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