Effect of mutation of a calmodulin binding site on Ca2+ regulation of inositol trisphosphate receptors

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Abstract
Several studies have shown that calmodulin (CaM) modulates d-myo-inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) channel activity and ligand binding to IP3Rs. It has been proposed that CaM may act as the Ca2+ sensor for mediating Ca2+ inhibition of IP3R channel activity. However, the functional role of CaM binding sites and the mechanism by which CaM regulates IP3R activities remains unclear. Tryptophan at position 1577 of type I IP3R has been shown to be part of a motif that is responsible for CaM binding to IP3Rs and we have mutated this residue to alanine in the long (neuronal) and short (peripheral) SII splice variants of the type I IP3R. CaM–Sepharose binding assays using COS-7 cell lysates confirmed that the W1577A mutant in both splice variants completely eliminated CaM binding. Functional measurements of IP3-mediated 45Ca2+ fluxes indicated that there was no change in the IP3 sensitivity of the channel induced by the W1577A mutation. Such measurements also indicated that the W1577A mutants of both splice variants have a dependence on external [Ca2+] that was indistinguishable from the corresponding wild-types. Although subtle differences in the Ca2+ and CaM sensitivity of [3H]IP3 binding were noted between wild-type and mutant receptors, our data suggest that the CaM binding motif involving the W1577A locus does not play a role in Ca2+ regulation of IP3R channel activity.