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

Multi-scale calcium (Ca ²⁺ ) dynamics, exhibiting wide-ranging temporal kinetics, constitutes a ubiquitous mode of signal transduction. We report a novel endoplasmic-reticulum (ER)-targeted Ca ²⁺ indicator, R-CatchER, which showed superior kinetics in vitro (k _off ≥2×10 ³  s ^-1 , k _on ≥7×10 ⁶  M ^-1  s ^-1 ) and in multiple cell types. R-CatchER captured spatiotemporal ER Ca ²⁺ dynamics in neurons and hotspots at dendritic branchpoints, enabled the first report of ER Ca ²⁺ oscillations mediated by calcium sensing receptors (CaSRs), and revealed ER Ca ²⁺ -based functional cooperativity of CaSR. We elucidate the mechanism of R-CatchER and propose a principle to rationally design genetically encoded Ca ²⁺ indicators with a single Ca ²⁺ -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 Ca ²⁺ dynamics, and drug discovery.
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