Ca2+ and calmodulin differentially modulate myo-inositol 1,4,5-trisphosphate (IP3)-binding to the recombinant ligand-binding domains of the various IP3 receptor isoforms

We have expressed the N-terminal 581 amino acids of type 1 myo-inositol 1,4,5-trisphosphate receptor (IP₃R1), IP₃R2 and IP₃R3 as recombinant proteins [ligand-binding site 1 (lbs-1), lbs-2, lbs-3] in the soluble fraction of Escherichia coli. These recombinant proteins contain the complete IP₃-binding domain and bound IP₃ and adenophostin A with high affinity. Ca²⁺ and calmodulin were previously found to maximally inhibit IP₃ binding to lbs-1 by 42±6 and 43±6% respectively, and with an IC₅₀ of approx. 200 nM and 3 μM respectively [Sipma, De Smet, Sienaert, Vanlingen, Missiaen, Parys and De Smedt (1999) J. Biol. Chem. 274, 12157-12562]. We now report that Ca²⁺ inhibited IP₃ binding to lbs-3 with an IC₅₀ of approx. 700 nM (37±4% inhibition at 5 μM Ca²⁺), while IP₃ binding to lbs-2 was not affected by increasing [Ca²⁺] from 100 nM to 25 μM. Calmodulin (10 μM) inhibited IP₃ binding to lbs-3 by 37±4%, while IP₃ binding to lbs-2 was inhibited by only 11±2%. The inhibition of IP₃ binding to lbs-3 by calmodulin was dose-dependent (IC₅₀≈ 2 μM). We conclude that the IP₃-binding domains of the various IP₃R isoforms differ in binding characteristics for IP₃ and adenophostin A, and are differentially modulated by Ca²⁺ and calmodulin, suggesting that the various IP₃R isoforms can have different intracellular functions.