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 (k_off ≥2×10³  s^-1 , k_on ≥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.