Faculty, Staff and Student Publications

Language

English

Publication Date

3-1-2025

Journal

Journal of Biological Chemistry

DOI

10.1016/j.jbc.2025.108302

PMID

39947469

PMCID

PMC11938044

PubMedCentral® Posted Date

2-11-2025

PubMedCentral® Full Text Version

Post-print

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+ is obligatory for IP3R channel opening. Ca2+ however regulates IP3R activity in a biphasic manner. Ca2+ binding to a high-affinity pocket formed by the third armadillo repeat domain and linker domain promotes IP3R channel opening without altering the Ca2+ dependency for channel inactivation. These data suggest that 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 second armadillo repeat domain and central linker domain of IP3R type 1, reported to coordinate Ca2+ in the cryo-EM structures of the IP3R type 3. This "CD Ca2+-binding site" is well conserved in all IP3R subtypes. 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 was augmented in cells stably expressing Ca2+-binding site mutants. The inhibitory effect of high [Ca2+] on single-channel open probability (Po) 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.

Keywords

Inositol 1, 4, 5-Trisphosphate Receptors, Binding Sites, Calcium, Humans, Animals, HEK293 Cells, Calcium Signaling, ATP; calcium; inositol 1, 4, 5-trisphosphate receptors; single-channel recording; TIRF imaging

Published Open-Access

yes

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