Spontaneous Channel Activity of the Inositol 1,4,5-Trisphosphate (InsP₃) Receptor (InsP₃R). Application of Allosteric Modeling to Calcium and InsP₃ Regulation of InsP₃R Single-channel Gating

The InsP₃R Ca²⁺ release channel has a biphasic dependence on cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i). InsP₃ activates gating primarily by reducing the sensitivity of the channel to inhibition by high [Ca²⁺]_i. To determine if relieving Ca²⁺ inhibition is sufficient for channel activation, we examined single-channel activities in low [Ca²⁺]_i in the absence of InsP₃, by patch clamping isolated Xenopus oocyte nuclei. For both endogenous Xenopus type 1 and recombinant rat type 3 InsP₃R channels, spontaneous InsP₃-independent channel activities with low open probability P_o (∼0.03) were observed in [Ca²⁺]_i < 5 nM with the same frequency as in the presence of InsP₃, whereas no activities were observed in 25 nM Ca²⁺. These results establish the half-maximal inhibitory [Ca²⁺]_i of the channel to be 1.2–4.0 nM in the absence of InsP₃, and demonstrate that the channel can be active when all of its ligand-binding sites (including InsP₃) are unoccupied. In the simplest allosteric model that fits all observations in nuclear patch-clamp studies of [Ca²⁺]_i and InsP₃ regulation of steady-state channel gating behavior of types 1 and 3 InsP₃R isoforms, including spontaneous InsP₃-independent channel activities, the tetrameric channel can adopt six different conformations, the equilibria among which are controlled by two inhibitory and one activating Ca²⁺-binding and one InsP₃-binding sites in a manner outlined in the Monod-Wyman-Changeux model. InsP₃ binding activates gating by affecting the Ca²⁺ affinities of the high-affinity inhibitory sites in different conformations, transforming it into an activating site. Ca²⁺ inhibition of InsP₃-liganded channels is mediated by an InsP₃-independent low-affinity inhibitory site. The model also suggests that besides the ligand-regulated gating mechanism, the channel has a ligand-independent gating mechanism responsible for maximum channel P_o being less than unity. The validity of this model was established by its successful quantitative prediction of channel behavior after it had been exposed to ultra-low bath [Ca²⁺].