Modulation of cytoplasmic free Ca2+ -induced InsP 3 R sequestration and inactivation can account for these observations. These results suggest that apparent heterogeneous ligand sensitivity can be generated in a homogeneous population of InsP 3 R channels, providing a mechanism for graded Ca 2+ release that is intrinsic to the InsP 3 R Ca 2+ release channel itself.

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.

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