The InsP3R Ca2+ release channel has a biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca 2+]i). InsP3 activates gating primarily by reducing the sensitivity of the channel to inhibition by high [Ca 2+]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 InsP 3, 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 Po (∼0.03) were observed in [Ca2+] i < 5 nM with the same frequency as in the presence of InsP 3, 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 InsP 3, 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 Ca 2+-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 Po 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+]i.
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