Tuning Protein Dynamics to Sense Rapid Endoplasmic-Reticulum Calcium Dynamics.

Multi-scale calcium (Ca2+ ) dynamics, exhibiting wide-ranging temporal kinetics, constitutes a ubiquitous mode of signal transduction. We report a novel endoplasmic-reticulum (ER)-targeted Ca2+ indicator, R-CatchER, which showed superior kinetics in vitro (koff ≥2×103  s-1 , kon ≥7×106  M-1  s-1 ) and in multiple cell types. R-CatchER captured spatiotemporal ER Ca2+ dynamics in neurons and hotspots at dendritic branchpoints, enabled the first report of ER Ca2+ oscillations mediated by calcium sensing receptors (CaSRs), and revealed ER Ca2+ -based functional cooperativity of CaSR. We elucidate the mechanism of R-CatchER and propose a principle to rationally design genetically encoded Ca2+ indicators with a single Ca2+ -binding site and fast kinetics by tuning rapid fluorescent-protein dynamics and the electrostatic potential around the chromophore. The design principle is supported by the development of G-CatchER2, an upgrade of our previous (G-)CatchER with improved dynamic range. Our work may facilitate protein design, visualizing Ca2+ dynamics, and drug discovery.