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

摘要

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+.