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 (IP3) receptor (IP3R) activity resulting in modulation of intracellular Ca2+ release. This regulation is thought to define the spatio-temporal patterns of Ca2+ signals necessary for the appropriate activation of downstream effectors. The binding of both IP3 and Ca2+ are obligatory for IP3R channel opening, however, Ca2+ regulates IP3R activity in a biphasic manner. Mutational studies have revealed that Ca2+ binding to a high-affinity pocket formed by the ARM3 domain and linker domain promotes IP3R channel opening without altering the Ca2+ dependency for channel inactivation. These data suggest a distinct low-affinity Ca2+ binding site is responsible for the reduction in IP3R activity at higher [Ca2+]. We determined the consequences of mutating a cluster of acidic residues in the ARM2 and central linker domain reported to coordinate Ca2+ in cryo-EM structures of the IP3R type 3. This site is termed the "CD Ca2+ binding site" and is well-conserved in all IP3R sub-types. We show that the CD site Ca2+ binding mutants where the negatively charged glutamic acid residues are mutated to alanine exhibited enhanced sensitivity to IP3-generating agonists. Ca2+ binding mutants displayed spontaneous elemental Ca2+ events (Ca2+ puffs) and the number of IP3-induced Ca2+ puffs was significantly augmented in cells stably expressing Ca2+ binding site mutants. When measured with "on-nucleus" patch clamp, the inhibitory effect of high [Ca2+] on single channel-open probability (Po) was reduced in mutant channels and this effect was dependent on [ATP]. These results indicate that Ca2+ binding to the putative CD Ca2+ inhibitory site facilitates the reduction in IP3R channel activation when cytosolic [ATP] is reduced and suggest that at higher [ATP], additional Ca2+ binding motifs may contribute to the biphasic regulation of IP3-induced Ca2+ release.