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.