Mechanistic basis of bell-shaped dependence of inositol 1,4,5-trisphosphate receptor gating on cytosolic calcium

Abstract

The inositol 1,4,5-trisphosphate (IP 3) receptor (IP 3R) is an intracellular Ca 2+ release channel, and its opening is controlled by IP 3 and Ca 2+. A single IP 3 binding site and multiple Ca 2+ 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 Ca 2+ is unknown. In this study, we visualized conformational changes in IP 3R evoked by various concentrations of ligands by using the FRET between two fluorescent proteins fused to the N terminus of individual subunits. IP 3 and Ca 2+ have opposite effects on the FRET signal change, but the combined effect of these ligands is not a simple summative response. The bell-shaped Ca 2+ dependence of FRET efficiency was observed after the subtraction of the component corresponding to the FRET change evoked by Ca 2+ alone from the FRET changes evoked by both ligands together. A mutant IP 3R containing a single amino acid substitution at K508, which is critical for IP 3 binding, did not exhibit this bell-shaped Ca 2+ dependence of the subtracted FRET efficiency. Mutation at E2100, which is known as a Ca 2+ sensor, resulted in ∼10-fold reduction in the Ca 2+ dependence of the subtracted signal. These results suggest that the subtracted FRET signal reflects IP 3R activity. We propose a five-state model, which implements a dual-ligand competition response without complex allosteric regulation of Ca 2+ binding affinity, as the mechanism underlying the IP 3-dependent regulation of the bell-shaped relationship between the IP 3R activity and cytosolic Ca 2+.