title: 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+).
authors.affiliation: Laboratory for Developmental Neurobiology, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan.
authors.forename: Tadashi
authors.initials: T
authors.lastname: Shinohara
authors.forename: Takayuki
authors.initials: T
authors.lastname: Michikawa
authors.forename: Masahiro
authors.initials: M
authors.lastname: Enomoto
authors.forename: Jun-Ichi
authors.initials: J
authors.lastname: Goto
authors.forename: Miwako
authors.initials: M
authors.lastname: Iwai
authors.forename: Toru
authors.initials: T
authors.lastname: Matsu-ura
authors.forename: Haruka
authors.initials: H
authors.lastname: Yamazaki
authors.forename: Akitoshi
authors.initials: A
authors.lastname: Miyamoto
authors.forename: Akio
authors.initials: A
authors.lastname: Suzuki
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Mikoshiba
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism*; D015640:Ion Channel Gating / Q000187:drug effects; D008024:Ligands; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008024:Ligands; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C527701:enhanced cyan fluorescent protein; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D002118:Calcium