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

Abstract

The regulation of inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R) activity is thought to define the spatiotemporal 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. Ca²⁺, however regulates IP₃R activity in a biphasic manner. 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 mutated a cluster of acidic residues in the ARM2 and central linker domain of IP₃R type-1, reported to coordinate Ca²⁺ in cryo-EM structures of the IP₃R type 3. This "CD Ca²⁺ binding site" is well-conserved in all IP₃R sub-types. CD site Ca²⁺ binding mutants where the negatively charged glutamic acid residues were mutated to alanine exhibited enhanced sensitivity to IP₃-generating agonists. Ca²⁺ binding mutants displayed spontaneous elemental Ca²⁺ puffs and the number of IP₃-induced Ca²⁺ puffs were augmented in cells stably expressing Ca²⁺ binding site mutants. The inhibitory effect of high [Ca²⁺] on single channel-open probability (P_o) was reduced in mutant channels and this effect was dependent on [ATP]. This indicates that Ca²⁺ binding to the putative CD Ca²⁺ inhibitory site facilitates the reduction in IP₃R channel activation at subsaturating, likely physiological cytosolic [ATP], and suggest that at higher [ATP], additional Ca²⁺ binding motifs may contribute to the biphasic regulation of IP₃-induced Ca²⁺ release.