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

A recombinant protein (Lbs-1) containing the N-terminal 581 amino acids of the mouse type 1 inositol 1,4,5-trisphosphate receptor (IP3R-1), including the complete IP3-binding site, was expressed in the soluble fraction of E, coli, The characteristics of IP3 binding to this protein were similar as observed previously for the intact IP3R-1, Ca2+ dose-dependently inhibited IP3 binding to Lbs-1 with an IC50 of about 200 nM. This effect represented a decrease in the affinity of Lbs-1 for IP3, because the K-d increased from 115 +/- 15 nM in the absence to 196 +/- 18 nM in the presence of 5 mu M Ca2+, The maximal effect of Ca2+ on Lbs-1 (5 mu M Ca2+, 42.0 +/- 6.4% inhibition) was similar to the maximal inhibition observed for microsomes of insect Sf9 cells expressing full-length IP3R-1 (33.8 +/- 10.2%), Conceivably, the two contiguous Ca2+-binding sites (residues 304-450 of mouse IP3R-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 Ca2+ on IP3 binding to IP3R-1, Calmodulin also dose-dependently inhibited IP3 binding to Lbs-1 with an IC50 of about 3 mu M Maximal inhibition (10 mu M calmodulin, 43.1 +/- 5.9%) was similar as observed for Sf9-IP3R-1 microsomes (35.8 +/- 8.7%). Inhibition by calmodulin occurred independently of Ca2+ and was additive to the inhibitory effect of 5 mu M Ca2+ (together 74.5 +/- 5.1%). These results suggest that the N-terminal Ligand-binding region of IP3R-1 contains a calmodulin-binding domain that binds calmodulin independently of Ca2+ and that mediates the inhibition of IP3 binding to IP3R-1.