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 ₃ ) receptor (IP ₃ R) activity resulting in modulation of intracellular Ca ²⁺ release. This regulation is thought to define the spatio-temporal patterns of Ca ²⁺ signals necessary for the appropriate activation of downstream effectors. The binding of both IP ₃ and Ca ²⁺ are obligatory for IP ₃ R channel opening, however, Ca ²⁺ regulates IP ₃ R activity in a biphasic manner. Mutational studies have revealed that Ca ²⁺ binding to a high-affinity pocket formed by the ARM3 domain and linker domain promotes IP ₃ R channel opening without altering the Ca ²⁺ dependency for channel inactivation. These data suggest a distinct low-affinity Ca ²⁺ binding site is responsible for the reduction in IP ₃ R activity at higher [Ca ²⁺ ]. We determined the consequences of mutating a cluster of acidic residues in the ARM2 and central linker domain reported to coordinate Ca ²⁺ in cryo-EM structures of the IP ₃ R type 3. This site is termed the “CD Ca ²⁺ binding site” and is well-conserved in all IP ₃ R sub-types. We show that the CD site Ca ²⁺ binding mutants where the negatively charged glutamic acid residues are mutated to alanine exhibited enhanced sensitivity to IP ₃ -generating agonists. Ca ²⁺ binding mutants displayed spontaneous elemental Ca ²⁺ events (Ca ²⁺ puffs) and the number of IP ₃ -induced Ca ²⁺ puffs was significantly augmented in cells stably expressing Ca ²⁺ binding site mutants. When measured with “on-nucleus” patch clamp, 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]. These results indicate that Ca ²⁺ binding to the putative CD Ca ²⁺ inhibitory site facilitates the reduction in IP ₃ R channel activation when cytosolic [ATP] is reduced and suggest that at higher [ATP], additional Ca ²⁺ binding motifs may contribute to the biphasic regulation of IP ₃ -induced Ca ²⁺ release.