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(3)R) 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(3)R 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(3)R 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(3)R 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(3)R activity and cytosolic Ca(2+).

SUBMITTER: Shinohara T 

PROVIDER: S-EPMC3174635 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature