Modulation of Inositol 1,4,5-Trisphosphate Binding to the Recombinant Ligand-binding Site of the Type-1 Inositol 1,4,5-Trisphosphate Receptor by Ca2+ and Calmodulin

• Published in: The Journal of biological chemistry 1999-04, Vol.274 (17), p.12157-12162
• Main Authors: Sipma, H, De Smet, P, Sienaert, I, Vanlingen, S, Missiaen, L, Parys, J B, De Smedt, H
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Binding Sites; Calcium - metabolism; Calcium Channels - metabolism; Calmodulin - metabolism; DNA Primers; Inositol 1,4,5-Trisphosphate - metabolism; Inositol 1,4,5-Trisphosphate Receptors; Mice; Protein Binding; Receptors, Cytoplasmic and Nuclear - metabolism; Recombinant Proteins - metabolism; Spodoptera
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.17.12157

A recombinant protein (Lbs-1) containing the N-terminal 581 amino acids of the mouse type 1 inositol 1,4,5-trisphosphate receptor (IP 3 R-1), including the complete IP 3 -binding site, was expressed in the soluble fraction of E. coli . The characteristics of IP 3 binding to this protein were similar as observed previously for the intact IP 3 R-1. Ca 2+ dose-dependently inhibited IP 3 binding to Lbs-1 with an IC 50 of about 200 n m . This effect represented a decrease in the affinity of Lbs-1 for IP 3, because the K d increased from 115 ± 15 n m in the absence to 196 ± 18 n m in the presence of 5 μ m Ca 2+ . The maximal effect of Ca 2+ on Lbs-1 (5 μ m Ca 2+ , 42.0 ± 6.4% inhibition) was similar to the maximal inhibition observed for microsomes of insect Sf9 cells expressing full-length IP 3 R-1 (33.8 ± 10.2%). Conceivably, the two contiguous Ca 2+ -binding sites (residues 304–450 of mouse IP 3 R-1) previously found by us (Sienaert, I., Missiaen, L., De Smedt, H., Parys, J.B., Sipma, H., and Casteels, R. (1997) J. Biol. Chem. 272, 25899–25906) mediate the effect of Ca 2+ on IP 3 binding to IP 3 R-1. Calmodulin also dose-dependently inhibited IP 3 binding to Lbs-1 with an IC 50 of about 3 μ m . Maximal inhibition (10 μ m calmodulin, 43.1 ± 5.9%) was similar as observed for Sf9-IP 3 R-1 microsomes (35.8 ± 8.7%). Inhibition by calmodulin occurred independently of Ca 2+ and was additive to the inhibitory effect of 5 μ m Ca 2+ (together 74.5 ± 5.1%). These results suggest that the N-terminal ligand-binding region of IP 3 R-1 contains a calmodulin-binding domain that binds calmodulin independently of Ca 2+ and that mediates the inhibition of IP 3 binding to IP 3 R-1.