Permeant calcium ion feed-through regulation of single inositol 1,4,5-trisphosphate receptor channel gating.

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+)) 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+) on homotetrameric rat type 3 InsP(3)R channel activity. In optimal Ca(2+) and subsaturating [InsP(3)], jumps of Ca(2+) from 70 nM to 300 µM reduced channel activity significantly. This inhibition was abrogated by saturating InsP(3) but restored when Ca(2+) was raised to 1.1 mM. In suboptimal Ca(2+), jumps of Ca(2+) (70 nM to 300 µM) enhanced channel activity. Thus, Ca(2+) effects on channel activity exhibited a biphasic dependence on Ca(2+). In addition, the effect of high Ca(2+) 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+), raising local Ca(2+) around the channel to regulate its activity through its cytoplasmic regulatory Ca(2+)-binding sites. Importantly, Ca(2+) regulation of InsP(3)R channel activity depended on cytoplasmic Ca(2+)-buffering conditions: it was more pronounced when Ca(2+) 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+) were completely abolished. Collectively, these results rule out Ca(2+) regulation of channel activity by direct binding to the luminal aspect of the channel.