title: Permeant calcium ion feed-through regulation of single inositol 1,4,5-trisphosphate receptor channel gating.
abstract: The ubiquitous inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP(3)R) Ca(2+) release channel plays a central role in the generation and modulation of intracellular Ca(2+) signals, and is intricately regulated by multiple mechanisms including cytoplasmic ligand (InsP(3), free Ca(2+), free ATP(4-)) binding, posttranslational modifications, and interactions with cytoplasmic and endoplasmic reticulum (ER) luminal proteins. However, regulation of InsP(3)R channel activity by free Ca(2+) in the ER lumen ([Ca(2+)](ER)) remains poorly understood because of limitations of Ca(2+) flux measurements and imaging techniques. Here, we used nuclear patch-clamp experiments in excised luminal-side-out configuration with perfusion solution exchange to study the effects of [Ca(2+)](ER) on homotetrameric rat type 3 InsP(3)R channel activity. In optimal [Ca(2+)](i) and subsaturating [InsP(3)], jumps of [Ca(2+)](ER) from 70 nM to 300 µM reduced channel activity significantly. This inhibition was abrogated by saturating InsP(3) but restored when [Ca(2+)](ER) was raised to 1.1 mM. In suboptimal [Ca(2+)](i), jumps of [Ca(2+)](ER) (70 nM to 300 µM) enhanced channel activity. Thus, [Ca(2+)](ER) effects on channel activity exhibited a biphasic dependence on [Ca(2+)](i). In addition, the effect of high [Ca(2+)](ER) was attenuated when a voltage was applied to oppose Ca(2+) flux through the channel. These observations can be accounted for by Ca(2+) flux driven through the open InsP(3)R channel by [Ca(2+)](ER), raising local [Ca(2+)](i) around the channel to regulate its activity through its cytoplasmic regulatory Ca(2+)-binding sites. Importantly, [Ca(2+)](ER) regulation of InsP(3)R channel activity depended on cytoplasmic Ca(2+)-buffering conditions: it was more pronounced when [Ca(2+)](i) was weakly buffered but completely abolished in strong Ca(2+)-buffering conditions. With strong cytoplasmic buffering and Ca(2+) flux sufficiently reduced by applied voltage, both activation and inhibition of InsP(3)R channel gating by physiological levels of [Ca(2+)](ER) were completely abolished. Collectively, these results rule out Ca(2+) regulation of channel activity by direct binding to the luminal aspect of the channel.
authors.affiliation: Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Horia
authors.initials: H
authors.lastname: Vais
authors.forename: J Kevin
authors.initials: JK
authors.lastname: Foskett
authors.forename: Ghanim
authors.initials: G
authors.lastname: Ullah
authors.forename: John E
authors.initials: JE
authors.lastname: Pearson
authors.forename: Don-On Daniel
authors.initials: DO
authors.lastname: Mak
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism*; D015640:Ion Channel Gating / Q000502:physiology*; D007700:Kinetics; D008024:Ligands; D051381:Rats
chemical_list: D053496:Inositol 1,4,5-Trisphosphate Receptors; D008024:Ligands; D002118:Calcium