Rapid ligand-regulated gating kinetics of single inositol 1,4,5-trisphosphate receptor Ca2+ release channels

The ubiquitous inositol 1,4,5-trisphosphate receptor (InsP3R) intracellular Ca2+ release channel is engaged by thousands of plasma membrane receptors to generate Ca2+ signals in all cells. Understanding how complex Ca2+ signals are generated has been hindered by a lack of information on the kinetic responses of the channel to its primary ligands, InsP3 and Ca2+, which activate and inhibit channel gating. Here, we describe the kinetic responses of single InsP3R channels in native endoplasmic reticulum membrane to rapid ligand concentration changes with millisecond resolution, using a new patch-clamp configuration. The kinetics of channel activation and deactivation showed novel Ca2+ regulation and unexpected ligand cooperativity. The kinetics of Ca2+-mediated channel inhibition showed the single-channel bases for fundamental Ca2+ release events and Ca2+ release refractory periods. These results provide new insights into the channel regulatory mechanisms that contribute to complex spatial and temporal features of intracellular Ca2+ signals

Redox-Regulated Heterogeneous Thresholds for Ligand Recruitment among InsP3R Ca2+-Release Channels

To clarify the molecular mechanisms behind quantal Ca2+ release, the graded Ca2+ release from intracellular stores through inositol 1,4,5-trisphosphate receptor (InsP3R) channels responding to incremental ligand stimulation, single-channel patch-clamp electrophysiology was used to continuously monitor the number and open probability of InsP3R channels in the same excised cytoplasmic-side-out nuclear membrane patches exposed alternately to optimal and suboptimal cytoplasmic ligand conditions. Progressively more channels were activated by more favorable conditions in patches from insect cells with only one InsP3R gene or from cells solely expressing one recombinant InsP3R isoform, demonstrating that channels with identical primary sequence have different ligand recruitment thresholds. Such heterogeneity was largely abrogated, in a fully reversible manner, by treatment of the channels with sulfhydryl reducing agents, suggesting that it was mostly regulated by different levels of posttranslational redox modifications of the channels. In contrast, sulfhydryl reduction had limited effects on channel open probability. Thus, sulfhydryl redox modification can regulate various aspects of intracellular Ca2+ signaling, including quantal Ca2+ release, by tuning ligand sensitivities of InsP3R channels. No intrinsic termination of channel activity with a timescale comparable to that for quantal Ca2+ release was observed under any steady ligand conditions, indicating that this process is unlikely to contribute