Functional investigation of a putative calcium-binding site involved in the inhibition of inositol 1,4,5-trisphosphate receptor activity.

The regulation of inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) activity is thought to define the spatiotemporal patterns of Ca(2+) signals necessary for the appropriate activation of downstream effectors. The binding of both IP(3) and Ca(2+) is obligatory for IP(3)R channel opening. Ca(2+) however regulates IP(3)R activity in a biphasic manner. Ca(2+) binding to a high-affinity pocket formed by the third armadillo repeat domain and linker domain promotes IP(3)R channel opening without altering the Ca(2+) dependency for channel inactivation. These data suggest that a distinct low-affinity Ca(2+)-binding site is responsible for the reduction in IP(3)R activity at higher [Ca(2+)]. We mutated a cluster of acidic residues in the second armadillo repeat domain and central linker domain of IP(3)R type 1, reported to coordinate Ca(2+) in the cryo-EM structures of the IP(3)R type 3. This "CD Ca(2+)-binding site" is well conserved in all IP(3)R subtypes. CD site Ca(2+)-binding mutants where the negatively charged glutamic acid residues were mutated to alanine exhibited enhanced sensitivity to IP(3)-generating agonists. Ca(2+)-binding mutants displayed spontaneous elemental Ca(2+) puffs, and the number of IP(3)-induced Ca(2+) puffs was augmented in cells stably expressing Ca(2+)-binding site mutants. The inhibitory effect of high [Ca(2+)] on single-channel open probability (P(o)) was reduced in mutant channels, and this effect was dependent on [ATP]. This indicates that Ca(2+) binding to the putative CD Ca(2+) inhibitory site facilitates the reduction in IP(3)R channel activation at subsaturating, likely physiological cytosolic [ATP], and suggest that at higher [ATP], additional Ca(2+)-binding motifs may contribute to the biphasic regulation of IP(3)-induced Ca(2+) release.