Mode Switching Is the Major Mechanism of Ligand Regulation of InsP₃ Receptor Calcium Release Channels

The inositol 1,4,5-trisphosphate (InsP₃) receptor (InsP₃R) plays a critical role in generation of complex Ca²⁺ signals in many cell types. In patch clamp recordings of isolated nuclei from insect Sf9 cells, InsP₃R channels were consistently detected with regulation by cytoplasmic InsP₃ and free Ca²⁺ concentrations ([Ca²⁺]_i) very similar to that observed for vertebrate InsP₃R. Long channel activity durations of the Sf9-InsP₃R have now enabled identification of a novel aspect of InsP₃R gating: modal gating. Using a novel algorithm to analyze channel modal gating kinetics, InsP₃R gating can be separated into three distinct modes: a low activity mode, a fast kinetic mode, and a burst mode with channel open probability (P_o) within each mode of 0.007 ± 0.002, 0.24 ± 0.03, and 0.85 ± 0.02, respectively. Channels reside in each mode for long periods (tens of opening and closing events), and transitions between modes can be discerned with high resolution (within two channel opening and closing events). Remarkably, regulation of channel gating by [Ca²⁺]_i and [InsP₃] does not substantially alter channel P_o within a mode. Instead, [Ca²⁺]_i and [InsP₃] affect overall channel P_o primarily by changing the relative probability of the channel being in each mode, especially the high and low P_o modes. This novel observation therefore reveals modal switching as the major mechanism of physiological regulation of InsP₃R channel activity, with implications for the kinetics of Ca²⁺ release events in cells.