Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 108, Issue 37, Pages 15486-15491Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1101677108
Keywords
calcium signal; channel gating; ion channel
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Funding
- RIKEN
- Ministry of Education, Culture, Sports, Science and Technology of Japan [20700344, 20370054, 20220007]
- Grants-in-Aid for Scientific Research [20700344, 20370054, 20220007] Funding Source: KAKEN
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The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) is an intracellular Ca2+ release channel, and its opening is controlled by IP3 and Ca2+. A single IP3 binding site and multiple Ca2+ binding sites exist on single subunits, but the precise nature of the interplay between these two ligands in regulating biphasic dependence of channel activity on cytosolic Ca2+ is unknown. In this study, we visualized conformational changes in IP3R evoked by various concentrations of ligands by using the FRET between two fluorescent proteins fused to the N terminus of individual subunits. IP3 and Ca2+ have opposite effects on the FRET signal change, but the combined effect of these ligands is not a simple summative response. The bell-shaped Ca2+ dependence of FRET efficiency was observed after the subtraction of the component corresponding to the FRET change evoked by Ca2+ alone from the FRET changes evoked by both ligands together. A mutant IP3R containing a single amino acid substitution at K508, which is critical for IP3 binding, did not exhibit this bell-shaped Ca2+ dependence of the subtracted FRET efficiency. Mutation at E2100, which is known as a Ca2+ sensor, resulted in similar to 10-fold reduction in the Ca2+ dependence of the subtracted signal. These results suggest that the subtracted FRET signal reflects IP3R activity. We propose a five-state model, which implements a dual-ligand competition response without complex allosteric regulation of Ca2+ binding affinity, as the mechanism underlying the IP3-dependent regulation of the bell-shaped relationship between the IP3R activity and cytosolic Ca2+.
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