Spontaneous Channel Activity of the Inositol 1,4,5-Trisphosphate (InsP(3)) Receptor (InsP(3)R). Application of Allosteric Modeling to Calcium and InsP(3) Regulation of InsP(3)R Single-channel Gating

The InsP(3)R Ca(2+) release channel has a biphasic dependence on cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)). InsP(3) activates gating primarily by reducing the sensitivity of the channel to inhibition by high [Ca(2+)](i). To determine if relieving Ca(2+) inhibition is sufficient for channel activation, we examined single-channel activities in low [Ca(2+)](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 InsP(3)R channels, spontaneous InsP(3)-independent channel activities with low open probability P (o) (∼0.03) were observed in [Ca(2+)](i) < 5 nM with the same frequency as in the presence of InsP(3), whereas no activities were observed in 25 nM Ca(2+). These results establish the half-maximal inhibitory [Ca(2+)](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 InsP(3)) are unoccupied. In the simplest allosteric model that fits all observations in nuclear patch-clamp studies of [Ca(2+)](i) and InsP(3) regulation of steady-state channel gating behavior of types 1 and 3 InsP(3)R isoforms, including spontaneous InsP(3)-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 InsP(3)-binding sites in a manner outlined in the Monod-Wyman-Changeux model. InsP(3) binding activates gating by affecting the Ca(2+) affinities of the high-affinity inhibitory sites in different conformations, transforming it into an activating site. Ca(2+) inhibition of InsP(3)-liganded channels is mediated by an InsP(3)-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 [Ca(2+)].