Sr 2ϩ and an inhibitory site that is nearly insensitive to Sr 2ϩ (15). One Ca 2ϩ -binding domain has been localized on the cytosolic side of the InsP 3 R-1 (16, 17), but whether this domain represents a stimulatory or an inhibitory interaction site is still unclear. Luminal Ca 2ϩ also regulates the InsP 3 -induced Ca 2ϩ release. Loading of the Ca 2ϩ stores results in a relatively more pronounced InsP 3 -induced Ca 2ϩ release (18 -21). A high affinity Ca 2ϩ -binding site was detected on the luminal loop (17), but its functional significance is not yet clear.The aim of the present work was two-fold. First, we wanted to investigate, at the structural level, the presence of direct Ca 2ϩ -binding sites on InsP 3 R-1. Second, we wanted to functionally demonstrate multiple Ca 2ϩ interactions on the InsP 3 R by a kinetic analysis of the effects of Ca 2ϩ and Sr 2ϩ on the InsP 3 -induced Ca 2ϩ release.For the first part of this study we constructed and expressed a number of GST fusion proteins that contain cytosolic fragments of the InsP 3 R sequence. The ability of these fusion proteins to bind ...

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(14,15).The N-terminal part of the IP 3 R contains all the structural determinants responsible for specific and selective binding of its physiological agonist, IP 3 (17-20). We have therefore expressed the N-terminal ligand-binding site (first 581 amino acids) of the mouse IP 3 R-1 in Escherichia coli, using a strategy of growth and expression at low temperatures, as described previously by Yoshikawa et al. (20). This protein contains a previously identified Ca 2ϩ -binding region located between amino acids 304 -450 (21). We now demonstrate that Ca 2ϩ and calmodulin can both inhibit IP 3 binding to this recombinant protein and that these inhibitors act independently and additively. Our data indicate that the N-terminal ligand-binding domain of IP 3 R-1 contains regulatory regions directly interacting with Ca 2ϩ and calmodulin. EXPERIMENTAL PROCEDURES Expression of IP 3 R-1 in Sf9Insect Cells-The full-length neuronal mouse IP 3 R-1 cDNA clone containing the S1 splice domain in p400C1 plasmid vector (22) was kindly provided by Drs. K. Mikoshiba and A. Miyawaki (University of Tokyo, Tokyo, Japan). The 5Ј-untranslated region of the original p400C1 clone was removed by polymerase chain reaction (PCR) by amplification of the 5Ј-terminal part up to the Cel-II restriction site (nucleotide 555), before subcloning the IP 3 R-1 cDNA in the baculovirus (Autographa californica) transfer vector pVL 1393 (Invitrogen). Recombinant virus was produced in Spodoptera frugiperda (Sf9) cells by cotransfection of the pVL 1393 IP 3 R-1 construct and the linearized A. californica nuclear polyhydrosis virus DNA (BaculoGold, Pharmingen). The recombinant viruses were purified by isolating individual plaques of transfected cells. These clonal viral populations were amplified by infecting Sf9 cells. The recombinant protein was harvested

1 is a second messenger used by many cell types to release Ca 2ϩ from internal stores (1). Cytosolic Ca 2ϩ has a bell-shaped effect on the IP 3 receptor (IP 3 R), with low concentrations stimulating the release and high concentrations inhibiting it (2-5). Calmodulin, a Ca 2ϩ -binding protein abundantly present in many cell types (6), binds to the modulatory region of the IP 3 R in a Ca 2ϩ -dependent way (7,8). Calmodulin also interacts with the IP 3 R in a Ca 2ϩ -independent way (9 -11), with one of the interaction sites located within the IP 3 -binding domain (11

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