Ca2+ and calmodulin differentially modulate myo-inositol 1,4,5-trisphosphate (IP)3-binding to the recombinant ligand-binding domains of the various IP3 receptor isoforms

We have expressed the N-terminal 581 amino acids of type I myo-inositol 1,4,5-trisphosphate receptor (IP(3)R1), IP(3)R2 and IP3RS 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 mu M 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 mu M Ca2+), while IP3 binding to lbs-2 was not affected by increasing [Ca2+] from 100 nM to 25 mu M. Calmodulin (10 mu M) 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 approximate to 2 mu M). 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.