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

A wide variety of factors influence inositol 1,4,5-trisphosphate (IP ) receptor (IP R) activity resulting in modulation of intracellular Ca release. This regulation is thought to define the spatio-temporal patterns of Ca signals necessary for the appropriate activation of downstream effectors. The binding of both IP and Ca are obligatory for IP R channel opening, however, Ca regulates IP R activity in a biphasic manner. Mutational studies have revealed that Ca binding to a high-affinity pocket formed by the ARM3 domain and linker domain promotes IP R channel opening without altering the Ca dependency for channel inactivation. These data suggest a distinct low-affinity Ca binding site is responsible for the reduction in IP R activity at higher [Ca ]. We determined the consequences of mutating a cluster of acidic residues in the ARM2 and central linker domain reported to coordinate Ca in cryo-EM structures of the IP R type 3. This site is termed the "CD Ca binding site" and is well-conserved in all IP R sub-types. We show that the CD site Ca binding mutants where the negatively charged glutamic acid residues are mutated to alanine exhibited enhanced sensitivity to IP -generating agonists. Ca binding mutants displayed spontaneous elemental Ca events (Ca puffs) and the number of IP -induced Ca puffs was significantly augmented in cells stably expressing Ca binding site mutants. When measured with "on-nucleus" patch clamp, the inhibitory effect of high [Ca ] on single channel-open probability (P ) was reduced in mutant channels and this effect was dependent on [ATP]. These results indicate that Ca binding to the putative CD Ca inhibitory site facilitates the reduction in IP R channel activation when cytosolic [ATP] is reduced and suggest that at higher [ATP], additional Ca binding motifs may contribute to the biphasic regulation of IP -induced Ca release.